Method for producing a nanoemulsion with encapsulated natural antioxidants for preserving fresh and minimally processed foods, and the nanoemulsion thus produced

Abstract

A process for producing a nanoemulsion formulation comprising the following main stages: (a) extraction of natural antioxidants from peels or seeds of fruits, vegetables or cereals, wherein the extraction is carried out with pure water, with a concentration of the extracted natural antioxidants with a vacuum distillation method between 0.5-15 inHg at 20-60° C. for 10-95 minutes until the concentration of the extracted natural antioxidants is between 10-50 wt % and then a tangential nanofiltration of the concentrated natural antioxidants; (b) encapsulation of the natural antioxidants; (c) formation of a nanoemulsion; and (d) cryodrying of the formed nanoemulsion.

Claims

1. A process for producing a nanoemulsion formulation with encapsulated natural antioxidants for the preservation of fresh fruits, vegetables, cereals, juices and minimally processed foods, comprising the following stages: a. performing an extraction of natural antioxidants from peels or seeds of fruits, vegetables or cereals, wherein the extraction is carried out with pure water, with a concentration of natural antioxidants with a vacuum distillation method between 0.5-15 inches of Hg at 20-60° C. for 10-95 minutes until a concentration of natural antioxidants is between 10-50 wt. % and then a tangential nanofiltration is performed on the concentration of natural antioxidants; b. performing an encapsulation of the concentration of natural antioxidants generated after the tangential nanofiltration to produce encapsulated natural antioxidants; c. forming a nanoemulsion with the encapsulated natural antioxidants; and d. cryodrying the nanoemulsion with the encapsulated natural antioxidants.

2. The process for producing a nanoemulsion formulation with encapsulated natural antioxidants for the preservation of fresh fruits, vegetables, cereals, juices and minimally processed foods, according to claim 1, wherein the extraction of natural antioxidants comprises the following stages: a. sorting, washing and disinfecting the peels or seeds of the fruits, vegetables or cereals to produce disinfected peels or seeds; b. dehydration of the disinfected peels or seeds in a conventional oven at a temperature of 30-60° C. for 2-6 hours with air in reflux, or by freeze-drying at a temperature of −30-−50° C. and an absolute vacuum pressure of 0.04 mbar for 10-15 hours; up to a humidity of 2-40 wt. % to obtain dehydrated peels or seeds; c. extraction with pure water, from the dehydrated peels or seeds, assisted by microwaves at a power of 100-400 W for 5-40 minutes, or ultrasound with vibration power of 20-60 kHz, for 10-40 minutes at a temperature of 30-60° C., for obtaining extracted natural antioxidants; d. concentration of the extracted natural antioxidants with a vacuum distillation method in a rotovap under vacuum between 0.5-15 inches of Hg at 20-60° C. for 10-95 minutes until obtaining concentrated natural antioxidants between 10-50 wt. %; and, e. tangential nanofiltration of the concentrated natural antioxidants, with a pH of 6-10.5, in two sequential filters with 10-100 nm nanopores and a minimum surface area of 0.01 m.sup.2, at a temperature of 30-60° C.; the concentrated natural antioxidants are pumped out at a pressure of 0.5-1 bar.

3. The process for producing a nanoemulsion formulation with encapsulated natural antioxidants for the preservation of fresh fruits, vegetables, cereals, juices and minimally processed foods, according to claim 1, wherein the encapsulation comprises the following stages: a. the concentration of natural antioxidants generated after the tangential nanofiltration mixed in a weight ratio of 1:1 with polysaccharides in an amount that replaces a percentage by weight of soluble solids measured with refractometry techniques to produce a mixture; the mixture is carried out at a speed of 500-2000 RPM, at a temperature between 20-60° C. for 1-3 hours; b. homogenization at 8000-1500 RPM for 1-15 minutes, to produce the encapsulation, which is stored at a temperature of 5-15° C.

4. The process for producing a nanoemulsion formulation with encapsulated natural antioxidants for the preservation of fresh fruits, vegetables, cereals, juices and minimally processed foods, according to claim 1, wherein the nanoemulsion formulation with the encapsulated natural antioxidants comprises the following stages: a. dilution of alginic acid in drinking water, at a temperature of 50-70° C., with constant movement at 500-1500 RPM for 1-3 hours; b. homogenization at a speed of 8000-15000 RPM for about 1-15 minutes of the alginic acid in a solution with an oily matrix, polysorbate, glycerol and the encapsulated natural antioxidant; c. microfluidization at high pressures of 100-200 MPa, for 3-5 cycles.

5. The process for producing a nanoemulsion formulation with encapsulated natural antioxidants for the preservation of fresh fruits, vegetables, cereals, juices and minimally processed foods, according to claim 1, wherein the cryodrying of the nanoemulsion comprises nanoemulsion concentration through cryodrying methods.

6. The process for producing a nanoemulsion formulation with encapsulated natural antioxidants for the preservation of fresh fruits, vegetables, cereals, juices and minimally processed foods, according to claim 5, wherein the cryodrying is performed through freeze-drying, with a vacuum pressure of 0.04 mbar and a temperature of −10 to −15° C.

7. The process for producing a nanoemulsion formulation with encapsulated natural antioxidants for the preservation of fresh fruits, vegetables, cereals, juices and minimally processed foods according to claim 1, wherein the encapsulated natural antioxidants come from a combination of peels or seeds of fruits, vegetables or cereals, and the combination is a functional formula.

8. The process for producing a nanoemulsion formulation with encapsulated natural antioxidants for the preservation of fresh fruits, vegetables, cereals, juices and minimally processed foods according to claim 4, wherein the oily matrix is a combination of coconut, canola, almond, avocado or peanut oil.

9. The process for producing a nanoemulsion formulation with encapsulated natural antioxidants for the preservation of fresh fruits, vegetables, cereals, juices and minimally processed foods according to claim 4, wherein the formulation replaces glycerol and polysorbate for an amount of 1-5 wt % of calcium ascorbate or a 1:1 weight ratio combination of ascorbic acid and citric acid, and the food to be preserved is a fruit cut under refrigeration conditions.

10. The process for producing a nanoemulsion formulation with encapsulated natural antioxidants for the preservation of fresh fruits, vegetables, cereals, juices and minimally processed foods according to claim 1, wherein the process encapsulates proteins, vitamins and minerals, which have been extracted from fruits, vegetables or cereals.

Description

BRIEF FIGURE DESCRIPTION

(1) FIG. 1. Flow chart of the process of the invention detailing its four main stages.

EXAMPLE OF PREFERRED EMBODIMENT OF THE INVENTION

(2) A. Process for Producing the Nanoemulsion with Encapsulated Natural Antioxidants

(3) A preparation of the nanoemulsion of the invention, by way of example, involves the selection, washing and disinfection of the peels and seeds of the mango fruit Mangifera indica L. with 100 ppm of sodium hypochlorite diluted in ozonized water; it is then dried with a tray freeze-dryer at a temperature of approximately −40° C. and an absolute vacuum pressure of at least 0.04 mbar, for 12 hours. Once the dehydration is completed, the degree of humidity of the raw material is measured and it is extracted with polar solvents by microwave assisted extraction at a power of 200 W for 20 minutes; the antioxidant extract is then partially concentrated using the simple distillation method in a rotovap under vacuum at 0.5-15 inHg at 40° C. for 60 minutes until a concentration of at least 15% is achieved, to pass through the tangential nanofiltration of the concentrated antioxidant solution in two sequential filters with 50 nm nanopores and minimum surface area of 0.01 m.sup.2, at the same temperature as the previous process, through which the pumped solution passes at pressures between 0.5 bar. In a separate process, the concentrated antioxidant extracts are mixed at a speed of 500 RPM, at 25° C. for 2 hours in a mixture in direct proportion to the percentage of Brix degrees of the modified corn starch concentrate; it is homogenized in an industrial mixer at a speed of 12,000 RPM for about 5 minutes to encapsulate the active compounds, the resulting antioxidant microemulsion is stored temporarily under refrigeration at a temperature of 5-15° C. The alginic acid is diluted in ozonized water at 50° C., with constant movement for 1 hour, and then it is homogenized with the rest of the inputs: oily matrix, polysorbate, glycerol and the antioxidant microemulsion. Then followed the microfluidization process at high pressures of 100 MPa for 3 cycles, which produced the fluid nanoemulsion, enhanced with natural antioxidants from fruit and/or vegetable waste. Finally, the nanoemulsion produced in the previous stage was cryodried at vacuum pressures of at least 0.04 mbar and temperatures of −40° C., which gives it a fine granulometry solid appearance.

(4) B. Application of the Nanoemulsion with Encapsulated Natural Antioxidants on Selected Foods

(5) The fruits and vegetables subjected to the experiment (mango, avocado, mandarin, strawberry and asparagus) were selected considering optimal quality characteristics, i.e. uniform sizes, shapes, colors and absence of post-harvest mechanical or phytosanitary damage, half-ripe, washed and disinfected with 100 ppm of sodium hypochlorite; then, the fluid nanoemulsion was applied in the form of a spray for 5 minutes and dried—drained for another 5 minutes—, at room temperature of 25±2° C. on harmless grids; then the food was packed in containers recommended by CODEX Alimentarium. Once the application protocol of the nanoemulsion of the invention was completed, accelerated controlled tests were carried out in triplicate, every 7 and 14 days under a temperature of 25±2° C. and relative humidity of 80-90%, with the exception of the strawberry and asparagus, which were refrigerated at 10±2° C., all the tests at the same product concentration. The samples were experimental lots of 1 kg of Mangifera indica L. mango, Persea americana avocado, Citrus reticulata mandarin, Fragaria vesca L. strawberry and Asparagus officinalis asparagus. The variables measured were weight differential, Brix degrees differential and hedonic scale, based on a Sensory Evaluation test. The evaluation was performed on a scale from 1 to 5, being 5 the scale of better acceptance according to the taste of the product, before a panel of 15 previously trained tasters. The results evidence that the experimental samples filmed with the invention show a remarkable improvement compared to the control samples without any treatment, since these—after 8-12 days—, depending on the fruit, were in a state of putrefaction. It should be noted that the tests were conducted at extreme temperatures, so that the samples at refrigeration temperatures between 5-10° C. increase their quality by at least 50%, which is beneficial for the agro-industrial sector, which manages even freezing temperatures and even controlled atmospheres to maintain the quality of the food product.

(6) TABLE-US-00001 Day Zero Sample °Brix Taste Δ pH Mango 10.0 ± 0.05 5.0 ± 0.35 Avocado  8.8 ± 0.05 5.0 ± 0.15 6.0 ± 0.05 Mandarin 10.8 ± 0.04 4.3 ± 0.40 3.15 ± 0.05  Strawberry  9.1 ± 0.05 5.0 ± 0.10 3.8 ± 0.03 Asparagus 5.0 ± 0.40 5.0 ± 0.03

(7) TABLE-US-00002 Day 7 Sample ΔWeight Δ°Brix Taste Δ pH Mango 0.04 ± 0.05 0.38 ± 0.05 4.5 ± 0.30 Avocado 0.12 ± 0.05 0.50 ± 0.03 4.4 ± 0.35 0.05 ± 0.02 Mandarin 0.10 ± 0.04 0.45 ± 0.05 4.0 ± 0.25 0.05 ± 0.01 Strawberry 0.12 ± 0.05 0.35 ± 0.04 4.4 ± 0.30 0.05 ± 0.02 Asparagus 0.10 ± 0.05 4.4 ± 0.30 0.15 ± 0.01

(8) TABLE-US-00003 Day 14 Sample ΔWeight Δ°Brix Taste Δ pH Mango 0.24 ± 0.04 0.82 ± 0.05 3.9 ± 0.20 Avocado 0.39 ± 0.05 1.10 ± 0.05 3.7 ± 0.30 0.35 ± 0.02 Mandarin 0.33 ± 0.05 0.85 ± 0.04 3.8 ± 0.25 0.30 ± 0.02 Strawberry 0.35 ± 0.05 0.85 ± 0.05 3.9 ± 0.25 0.25 ± 0.01 Asparagus 0.30 ± 0.05 3.8 ± 0.35 0.30 ± 0.01

(9) TABLE-US-00004 Day 14 CONTROL SAMPLE °Brix Taste Δ pH Mango 20.2 ± 0.04 0 ± 0.0 Avocado NC 0 ± 0.0 NC Mandarin 15.6 ± 0.05 0.8 ± 0.25  NC Strawberry NC 0 ± 0.0 NC Asparagus 0 ± 0.0 NC

(10) The biochemical degradation of food, caused largely by oxidative processes, is the main non-microbial factor where the free radicals formed initiate spoilage reactions that act mainly on lipids and proteins (Descalzo, Rizzo, Rossetti, Negri, Paéz, Costabel and Taverna, 2010), which can be counteracted with the invention containing properly encapsulated antioxidants.

(11) Therefore, the experimental results obtained for asparagus can be extrapolated to cereals, since asparagus, a representative of vegetables, has similar proportions of essential amino acids, which reflects the protein similarity between both classes of foods.

(12) TABLE-US-00005 TABLE Amino Acid Content in Representative Cereals and Asparagus Quinoa Kiwicha Corn Asparagus Essential (g/100 g (g/100 g (g/100 g (g/100 g Amino Acids protein).sup.3 protein).sup.3 protein).sup.1 protein).sup.2 Isoleucine 6.9 5.2 4.0 6.9 Leucine 6.7 4.6 12.5 8.3 Lysine 6.8 6.7 2.9 8.9 Methionine 3.3 3.5 4.0 2.7 Phenylalanine 6.9 3.5 8.6 6.0 Threonine 4.5 5.1 3.8 5.7 Tryptophan 1.3 1.1 0.7 2.3 Valina 4.5 4.5 5.0 9.1 .sup.1Source: FAO (2013), Dietary protein quality evaluation in human nutrition .sup.2Source: Alimentos.org.es (s/n), Aminoácidos de los Espárragos .sup.3Source: Ayala, G. (2014), Aporte de los cultivos andinos a la nutrición humana

(13) On the other hand, samples of minimally processed foods and juices were evaluated, in experimental lots in triplicate, of 500 g of peeled and cut Malus communis apples and 500 ml of Musa paradisiaca banana juice with a minimum of 15-20% fruit, under controlled conditions of 20±2° C. and 70% relative humidity. The measurement was performed using the hedonic scale, based on a Sensory Evaluation test, with a scale from 1 to 5, being 5 the scale of best acceptance. The sensory criteria were taste, smell and sight, before a panel of 15 previously trained tasters, and pH to analyze the degree of acidity taken by the fruit. The experimental samples show a considerable improvement over the untreated control samples and the pH ranges remain within the fruit standard.

(14) TABLE-US-00006 Day Zero Sample Taste Sight Smell pH Apple 4.5 ± 0.5 White Color 5.0 ± 0.5 4.5 ± 0.05 Banana Juice 5.0 ± 0.5 Light Yellow Color 5.0 ± 0.1 5.0 ± 0.04

(15) TABLE-US-00007 Day 5 Sample Taste Sight Smell pH Apple 3.4 ± 0.4 White Color with 3.7 ± 0.4 4.1 ± 0.05 Light Brown Borders Banana Juice 3.1 ± 0.3 Slightly Light Yellow 3.5 ± 0.5 4.7 ± 0.05 Color

(16) TABLE-US-00008 CONTROL Day 5 SAMPLE Taste Sight Smell pH Apple 0.0 Dark Brown Color 0.0 NC with Mold Banana Juice 0.0 Dark Yellow Color 0.0 NC with Mold

(17) C. Quality Parameters of the Nanoemulsion with Encapsulated Natural Antioxidants

(18) 1. Encapsulation Efficiency

(19) This is measured as a percentage; it was performed through a relationship between the output and input of the efficient encapsulation process, taking an average of the tests performed with a standard deviation that shows the uncertainty of the repetitions in each run. An evaluation was considered of the best results obtained from the present invention, with respect to the best result stated in patent

(20) TABLE-US-00009 Comparative table-encapsulation efficiency (%) KR20160005182A INVENTION 77.87 ± 0.47 81.32 ± 0.16

(21) The results show that patent KR20160005182A, at a concentration of 1.6% cinnamon oil, had an encapsulation efficiency of 77.87%, while with the patent filed, the antioxidant, at a concentration of 1.5%, had 81.32% efficiency.

(22) 2. Acute Toxicity

(23) The ability of a substance to be lethal in low doses in humans (SINIA, 2017). The nanoemulsion mentioned in the invention was subjected to the oral LD50 ingestion toxicity test, according to the OECD Test Guideline 423, which consisted of a single dose to laboratory rats administering 2000 mg/kg of body weight, being observed for 14 days, a period of time that did not induce toxic damage and presented an LD50 higher than 2000 mg/kg of body weight, so the final product can be considered not classifiable as toxic or low intrinsic toxicity.

(24) 3. Nanoemulsion Particle Size Measurement

(25) It was measured on a Mastersizer laser analyzer (<100 nm to >2 mm). The colloidal sample is placed on the optical bench of the measuring instrument, where a light beam illuminates the particles and the measurement is generated from different angles scattering the light throughout the sample. The invention presents an average particle size between 90-100 nm, at the 90th percentile.

(26) 4. Measurement of Zeta Potential (mV)

(27) The measurement of the zeta potential of the nanoemulsion is a measurement of the electrical potential on the interfacial surface of suspensions; this is measured in electrophoretic cells with two electrodes connected to a source of energy (Kosegarten & Jimenez, 2012); it is associated with the pH value, as it associates the charge of the particles. A zeta-meter was used as a measurement instrument for the nanoemulsion in the invention; it yielded a value between −20 mV to −40 mV, depending on the concentrations of the inputs, and the pH was around 6.5 to 10, which shows adequate stability within the permitted range <−30 mV. Patent KR20160005182A, presents zeta potentials of about 0.5 to almost 6 mV, which could mean a high degree of dissociation.

(28) 5. Sanitary Microbiological Analysis

(29) The sanitary microbiological analysis of the nanoemulsions applied to minimally processed foods was performed considering the growth of microorganisms such as mesophilic aerobics over time, under temperature conditions between 15-30° C. The colony count at 30° C. through the surface sowing technique resulted—at 14 days, in mangoes, avocados, mandarins (shelf at 30° C.); and 10 days in strawberries and camu-camu (shelf at 15° C.)—in favorable treatments that were in a ratio between 450-600 CFU/g of total aerobes. This indicator shows an improvement of between 200 and 250% for the same fruits compared to the blank which does not have any film.

(30) 6. Sensory Analysis

(31) This is an experimental method that analyzes the organoleptic characteristics of a product, based on a panel of judges who perceive and qualify according to their criteria. The analysis of fresh mangoes filmed with the antioxidant-enriched nanoemulsion from mango waste was carried out in triplicate with a panel of 15 trained judges. The results of the above-mentioned test had an average score, from 1 to 5, of 5.0 points, which shows a slight improvement in the flavor of the product since the mango without application (control) obtained a score of 4.8 due to the enhancement of the fruity aroma. The results become more interesting when at 14 days the mango with the film obtains an average score of 3.9 points, while the control sample has a score of 0.0 due to its level of decomposition.