Method for microencapsulating sulforaphane (SFN) to stabilise same and emulsion, microcapsules and additive

Abstract

The present invention relates to a method for preparing stabilised SFN microcapsules, comprising: a) obtaining an SFN-rich extract from a.1) broccoli seeds ground to a flour, dissolving in water in a ratio (w / v) of 5:4, and obtaining a homogenate, which is incubated at 45°C; a.2) the double organic extraction of said homogenate with methylene chloride, and filtering, wherein the separated SFN-rich organic phase is concentrated at 30°C; a.3) resuspending said organic phase in ethanol (50% v / v), and obtaining an SFN-rich ethanolic extract; and b) preparing stabilised SFN microcapsules from b.1) an oil-in-water (O / W) emulsion, mixing said ethanolic extract (oily phase) under stirring at 20°C with gum arabic (GA), 20% (aqueous phase), and a 7% w / w surfactant, obtaining a homogenate with an O / W emulsion ratio of SFN in petroleum jelly and GA (μg / mg) of 0.7 and a surfactant and oily phase ratio (SOR) of 1; and b.2) drying and grinding.

Description

[0001]SULFORAPHANE (SFN) MICROENCAPSULATION METHOD FOR STABILIZING IT AND EMULSION, MICROCAPSULES, AND ADDITIVE The present invention relates to the area of ​​chemistry, and in particular to a method for increasing the thermal stability of sulforaphane (SFN), and more particularly still, a microencapsulation method of SFN obtained from broccoli seeds, and oil-in-water emulsion of stabilized SFN, microcapsules of stabilized SFN and food, pharmacological, cosmetic, nutraceutical, veterinary additive containing them. Background It is known that antioxidant compounds have the ability to inhibit or delay oxidation reactions that cause damage to the body's tissues via free radicals, which can cause multiple diseases (Moon, J.-K., & Shibamoto, T. (2009). Antioxidant Assays for Plant and Food Components. J. Agric. Food Chem, 1655–1666).Therefore, a diet rich in antioxidants that includes polyphenolic compounds, carotenoids, vitamins E and C, is highly desirable for human health. The antioxidant activity of different food matrices has been studied (Huang, D., Ou, B., & Prior, R. (2005). The Chemistry behind Antioxidant Capacity Assays. J. Agric. Food Chem., 1841-1856), particularly that of cruciferous vegetables, which have been reported as food matrices rich in phenolic compounds and carotenoids (Ramirez, D., Abellán-Victorio, A., Beretta, V., & Camargo, A. (2020). Functional Ingredients From Brassicaceae Species: Overview and Perspectives. Int. J. Mol. Sci., 1998). It is also known that polyphenols, which are present in a wide variety of vegetables, are powerful antioxidants and confer multiple benefits to human health, for example, as protectors against heart disease and cancer (Cartea, M., Francisco, M., Soengas, P., & Velasco, P. (2011).Phenolic Compounds in Brassica Vegetables. Molecules, 251-280). In the Brassicaceae family (cruciferous vegetables), flavonoids and hydroxycinnamic acids stand out as the most important phenolic compounds (Ramirez, D., Abellán-Victorio, A., Beretta, V., & Camargo, A. (2020). Functional Ingredients From Brassicaceae Species: Overview and Perspectives. Int. J. Mol. Sci., 1998). In the Brassicaceae family are broccoli, cabbage, mustard and watercress, which have a high content of glucosinolates, which once hydrolyzed generate isothiocyanates, compounds that have shown anticarcinogenic properties in various animal tests (Vermeulen, M. (2009). Isothiocyanates from Cruciferous Vegetables: Kinetics, Biomarkers and Effects. Wageningen, Netherlands: Wageningen University).Within this family, broccoli (Brassica oleracea italica) stands out for its high content of polyphenols, ascorbic acid, glucosinolates and isothiocyanates and its high antioxidant activity, and the isothiocyanate sulforaphane (SFN) stands out as a mediator of most of the properties present in broccoli (Yagishita, Y., Fahey, J., Dinkova-Kostova, AT, & Kensler, TW (2019). Broccoli or Sulforaphane: Is It the Source or Dose That Matters? Molecules, 3593). SFN comes from the enzymatic hydrolysis of the glucosinolate glucoraphanin by the action of myrosinase (Zhen-xin, G., Qiang-hui, G., & Ying-juan, G. (2012). Factors Influencing Glucoraphanin and Sulforaphane Formation in Brassica Plants: A Review. Journal of Integrative Agriculture, 1804-1818), but grinding or chewing is required for this reaction to take place when the substrate and enzyme come into contact (Mahn, A., & Castillo, A. (2021). Potential of Sulforaphane as a Natural Immune System. Molecules, 752-764).It is known that the cooking that broccoli undergoes to be consumed causes a decrease in the concentration of glucosinolates, the activity of myrosinase and the bioavailability of SFN in the vegetable (Pérez, C., Barrientos, H., Román, J., & Mahn, A. (2014). Optimization of a blanching step to maximize sulforaphane synthesis in broccoli florets. Food Chemistry, 264-271). On the other hand, it has been reported that SFN is an unstable compound, susceptible to being degraded by high temperatures and aqueous phases, and therefore, to take advantage of its properties it is necessary to design a process that stabilizes it and maximizes its concentration (Zhang, S., Ying, D., Cheng, L., Bayrak, M., Jegasothy, H., Sanguansri, L., & Augustin, M. (2020). Sulforaphane in broccoli-based matrices: Effects of heat treatment and addition of oil. LWT - Food Science and Technology, 19443, Mahn, A., & Castillo, A. (2021). Potential of Sulforaphane as a Natural Immune System. Molecules, 752-764).Thus, the use of microencapsulation processes emerges as an alternative to stabilize it and preserve its properties and incorporate it into food matrices (Lakkis, J. (2016). Encapsulation and Controlled Release Technologies in Food Systems. Barcelona, ​​Spain: Wiley Blackwell). There is very little literature addressing the encapsulation of SFN and this literature shows low efficiency or encapsulation requires a dilution of SFN in the organic phase, and also lyophilization to obtain a dry product, which increases the cost. There is more literature for encapsulation processes of other types of isothiocyanates, and they have better efficiencies than those achieved so far with SFN. Therefore, the adverse effect of temperature and the degradation of SFN in the aqueous phase have not been overcome. Therefore, the technological challenge related to the instability of SFN, which is susceptible to degradation by various factors, such as light, heat and water, remains pending.Table 1 summarizes the yield and efficiency of SFN encapsulation processes from scientific publications, including spray-drying encapsulation (Wu, Y., Zou, L., Mao, J., Huang, J., & Liu, S. (2014). Stability and encapsulation efficiency of sulforaphane microencapsulated by spray drying. Carbohydrate Polymers, 497–503) and vacuum oven-drying coacervation (García-Saldaña, J., Campas-Baypoli, O., López-Cervantes, J., Sánchez-Machado, D., CantúSoto, E., & Rodríguez-Ramírez, R. (2016). Microencapsulation of sulforaphane from broccoli seed extracts by gelatin / gum arabic and gelatin / pectin complexes. Food Chemistry, 94–100). The efficiencies with both methodologies are very low (30-40% and 10-20%, respectively). Meanwhile, Manjili, Sharafi, Attari, & Danafar, 2017 (Manjili, HK, Sharafi, A., Attari, E., & Danafar, H. (2017). Pharmacokinetics and in vitro and in vivo delivery of sulforaphane by PCL–PEG–PCL copolymeric-based micelles.Artificial Cell, Nanomedicine, and Biotechnology, 1728–1739) investigated the pharmacokinetics and release of SFN micelles obtained by lyophilization, obtaining an encapsulation efficiency of 87.1%, which is significantly higher than those obtained by spray drying or vacuum oven. While Danafar, Sharafi, Manjili, & S. (2016) (Danafar, H., Sharafi, A., Manjili, HK, & S., A. (2016). Sulforaphane delivery using mPEG–PCL copolymer nanoparticles to breast cancer cells. PharmDev Technol, 1–10) encapsulated SFN using monomethoxypoly (ethylene glycol)-poly ( -caprolactone) (mPEG PCL), obtaining SFN micelles with an encapsulation efficiency of 86 ± 1.58%, and where the SFN is not diluted in water, but in an organic solution. On the other hand, the methods that use GRAS compounds described so far, reach efficiencies less than 40%. Table 1. Efficiencies of known SFN microencapsulation methods.The method of the present invention achieves an encapsulation efficiency equal to 90%. The significant differences between the stability of free SFN and encapsulated SFN are well known. Wu, Y., Mao, J., Mei, L., & Liu, S. (2013). Kinetic studies of the thermal degradation of sulforaphane and its hydroxypropyl-β-cyclodextrin inclusion complex. Food Res. Int., 529–533, studied the thermal degradation kinetics of free SFN and its inclusion complex with hydroxypropyl-β-cyclodextrin at different pH and temperatures between 60 and 90°C, showing that pH has a significant effect on SFN stability and that low pH reduces the SFN depletion rate. The activation energy of free SFN and encapsulated SFN is also known to be higher at lower pH regardless of temperature, Fahey, J., Wade, K., Wehage, S., Holtzclaw, W., Liu, H., Talalay, P., Stephenson, K. (2017). Stabilized sulforaphane for clinical use: phytochemical delivery efficiency. Mol. Nutr. Food Res., 1600766.The activation energy was 56 [kJ / mol], being similar to that reported in Mahn, A., Martin, C., Reyes, A., & Saavedra, A. (2016). Evolution of sulforaphane content in sulforaphane-enriched broccoli during tray drying. Journal of Food Engineering, 27-33, which was 58 [kJ / mol] for SFN degradation during tray drying and 103.02 [kJ / mol] for encapsulated SFN, demonstrating that α-cyclodextrin microencapsulation significantly increased the stability of SFN. Wu, H., Liang, H., Yuan, Q., Wang, T., & Yan, X. (2010). Preparation and stability investigation of the inclusion complex of sulforaphane with hydroxypropyl- β-cyclodextrin. Carbohydrate Polymers, 613–617) evaluated the stability of the SFN inclusion complex in hydroxypropyl-β-cyclodextrin at 50°C, and when calculating the degradation kinetic constant, obtained a value of 0.002 [h. -1], which is consistent with Fahey, J., Wade, K., Wehage, S., Holtzclaw, W., Liu, H., Talalay, P., ... Stephenson, K. (2017). Stabilized sulforaphane for clinical use: phytochemical delivery efficiency. Mol. Nutr. Food Res., 1600766) at the same temperature but using α-cyclodextrin as the encapsulating agent. However, none of these methods use compounds that are not accepted by the RSA or the FDA for use in food or drugs (NON-GRAS compounds). Furthermore, the cost of these compounds is very high compared to the compounds used in the present invention. Regarding encapsulation and controlled release systems that seek to protect bioactive compounds from undesirable interactions, improving their stability, functionality and bioavailability (Vincekovic, M., Viski, M., Juri, S., Giacometti, J., Kovacevic, D., Putnik, P.,. Jambrak, A. (2017). Innovative technologies for encapsulation of Mediterranean plants extracts.Trends in Food Science & Technology, 1-12), the core contains the active substance and this is surrounded by a wall material called encapsulating agent, coating, membrane, shell, capsule, carrier material, external phase or matrix (Fang, Z., & Bhandari, B. (2010). Encapsulation of polyphenols - a review. Trends in Food Science & Technology, 510-523). There are different morphologies and structures of microcapsules. The capsule shows a continuous phase with the substance enclosed by a shell. The matrix, microsphere or multicore shows a solid phase where the encapsulated substance is distributed (Lakkis, J. (2016). Encapsulation and Controlled Release Technologies in Food Systems. Barcelona, ​​Spain: Wiley Blackwell).Microcapsules can be obtained by (i) physicochemical processes (simple or complex coacervation, ionic gelation, emulsification, liposome encapsulation, nanoemulsion, solvent evaporation / extraction); (ii) mechanical methods (spray drying / cooling / freezing, fluidized bed, extrusion-spherification, centrifugal extrusion, supercritical fluid processes); and (iii) chemical processes (interfacial polycondensation, cross-linking polymerization and in situ polymerization) (Vincekovic, M., Viski, M., Juri, S., Giacometti, J., Kovacevic, D., Putnik, P., Jambrak, A. (2017). Innovative technologies for encapsulation of Mediterranean plants extracts. Trends in Food Science & Technology, 1-12; Ozkan, G., P., F., De Marco, I., Xiaoc, J., & Capanoglua, E. (2019). A review of microencapsulation methods for food antioxidants: Principles, advantages, drawbacks and applications. Food Chemistry, 494–506).In general, microencapsulation involves a physicochemical or chemical process to entrap the bioactive compound within a matrix or wall material, followed by a mechanical process to obtain the final product, in powder or gel form. Emulsions are a mixture of two immiscible liquids, one of which is dispersed as droplets ("dispersed phase") in the other ("continuous phase"). Depending on the droplet size, they are classified as macroemulsions (0.1–100 μm), nanoemulsions (20–100 nm), and microemulsions (5–50 nm). The first two are thermodynamically unstable, with the former being opaque while the latter is almost transparent. Microemulsions are usually transparent and thermodynamically stable (McClements, M. (2010). Emulsion Design to Improve the Delivery of Functional Lipophilic Components. Annu. Rev. Food Sci. Technol., 241–269).Droplet size is closely related to the stability of an emulsion. A smaller droplet size implies greater stability to gravitational separation and aggregation in emulsions (Tadros, T.I. (2004). Formation and stability of nano-emulsions. Advances in Colloid and Interface Science, 303–318; Tadros, T., Izquierdo, P., Esquena, J., & Solans, C. (2004). Formation and stability of nanoemulsions. Advances in Colloid and Interface Science, 303–318). The breakup and coalescence rates of dispersed droplets in the emulsion determine the temporal evolution of the droplet size distribution, and are affected by the physicochemical properties of the two phases, including interfacial tensions, viscosities and densities, volume fraction of the phases, type and concentration of the stabilizer, and the quality of agitation.Droplet breakup and coalescence can be controlled by appropriately selected emulsifiers or stabilizers (surfactants, small polymeric molecules, surfactants) that decrease interfacial tension and reduce the mechanical energy required to achieve the desired droplet sizes (Mishra, M. (2016). Handbook of Encapsulation and Controlled Release. Boca Raton, United States: Taylor & Francis Group). Emulsifiers adsorb at the interface between the two phases, forming a protective film that prevents droplet coalescence and reduces immediate coalescence between them. Emulsions are basically composed of 3 components: 2 immiscible liquids, generally an oily phase and an aqueous phase; and a surfactant, and depending on the proportion of each component present, different types of emulsions are generated (Mishra, M. (2016). Handbook of Encapsulation and Controlled Release.Boca Raton, United States: Taylor & Francis Group), and the selection of components generally seeks low or no toxicity, high biocompatibility, and that they are clinically acceptable and considered generally safe (GRAS, Generally Recognized As Safe). Among the compounds selected for the oily phase of the emulsion, which is the most important (Mishra, M. (2016). Handbook of Encapsulation and Controlled Release. Boca Raton, United States: Taylor & Francis Group), the most common are castor oil, corn oil, glyceryl monostearate, lanolin, medium-chain monoglycerides, medium-chain triglycerides, petrolatum, sesame oil, among others (Ruiz, JC, & Segura, MR (2017). New polymers for encapsulation of nutraceutical compounds. West Sussex, United Kingdom: John Wiley & Sons, Ltd). Meanwhile, surfactants, emulsion stabilizing substances and facilitators of emulsion formation (McClements, D., & Jafari, S. (2018).Improving emulsion formation, stability and performance using mixed emulsifiers: A review. Advances in Colloid and Interface Science, 55–79), are preferably selected from amphiphilic compounds, which have a hydrophilic part soluble in polar solvents (head) and another hydrophobic part insoluble in the polar solvent (tail), and therefore, they adsorb on surfaces and interfaces, which decreases surface tension, allows the formation of aggregates within the solutions, and thus, the formation of emulsions (Mishra, M. (2016). Handbook of Encapsulation and Controlled Release. Boca Raton, United States: Taylor & Francis Group). The HLB (Hydrophilic-Lipophilic-Balance) is an indicator of the emulsifying characteristics of a surfactant, although not necessarily of its efficiency. Surfactants that have a high HLB promote the formation of O / W emulsions (Schramm, L., Stasiuk, E., & Marangoni, D. (2003). Surfactants and their applications. Annu. Rep. Prog. Chem., 3-48). A mixture of surfactants is often used (Li, Y., Teng, Z., Chen, P., Song, Y., Luo, Y., & Q., W. (2015). Enhancement of aqueous stability of allyl isothiocyanate using nanoemulsions prepared by an emulsion inversion point method. Journal of Colloid and Interface Science, 130–137). Among the most commonly used surfactants in O / W emulsions are Tween 80 (HLB: 15) and Tween 20 (HLB: 16.7). One of the factors affecting the stability of an emulsion is the surfactant-to-oil ratio (SOR), with the SOR being reported to be closely related to the final droplet size of an allyl isothiocyanate-loaded nanoemulsion system (Li, Y., Teng, Z., Chen, P., Song, Y., Luo, Y., & Q., W. (2015). Enhancement of aqueous stability of allyl isothiocyanate using nanoemulsions prepared by an emulsion inversion point method. Journal of Colloid and Interface Science, 130–137).Additionally, the aqueous phase is also very relevant when formulating an emulsion and can have stabilizing, gelling or stabilizing functions depending on the type of polymer (Lakkis, J. (2016). Encapsulation and Controlled Release Technologies in Food Systems. Barcelona, ​​Spain: Wiley Blackwell). One of the most widely used polymers is gum arabic (GA), which is a highly branched and complex polymer, formed mainly by D‐galactose, L‐rabinose units and small proportions of 4‐O‐methyl‐D‐glucuronate and L‐rhamnose. GA contains 1–2% protein and is the most widely used wall material for the encapsulation of lipophilic compounds due to its excellent emulsifying property, film-forming ability, high solubility and low viscosities in aqueous systems, and its dual function as surfactant and drying matrix (Anandharamakrishnan, C., & Ishwarya, P.S. (2015). Spray drying techniques for food ingredient encapsulation.West Sussex, UK: John Wiley & Sons, Ltd). Emulsions are mainly divided into two categories: 1) Oil-in-water (O / W), b) Water-in-oil (W / O). The first one is formed when an internal oil phase is emulsified with a continuous aqueous phase (Mishra, M. (2016). Handbook of Encapsulation and Controlled Release. Boca Raton, USA: Taylor & Francis Group), while the second one, an internal aqueous phase is emulsified with a continuous oil phase (Anandharamakrishnan, C., & Ishwarya, P.S. (2015). Spray drying techniques for food ingredient encapsulation. West Sussex, UK: John Wiley & Sons, Ltd). In a conventional O / W emulsion, oil droplets are dispersed in a continuous aqueous phase and surrounded by a thin interfacial layer of emulsifying molecules, and corresponds to the most common form of emulsion currently used in the food industry. (McClements, M. (2010). Emulsion Design to Improve the Delivery of Functional Lipophilic Components.Annu. Rev. Food Sci. Technol., 241–269). The most conventional way to prepare an O / W emulsion is to homogenize the oil phase and the water phase in the presence of a water-soluble surfactant. A variety of homogenizers are available, including high-shear mixers, high-pressure homogenizers, ultrasonic homogenizers, and membrane homogenizers (Walstra, P. (1993). Principles of emulsion formation. Chemical Engineering Science, 333–349). In food production and encapsulation processes, the most important factor for an emulsion is its initial stability. Two-phase emulsions tend to be thermodynamically unstable, and therefore the phases will separate over time (Lakkis, J. (2016). Encapsulation and Controlled Release Technologies in Food Systems. Barcelona, ​​Spain: Wiley Blackwell) either by gravitational separation, flocculation, coalescence and Ostwald ripening.Gravitational separation is the most common mechanism of instability (McClements, M. (2010). Emulsion Design to Improve the Delivery of Functional Lipophilic Components. Annu. Rev. Food Sci. Technol., 241–269), with the difference in density being what tends to separate the oily phase from the aqueous phase by the effects of gravity. In flocculation, on the other hand, there is a tendency for the droplets that make up an emulsion to aggregate or associate, keeping their interfacial layer intact. Flocculation occurs when the attraction energy between the droplets increases (Lakkis, J. (2016). Encapsulation and Controlled Release Technologies in Food Systems. Barcelona, ​​Spain: Wiley Blackwell). In coalescence, there is a break in the interface between adjacent drops, increasing the drop size distribution in the emulsion, which is very common in the absence of emulsifiers or surfactants (Lakkis, J. (2016). Encapsulation and Controlled Release Technologies in Food Systems.Barcelona, ​​Spain: Wiley Blackwell). Finally, Ostwald ripening is similar to coalescence and droplets diffuse from small to large droplets, even in the presence of surfactant, as the interfacial area (energy) is reduced (Mishra, M. (2016). Handbook of Encapsulation and Controlled Release. Boca Raton, United States: Taylor & Francis Group).As for patent documents, CN102688219A (Ma Jianhua) discloses a microencapsulation method comprising a) extracting SFN by digesting cruciferous raw material by extraction, and where previously the cruciferous seeds are ground and degreased by adding petroleum ether and the pH value is regulated to a value 7 with phosphate buffer and then performing an enzymolysis and then adding an extractant and sodium sulfate and sodium chloride are added respectively, and then filtering and concentrating under vacuum to remove the organic solvent, setting the volume to 5000 ml with ethanol; filtering with a 0.22 micron microfiltration membrane and measuring the SFN content by liquid chromatography; and II, microencapsulating the SFN by adding monoglyceride and sodium alginate to the composite wall material, adding the deionized aqueous solution, adding SFN with stirring, spray drying and collecting the product.Thus, extremely unstable SFN in liquid is synthesized into solid powder particles through microencapsulation, achieving perfect stability. CN109043533B (Zhejiang University ZJU) refers to a method of preparing microencapsulated SFN by emulsification and spray-drying method, which takes broccoli seeds as raw materials, which are supercritically ground and degreased, SFN is extracted by endogenous enzyme hydrolysis, SFN is purified by solvent extraction and macroporous resin, then emulsifier, antioxidant and oil are added into composite capsule materials to prepare water phase, the oil phase is added into the water phase while stirring according to the proportion of the core material, high-speed shear is conducted, and finally an emulsion is obtained, which is spray-dried to prepare SFN microcapsules.SFN microcapsules have uniform size and smooth surface; the embedding rate reaches 80.59%, and the microcapsule particle size is 1.03-3.38 µm; SFN is placed at a high temperature of 90℃ for 5 hours, and the SFN retention rate can reach more than 90%. The method is simple, low-cost, improves the high-temperature resistance of SFN, and is suitable for industrial application. US9603828B2 (PharmAgra Labs Inc) discloses a method for isolating and purifying SFN from natural sources by forming high-purity complexes with cyclodextrin. CN107569470B (Yangtze Normal University) discloses a preparation of SFN microcapsules. Mesona chewing gum, serving as a special wall material, has good resistance to light, heat, acid and alkali, and good antioxidant effect, so the bioavailability and stability of SFN are effectively improved, SFN can be stored for a longer time in a natural environment, and the application of SFN in food is facilitated.The prepared SFN microcapsules have uniform size, regular shape and good encapsulation effect, the imbibing rate reaches 71.68%, the average grain diameter is 22.45 µm, and the SFN retention rate can reach over 86% under different storage conditions. CN112617178B (Northwest University) discloses a method for preparing SFN microcapsules with a slow-releasing effect under in vitro simulated gastrointestinal release condition, where the SFN degradation rate is less than 10% when stored for 180 days at 25 ℃. It adopts double imbibing, also improves the storage stability by secondary imbibing, which obviously delays the release of SFN in the process of digestion, can continuously maintain a certain concentration of SFN in blood plasma.SFN is embedded by adopting an electrostatic spraying method so that the SFN can be dried at room temperature and the loss under high drying temperature is avoided. The particle size of the microcapsule is reduced by more than 60% through electrostatic spraying, and the microcapsule obtained finally has an average particle size less than 200 µm after freeze-drying, thus being beneficial for application in food systems. CN111601896A (Commonwealth Scientific and Industrial Research Organization CSIRO) discloses a method for producing isothiocyanate-containing products from cruciferous materials (Brassicaceae) and lactic acid bacteria.CN115364070A (Pioneer Herb Industrial Co ltd) discloses double-embedded SFN microcapsule powder and preparation method, from an aqueous solution of SFN and polyol, by enrobing the SFN solution with polyglycerol polyricinoleate and adding edible oil and vitamin E and then heating at 70-80. oC, and subjected to high speed shear to obtain an oil-soluble SFN solution, and prepare an aqueous solution of sodium starch octenylsuccinate (SSO) with a SSO to maltodextrin to water ratio of 2:1-5:1; and heated to 60-80 ℃, adding the oily SFN solution and perform high speed shear, and then spray dried to obtain the SFN double-embedded microcapsule powder, where the stability of the latter improves during storage, as well as its biological activity. In this way, a method for thermally stabilizing sulforaphane (SFN) is still pending in the state of the art, and this is useful for applications in the food, pharmaceutical, cosmetic or nutraceutical industries as a food, pharmacological, cosmetic, and nutraceutical additive.Brief Description of the Invention The present invention relates to a method for microencapsulating SFN using an oil-in-water (O / W) emulsion system that increases the thermal stability of SFN compared to unencapsulated SFN, where SFN is extracted from broccoli seeds. The method presents conditions that confer high performance and efficiency. Emulsion of stabilized SFN, microcapsules of stabilized SFN and food, pharmacological, cosmetic, nutraceutical, veterinary additive that comprise them. In this way, the present method makes it possible to increase the thermal stability of this anticancer compound (SFN), with high encapsulation efficiency and high performance. SFN begins to degrade at 40°C, which is why its industrial application has been limited until now.The present invention relates to a method for microencapsulating SFN using an oil-in-water (O / W) emulsion system that increases the stability of SFN relative to unencapsulated SFN, where the SFN is extracted from broccoli seeds. The method features conditions that provide high yield and efficiency. The SFN microcapsules display a characteristic composition and are thermally stable. The present method for preparing thermally stabilized SFN microcapsules, comprising: a) obtaining an SFN-rich extract from broccoli seeds from a.1) grinding broccoli seeds into a flour which is then dissolved in ultra-pure water, the weight ratio (mg) of broccoli seed flour to the volume (ml) of water being 5:4, and obtaining an SFN homogenate which is incubated at 45°C, preferably in a water bath; a.2) the double organic extraction of the incubated SFN homogenate with methylene chloride in a sonication bath and vacuum filtering the sonicated SFN organic extract to separate the SFN-rich organic phase which is then dried under vacuum at 30°C by rotary evaporation, a.3) resuspending the SFN-rich organic phase dried by rotary evaporation in ethanol (50% v / v) and obtaining an SFN-rich ethanolic extract, and b) preparing stabilized SFN microcapsules from: b.1) preparing an oil-in-water emulsion by mixing the SFN-rich ethanolic extract with petrolatum, preferably oral liquid petrolatum, as the oily phase of the emulsion, under stirring and in a water bath at 20°C, and then adding 20% ​​gum arabic (GA) as the aqueous phase, and in the presence of a 7% surfactant (mg surfactant / mg emulsion), preferably Tween 80, obtaining an O / W emulsion homogenate of SFN in petrolatum and GA, where the O / W emulsion ratio of SFN in petrolatum and GA (μg / mg) is 0.7, and the oil phase to surfactant ratio (SOR) is 1; and b.2) dry by dehydrating the O / W emulsion of SFN in petroleum jelly and GA, in layers no greater than 1 mm, under vacuum and at 37°C, preferably in an oven, and grind the O / W emulsion of SFN in petroleum jelly and GA to obtain powdered microcapsules.The present invention also relates to an oil-in-water emulsion of stabilized SFN, microcapsules of stabilized SFN, and food, pharmacological, cosmetic, or nutraceutical compositions or formulations containing them. The present method has potential application in the food sector, since SFN is a naturally occurring compound that offers various health benefits, including the prevention of some types of cancer. The inclusion of stabilized SFN would allow for the development of alternative functional foods or products for people with special dietary needs, such as vegans and celiac patients, among others.This method also has high potential in the food, pharmaceutical, cosmetics and nutraceutical industries since, for example, SFN has been tested in clinical studies as a treatment for breast and ovarian cancer, obtaining promising results. In addition, there is interest in producing SFN as a medicine by multinational pharmaceutical companies, and recognizing it as a drug in the European Union (EU). The main advantage of this method lies in the possibility of microencapsulation of SFN using an oil-in-water emulsion with two organic phases (methylene chloride and petrolatum), which increases the recovery of SFN. A microcapsule washing step is also included to improve their dehydration and obtain a homogeneous powder as the final product. Brief Description of the Figures Figures 1A and 1B. Micrograph of the microcapsules obtained by the O / W emulsion method. Fig. 1A: liquid emulsion sample and Fig. 1B: dry sample resuspended in water.Figures 2A-2H. Images obtained by optical microscope: Fig. 2A: Ethanol / water extract of SFN; Fig. 2B: GA solution; Fig. 2C: Ethanol / water extract of SFN and GA aqueous solution; Fig. 2D: Tween 80 aqueous solution; Fig. 2E: Tween 80 aqueous solution and SFN ethanol / water extract; Fig. 2F: Petrolatum aqueous solution; Fig. 2G: Petrolatum aqueous solution and SFN ethanol / water extract; Fig. 2H: Microcapsule from liquid emulsion. Figures 3A-3E. FTIR spectra of SFN-GA-Tween-Petrolatum complex (Fig. 3A); GA (Fig. 3B), SFN extract (Fig. 3C), Petrolatum (Fig. 3D) and Tween 80 (Fig. 3E). Detailed Description of the Invention The present invention proposes a method for microencapsulating SFN using an oil-in-water (O / W) emulsion system where the SFN is diluted in an oily phase, and by applying low-temperature vacuum drying, its stability is increased when compared to unencapsulated SFN.The present invention also relates to the O / W emulsion of stabilized SFN, microcapsules of stabilized SFN and food, pharmacological, cosmetic and nutraceutical additive comprising them. Microencapsulation protects SFN and improves its application in food, pharmacological, cosmetic and nutraceutical matrices, and contributes to a more efficient incorporation of its beneficial properties in the preparation as food, pharmacological, cosmetic and nutraceutical additives, in various food, pharmacological, cosmetic and nutraceutical formulations and, likewise, provides a method that maximizes the efficiency and stability of SFN.In another aspect, the present invention relates to a food additive comprising the stabilized SFN microcapsules obtained by the method of the present invention, which may be added to a food formulation that may also comprise one or more flavoring agents, sweetening agents, gasifying agents, thickening agents, leavening agents, agglomerating agents, coloring agents, pH adjusting agents, preserving agents, edible vehicles, humectant agents, emulsifying agents, vitamins, amino acids, among others.The present food additive can be added to different types of food formulations, including functional foods, processed foods, baby foods, foods for the elderly, gluten-free foods, dietetic foods, foods to restore intestinal flora, low-protein foods, high-protein foods, vegan foods, foods for athletes, fortified foods, nutraceutical foods, among others. The present food additive can be added directly or together with other known food ingredients according to methods known in the art. The amount of additive to be added may vary depending on the purpose for which it is added. The food formulation to which the present additive can be added can be a solid or liquid food formulation.The solid feed formulation can be selected from one or more of sausages, bread, meats, snacks, cookies, sweets, cereals, ice cream, smoothies, soups, creams, yogurts, cheeses, sauces, jellies, among others. The liquid feed formulation can be selected from soft drinks, juices, sports drinks, among others. The feed formulation can be selected from a formulation for animal feed or for human consumption. The animal feed formulation can be selected from feed for farm animals, companion animals, aquatic animals, animals raised in industrial farms, animals raised in aquaculture farms, zoo animals, wild animals, among others. The farm animals or those raised in industrial farms can be selected from poultry, including hens, roosters, chickens, turkeys, geese, ducks, pheasants, quails, among others; and pigs, goats, sheep, cows, calves, oxen, horses, foals, among others.Pets can be dogs, cats, hamsters, gerbils, rabbits, among others. Aquatic animals and farm animals can be salmon, trout, among others. Zoo animals can be monkeys, apes, gorillas, among others. The present feed additive can be administered separately or combined with the feed formulation. The present feed additive can be formulated as an immediate- or sustained-release additive. The present additive can be formulated as a tablet, capsule, softgel, powder, syrup, liquid solution, liquid suspension, emulsion, pellet, granule, among others. The present additive can be added as a powder, suspension, emulsion, or liquid solution. The present additive can be added by spraying, mixing, immersion, among others.In yet another aspect, the present invention relates to a pharmacological additive, veterinary additive or nutraceutical additive comprising the stabilized SFN microcapsules obtained by the method of the present invention, which may be added to a pharmaceutical formulation, veterinary formulation or a nutraceutical formulation which may also comprise one or more flavoring agents, sweetening agents, gasifying agents, thickening agents, agglomerating agents, coloring agents, pH adjusting agents, preserving agents, wetting agents, emulsifying agents, vitamins, amino acids, among others.The present pharmacological additive, veterinary additive or nutraceutical additive can be added to different types of pharmacological formulations, veterinary formulations or nutraceutical formulations, including the same magistral formulations, antioxidant supplements, antioxidant pharmaceutical formulations, antioxidant veterinary formulations, antioxidant nutraceuticals, among others. The present pharmacological additive, veterinary additive or nutraceutical additive can be added directly or together with other pharmaceutical excipients, veterinary excipients or nutraceutical excipients, correspondingly, known and according to methods known in the art. The amount of additive to be added may vary depending on the objective for which it is added.The pharmacological formulation, veterinary formulation, or nutraceutical formulation to which the present additive can be added can be a solid, semi-solid, or liquid pharmacological formulation, veterinary formulation, or nutraceutical formulation. The pharmacological formulation, veterinary formulation, or solid nutraceutical formulation can be selected from one or more of pills, tablets, capsules, granules, pellets, wafers, soft capsules, hard capsules, powder for reconstitution, among others. The pharmacological formulation, veterinary formulation, or liquid nutraceutical formulation can be selected from one or more of syrups, solutions, suspensions, emulsions, creams, ointments, among others. The veterinary formulation or solid nutraceutical formulation can be selected from a veterinary formulation or nutraceutical formulation for administration to animals or humans. The veterinary formulation or solid nutraceutical formulation can be administered orally or topically.The veterinary formulation or nutraceutical formulation may be selected from a veterinary formulation or nutraceutical formulation for farm animals, companion animals, aquatic animals, animals raised in factory farms, animals raised in aquaculture farms, zoo animals, wild animals, among others. Farm animals or animals raised in factory farms may be selected from poultry, including hens, roosters, chickens, turkeys, geese, ducks, pheasants, quails, among others; and pigs, goats, sheep, cows, calves, oxen, horses, foals, among others. Companion animals may be selected from dogs, cats, hamsters, gerbils, rabbits, among others. Aquatic animals and animals raised in aquaculture farms may be selected from salmon, trout, among others. Zoo animals may be selected from monkeys, apes, gorillas, among others.The present pharmacological additive, veterinary additive, or nutraceutical additive can be administered separately or in combination with the pharmacological formulation, veterinary formulation, or nutraceutical formulation. The present pharmacological additive, veterinary additive, or nutraceutical additive can be manufactured as an immediate- or sustained-release additive. The present additive can be manufactured as a tablet, capsule, softgel, powder, syrup, liquid solution, liquid suspension, emulsion, pellet, granule, among others. The present additive can be added as a powder, suspension, emulsion, or liquid solution. The present additive can be added by spraying, mixing, immersion, among others.In yet another aspect, the present invention relates to a cosmetic additive comprising the stabilized SFN microcapsules obtained by the method of the present invention, which may be added to a cosmetic formulation and may also comprise one or more of perfuming agents, sweetening agents, thickening agents, agglomerating agents, coloring agents, pH adjusting agents, preserving agents, humectant agents, emulsifying agents, vitamins, amino acids, among others. The present cosmetic additive may be added to different types of cosmetic formulations, including those that may be selected from facial cosmetic formulations, hair cosmetic formulations, body cosmetic formulations, hand cosmetic formulations, foot cosmetic formulations, personal hygiene cosmetic formulations, among others.The present cosmetic additive is added together with other known cosmetic excipients according to methods known in the art. The amount of additive to be added may vary depending on the purpose for which it is added. The cosmetic formulation to which the present additive can be added can be a solid, semi-solid, or liquid cosmetic formulation. The cosmetic formulation can be selected from solid, semi-solid, or liquid formulations. The solid and semi-solid cosmetic formulation can be selected from one or more of creams, ointments, powders, soaps, shampoos, balms, masks, waxes, dyes, among others. The liquid cosmetic formulation can be selected from oils, emulsions, solutions, suspensions, liquid soaps, perfumes, lotions, among others. The present additive can be added as a dry powder or liquid solution. The present additive can be added by spraying, mixing, immersion, among others.For the implementation of the present method, stirring time (RT), SFN / gum arabic ratio, surfactant to oil ratio (SOR), and temperature (T) were established to prepare SFN microcapsules that could be used, for example, in a food formulation such as juice. The stability of the microencapsulated SFN was improved compared to unencapsulated SFN. The microencapsulation efficiency (EE, µg final SFN / µg loaded SFN) and yield (RE, mg final powder / mg encapsulating material used) were measured. SFN was obtained from purchased broccoli (Brassica oleracea var. Waltham 29) seeds, and the extraction method was proposed by García-Saldaña et al. (2016) (García-Saldaña, J., Campas-Baypoli, O., López-Cervantes, J., Sánchez-Machado, D., CantúSoto, E., & Rodríguez-Ramírez, R. (2016). Microencapsulation of sulforaphane from broccoli seed extracts by gelatin / gum arabic and gelatin / pectin complexes. Food Chemistry, 94–100) modified.The SFN content was determined by reverse phase HPLC, according to the method proposed by Liang, Yuan, Dong, & Liu (2006) (Liang, H., Yuan, Q., Dong, H., & Liu, Y. (2006). Determination of sulforaphane in broccoli and cabbage by high-performance liquid chromatography. Journal of Food Composition and Analysis, 473–476) modified (Pérez, C., Barrientos, H., Román, J., & Mahn, A. (2014). Optimization of a blanching step to maximize sulforaphane synthesis in broccoli florets. The characterization of the process was carried out according to that carried out by García-Saldaña, et al (2016) (García-Saldaña, J., Campas-Baypoli, O., López-Cervantes, J., Sánchez-Machado, D., CantúSoto, E., & Rodríguez-Ramírez, R. (2016). Microencapsulation of sulforaphane from broccoli seed extracts by gelatin / gum arabic and gelatin / pectin complexes. Food Chemistry, 94–100) modified.Process efficiency was obtained by calculating the ratio of the powder mass obtained at the end of the process and the encapsulating material used at the beginning of the process (gum arabic, oil phase, surfactant, SFN). Microencapsulation efficiency is given by the ratio of the SFN concentration in the microcapsules to the SFN loaded at the beginning of the process. To measure the SFN in the microcapsules, a quantity of powdered microcapsules was taken and diluted in methylene chloride. The solution was then placed in an ultrasonic bath for 30 minutes. It was allowed to stand for 1 hour, and the SFN was subsequently measured according to the method described above. Li et al. (2015) (Li, Y., Teng, Z., Chen, P., Song, Y., Luo, Y., & Q., W. (2015). Enhancement of aqueous stability of allyl isothiocyanate using nanoemulsions prepared by an emulsion inversion point method.Journal of Colloid and Interface Science, 130–137) report the effect of the combination of Span 20™ and Tween 80™ on the stability and droplet size of emulsions. No significant differences were observed for the SFN solution / oil phase ratio (ml / ml) and the type of oil phase on the average particle size. The EE was higher than most previously reported. García-Saldaña, et al. (2016) (García-Saldaña, J., Campas-Baypoli, O., López-Cervantes, J., Sánchez-Machado, D., CantúSoto, E., & Rodríguez-Ramírez, R. (2016). Microencapsulation of sulforaphane from broccoli seed extracts by gelatin / gum arabic and gelatin / pectin complexes. Food Chemistry, 94– 100) used complex coacervation for the microencapsulation of SFN using gelatin-gum arabic and gelatin-pectin systems, reporting an EE of 12.2 ± 0.1 and 17.9 ± 1.3%, respectively. Wu, Zou, Mao, Huang, & Liu (2014) (Wu, Y., Zou, L., Mao, J., Huang, J., & Liu, S. (2014).Stability and encapsulation efficiency of sulforaphane microencapsulated by spray drying. Carbohydrate Polymers, 497-503) encapsulated SFN by simple coacervation, using maltodextrin, gum arabic and k-carrageenan, and also by complex coacervation using maltodextrin-gum arabic and gum arabic-β-cyclodextrin. After chemical treatment, the microcapsules were subjected to spray drying. The lowest encapsulation efficiency was obtained with k-carrageenan (12%); whereas the other systems gave significantly higher values ​​(30-40%). These authors dissolved SFN in the aqueous phase, which could probably be a factor reducing the EE given the low water solubility of SFN, whereas in this work SFN was dissolved in ethanol (50% v / v). Manjili, Sharafi, Attari, & Danafar (2017) (Manjili, H.K., Sharafi, A., Attari, E., & Danafar, H. (2017).Pharmacokinetics and in vitro and in vivo delivery of sulforaphane by PCL–PEG–PCL copolymeric-based micelles. Artificial Cell, Nanomedicine, and Biotechnology, 1728–1739) reported EE of 87.1% in SFN micelles using lyophilization and a poly(caprolactone)-poly(ethylene glycol)-poly(caprolactone) copolymer (PCL-PEG-PCL) as the wall material, where SFN was diluted in acetone. In the same field, (Danafar, Sharafi, Manjili, & S., 2016) (Danafar, H., Sharafi, A., Manjili, HK, & S., A. (2016). Sulforaphane delivery using mPEG–PCL copolymer nanoparticles to breast cancer cells. Pharm Dev Technol, 1–10) encapsulated SFN by preparing micelles with monomethoxypoly (ethylene glycol)-poly (e.g., caprolactone)(mPEG – PCL) and with lyophilization drying at -78 ° C, they reported an efficiency of 86 ± 1.6 %.Table 1 shows the efficiencies (EE) and yields (RE, SFN content in the microcapsules) of different preliminary test mixtures, where an ANOVA test was used to analyze the effect of using petrolatum as the oil phase and temperature on EE and RE, showing that there are no significant differences in EE and RE with the different temperatures or the use of petrolatum as the oil phase. In fact, the SFN content in the microcapsules is slightly higher when petrolatum is used compared to without, with a maximum EE of 41% SFN, and the w / w SFN (μg) / GA (mg) ratio being 1.2. On the other hand, the EE value of SFN obtained in the microcapsules in the absence of one or more surfactants was 12.2 ± 0.1% for microcapsules dried under vacuum (in an oven) by the complex coacervation method with Gelatin / GA as wall material and SFN extract (methodology based on García-Saldaña, JS, Campas-Baypoli, ON, López-Cervantes, J., Sánchez-Machado, DI, CantúSoto , EU , & Rodriguez-Ramirez , R. ( 2016 ). Microencapsulation of sulforaphane from broccoli seed extracts by gelatin / gum Arabic and gelatin / pectin complexes. Food Chemistry, 201, 94–100. and was 39.8 ± 1.5%. using GA as wall material and spray drying in Wu, Y., Zou, L., Mao, J., Huang, J., & Liu, S. (2014). Stability and encapsulation efficiency of microencapsulated sulforaphane by spray drying. Carbohydrate Polymers, 102(1), 497–503. An SOR of 1 was used considering that the ratio and it is for the stability of the O / W emulsion (as established by Mujica Álvarez, J., Matiacevich, S., & Bustos, R. (2019). Studies of the optimal conditions to maximize physical and oxidative stability of a nanoencapsulated vitamin ingredient. Mundo Nano. Interdisciplinary Journal of Nanoscience and Nanotechnology, 12(23), 1. https: / / doi.org / 10.22201 / ceiich.24485691e.2019.23.67653). The resulting microcapsule had a wetter appearance when using petroleum jelly compared to those in the absence of petroleum jelly, making it difficult to obtain a fine powder. The results obtained when using a temperature of 20°C or 40°C were not statistically significant, and therefore, the tests continued to be carried out at 20°C to reduce the degradation of SFN, which begins to degrade above 40°C (Mahn, A., Saavedra, A., & Paz Rubio, M. (2018). Kinetic study of sulforaphane stability in blanched and un-blanched broccoli (Brassica oleracea var.italica) florets during storage at low temperatures. Journal of Food Science and Technology, 55(11), 4687-4693. https: / / doi.org / 10.1007 / s13197-018-3395-4). RE values ​​between 55 and 70% were obtained, thus demonstrating that the conditions described above have no significant effect on RE. Table 2: Preliminary encapsulation tests for different temperatures and oil phase by the O / W emulsion method. Volume: 10 ml; RT: 15 minutes; SFN / GA ratio (μg / mg): 1.2; Stirring speed: 8000 rpm; Wall material: GA; surfactant: Tween 80 (8%) and SOR (mg Tween80™ / mg petrolatum): 1.3. The behavior of the system with and without petrolatum as the oil phase was then evaluated at different stirring times (RT) and SFN / GA ratio, maintaining a SOR = 1. The results are shown in Table 3. Table 3. Preliminary SFN microencapsulation tests for different emulsion compositions by O / W emulsion. Volume: 10 ml; Stirring speed: 8000 rpm, Wall material: GA; Surfactant: Tween 80 (6%), Temperature: 20°C, SOR: 1.0, Oil phase: Petrolatum. Table 3 shows an increase in EE when compared to the results in Table 2, mainly as a result of the SOR value equal to 1 and a surfactant concentration of 7%. Then, from then on, the efficiency (EE) was tested with SOR = 1 and different surfactant concentrations considering that an excess of surfactant causes an increase in the polydispersity index, and thus, avoid the heterogeneous distribution of the particle size that arises with high polydispersity indices and that make the emulsion more unstable (Mujica Álvarez, J., Matiacevich, S., & Bustos, R. (2019). Studies of the optimal conditions to maximize physical and oxidative stability of a nanoencapsulated vitamin ingredient. Mundo Nano. Interdisciplinary Journal of Nanosciences and Nanotechnology, 12 (23), 1. https: / / doi.org / 10.22201 / ceiich.24485691e.2019.23.67653).Furthermore, it was confirmed that the efficiencies (EE) for the samples without petroleum jelly were lower than those for the samples with petroleum jelly, and it was also shown that there were no significant differences in EE, with the SFN / GA ratio and TA. Thus, the tests continued to maintain SOR = 1 and a temperature of 20. o C, with a variable ratio of SFN / GA, TA and Tween 80 concentration (CT, different proportions of Tween 80 in the emulsion) to determine the resulting EE, RE and FSL. A Box-Behnken design was used as shown in Table 4. Table 4. Independent experimental variables; stirring time, SFN / GA ratio and Tween 80 concentration and their effect on microencapsulation EE, RE and FSL Table 4 shows that there is a significant positive effect of the CT parameter and a significant negative effect for the SFN / GA ratio on the EE of microencapsulation. The significant effect of CT on the emulsion confirms what was proposed by Zhang, S., Ying, DY, Cheng, LJ, Bayrak, M., Jegasothy, H., Sanguansri, L., & Augustin, MA (2020). Sulforaphane in broccoli- based matrices: Effects of heat treatment and addition of oil. Lwt, 128 (October 2019), 109443. Yang, Q., Zhang, N., Du, Y., & Zhu, H. (2015). Preparation and characterization of inclusion complex of benzyl isothiocyanate extracted from papaya seed with β-cyclodextrin. Food Chemistry, 184, 99–104. where it is indicated that a Tween 80 surfactant concentration of 2 and 5% respectively in the emulsion managed to stabilize it, and therefore, obtain higher efficiencies. Meanwhile, very low values ​​of Tween 80 (3%) generate efficiencies less than 70%, the CT with the highest efficiency being 7%. At higher SFN loads, the lower its retention in the microcapsule, showing the negative effect of the SFN / GA ratio on the EE, marking a limit of loaded SFN content. In the case of TA, there is no significant difference. The highest efficiency value (90 ± 4%) is very close to that obtained by Wu, H., Xue, N., Hou, C.L., Feng, J.T., & Zhang, X. (2015). Microcapsule preparation of allyl isothiocyanate and its application on mature green tomato preservation. Food Chemistry, 175, 344–349. https: / / doi.org / 10.1016 / j.foodchem.2014.11.149, who used lyophilization as a drying method for AITC microcapsules obtained by complex coacervation with gelatin / GA, achieving an efficiency of 94.2 ± 2.3%. The best conditions for the highest EE were: TA: 11 minutes, SFN / GA ratio: 0.7 and CT: 7% with a predicted response of 91%. It was observed that when CT decreases the efficiency (EE) decreases, confirming that the amount of surfactant should be sufficient to keep the emulsion stable and that GA has a dual function, wall material and surfactant due to the higher efficiencies (EE) for low SFN / GA ratio values, the minimum value being 0.7. The optimal TA value within the ranges studied is between 11 and 13 minutes or 9 and 11 minutes, which could show that an increase in TA could further decrease the efficiency.The effect of these conditions on the ER was also evaluated, and it was found that the SFN / GA ratio has a significant negative effect on the ER; as the amount of GA added decreases, the microencapsulation ER decreases. Whereas, if CT increases, the ER decreases. The data in Table 4 show values ​​similar to those obtained by García-Saldaña, JS, Campas-Baypoli, ON, López-Cervantes, J., Sánchez-Machado, DI, Cantú-Soto, EU, & Rodríguez-Ramírez, R. (2016). Microencapsulation of sulforaphane from broccoli seed extracts by gelatin / gum Arabic and gelatin / pectin complexes. Food Chemistry, 201, 94–100. which is 85.13 ± 0.71%, and higher than that obtained by Sánchez, FM, García, F., Calvo, P., Bernalte, MJ, & González-gómez, D. (2016). Optimization of broccoli microencapsulation process by complex coacervation using response surface methodology. Innovative Food Science and Emerging Technologies, 34, 243–249. which is 64.5% where Gelatin / GA was used as in broccoli by complex coacervation while Wu, Y., Zou, L., Mao, J., Huang, J., & Liu, S. (2014). Stability and encapsulation efficiency of sulforaphane microencapsulated by spray drying. Carbohydrate Polymers, 102(1), 497–503. https: / / doi.org / 10.1016 / j.carbpol.2013.11.057 obtained an RE of 69.2 ± 1.4% by simple coacervation with GA as wall material for SFN microencapsulation and obtaining microcapsules by lyophilization. The SFN / GA ratio would have an optimal value between 1 and 1.4. In the microencapsulation process, losses may be related to the degradation of SFN during the process or the non-encapsulation of the compound remaining on the surface of the microcapsule, and therefore, the microcapsules were previously washed with ethanol, to then quantify the amount of SFN. CT has an effect on FSL that shows a significant positive difference. This agrees with what was established by Mujica Álvarez, J., Matiacevich, S., & Bustos, R. (2019).Studies of the optimal conditions for maximizing the physical and oxidative stability of a nanoencapsulated vitamin ingredient. Mundo Nano. Interdisciplinary Journal of Nanoscience and Nanotechnology. which indicates that excess surfactant produces an increase in the polydispersity index, the latter being closely related to the particle size, and where high values ​​of the polydispersity index indicate a heterogeneous particle size distribution, resulting in an unstable emulsion and a lower encapsulation efficiency (EE). The optimal conditions were found to be: TA: 14 minutes, SFN / GA: 2.1 and CT: 3%. As the SFN / GA ratio increases, so does the FSL. TA has an optimal value within the ranges 11 and 13 minutes. While when CT decreases, the FSL also decreases, behavior given by the polydispersity generated in the emulsion. These results are consistent with those obtained from the encapsulation efficiency (EE), where CT has the greatest effect on EE.However, although increasing CT results in greater efficiency, FSL increases in the microcapsule, meaning that more free SFN is found in the dry powder than in the encapsulated powder. Therefore, as the SFN / GA ratio decreases, EE increases. The above results were confirmed by performing an extension that considers only CT and the SFN / GA ratio, since TA does not have a significant effect, as shown before, and the shortest time was taken to decrease the energy required to obtain microcapsules. An increase in the SFN / GA or CT ratio does not increase EE, but it does increase FSL, demonstrating the higher free SFN content in the powdered microcapsule. Furthermore, although RE does not vary significantly, a decrease is noted with increasing surfactant. Therefore, the evaluated extension does not improve EE or RE, since FSL decreases with increasing surfactant, and therefore, there would be a decrease in SFN incorporated into the system.In summary, the conditions were defined as: SFN / GA ratio: 0.7%, CT: 7% and TA: 7 min, they were validated in triplicate, obtaining an efficiency of 90 ± 3%, an RE of 86 ± 5% and an FSL of 37 ± 8% as shown in Table 5. Table 5. Table 6. Description of the procedure Thus, the present method for preparing stabilized SFN microcapsules comprises: a) obtaining an extract rich in SFN from broccoli seeds from: a.1) grinding broccoli seeds until obtaining a flour which is then dissolved in ultrapure water, the proportion by weight (mg) of broccoli seed flour to the volume (ml) of water being from 8:7 to 3:2, and preferably is 5:4, and obtaining an SFN homogenate which is incubated at a temperature in the range of 40-50ºC, and preferably is 45ºC, preferably in a water bath; a.2) the double organic extraction of the incubated SFN homogenate with methylene chloride in a sonication bath and vacuum filtration of the sonicated SFN organic extract to separate the SFN-rich organic phase which is then vacuum dried at a temperature in the range of 25-35ºC, and preferably at 30ºC, by rotary evaporation, and a.3) resuspending the SFN-rich organic phase dried by rotary evaporation in ethanol (50% v / v) and obtaining an SFN-rich ethanolic extract, and b) preparing stabilized SFN microcapsules from: b.1) preparing an oil-in-water emulsion by mixing the SFN-rich ethanolic extract with petrolatum, preferably oral liquid petrolatum, as the oily phase of the emulsion, under stirring and in a water bath at a temperature in the range of 15 to 25°C, and preferably at 20°C, and then adding 20% ​​gum arabic (GA) as the aqueous phase, and in the presence of a surfactant, preferably a 7% surfactant (mg surfactant / mg emulsion), and more preferably in the presence of Tween 80, obtaining an O / W emulsion homogenate of SFN in petrolatum and GA, where the O / W emulsion ratio of SFN in petrolatum and GA / GA (μg / mg) is 0.7, and the oil phase to surfactant ratio (SOR) is 1; and b.2) drying by dehydrating the O / W emulsion of SFN in petroleum jelly and GA, in layers no greater than 1 mm, under vacuum and at 37°C, preferably in an oven, and grinding the O / W emulsion of SFN in petroleum jelly and GA to obtain powdered microcapsules. The present invention also relates to an oil-in-water emulsion of sulforaphane (SFN) comprising 20% ​​w / w gum arabic (GA) as a wall component, containing an oil-in-water, O / W emulsion, comprising petroleum jelly as the oil phase and an extract of SFN in 50% v / v ethanolic solution as the aqueous phase and Tween 80. TMas a surfactant, where the ratio of SFN extract to GA is 0.7 and the surfactant concentration is 7%. Another aspect of the present invention corresponds to the microcapsules with stabilized SFN that comprise the aforementioned emulsion and that are useful in the food, cosmetics, nutraceutical and pharmaceutical industries. Yet another aspect of the present invention corresponds to a food additive comprising the microcapsules with stabilized SFN described above and one or more food agents; a pharmacological additive comprising the microcapsules with stabilized SFN described above and one or more pharmacological agents; a cosmetic additive comprising the microcapsules with stabilized SFN described above and one or more cosmetic agents; or a nutraceutical additive comprising the microcapsules with stabilized SFN described above and one or more nutraceutical agents.Yet another aspect of the present invention relates to the use of the aforementioned additives in food formulations, pharmacological formulations, cosmetic formulations, or nutraceutical formulations. Example 1: Obtaining SFN and Quantification 25 g of seeds were ground into flour and homogenized in 20 ml of ultrapure water. To induce glucoraphanin hydrolysis, the mixture was incubated for 3 hours in a 45°C water bath. Excess water was removed with sodium hydroxide, and the seeds were dissolved twice with 100 ml of methylene chloride using a sonication bath for 30 minutes to extract the SFN formed. The SFN-rich organic phase remains at the bottom due to gravity. The mixture was vacuum filtered, the organic phase separated, and the aqueous phase discarded.10 ml of the organic phase were dried at 30°C under vacuum in a rotary evaporator (model RE300, Stuart) and the residue was dissolved in 10 ml of ethanol (50% v / v) to be loaded into the microencapsulation process. The SFN content was determined by reversed-phase HPLC. 1 ml of the extract diluted in methylene chloride was filtered through a filter paper to separate residues and the remaining methylene chloride was dried at 30°C under vacuum in a rotary evaporator (model RE300, Stuart). The residue was then dissolved in 2 ml of acetonitrile and filtered, with a 0.22 micron syringe filter, to be analyzed by HPLC. The samples were analyzed using a Shimadzu HPLC-DAD system (Tokyo, Japan), using a C18 reversed-phase column (5 μm, 250 mm × 4.6 mm) (Agilent Technologies, Santa Clara, CA, USA).The mobile phase consisted of 60% acetonitrile in water for 2 minutes; this solution was then linearly changed to 70% acetonitrile over 4 minutes, held for another 4 minutes, and held at 100% acetonitrile for 5 minutes to purge the column. The equipment was operated at 30°C, flow rate 1 ml / min, and injection volume was 20 μl. SFN was detected by absorbance at 254 nm with a retention time of 2.5 minutes. Example 2: Stabilization of SFN The following system components were used: oral liquid petrolatum (oil phase), Tween 80 (surfactant), ethanolic extract of SFN (active component), and 20% w / w gum arabic (GA) solution (aqueous phase). The validated parameters for the emulsion process were an active compound / polymer ratio (μg / mg) of 0.7, a Polysorbate 80 concentration of 7% (mg of Tween 80 / mg of emulsion), a SOR (Surfactant / Oil Phase Ratio) of 1, and a stirring time (TA) of 7 min.The mixture of surfactant, petrolatum, and SFN in ethanol obtained from broccoli seeds was poured into a beaker and stirred for one minute before adding the 20% Gum Arabic solution. The entire mixture was placed in a water bath at 20°C while stirring at 8000 rpm for 7 minutes in a homogenizer (T 25 D, IKA, Germany). The mixture was placed in a container that allows the formation of an emulsion layer that does not exceed 1 mm in thickness to ensure correct dehydration in a vacuum oven (-0.6 MPa) at 37 ± 2°C for 24 h. The free SFN found in the microcapsules obtained in powder form was quantified by weighing 300 mg of dry microcapsules, which were dissolved in 5 ml of absolute ethanol to solubilize any SFN that may have remained on their surface. The extract was filtered through Whatman No. 41 paper, and the solvent was evaporated on a rotary evaporator at 37 ± 2°C and reconstituted in 2 ml of HPLC-grade acetonitrile. SFN was quantified by HPLC.The SFN extract obtained from the seed contains other compounds, mainly unsaturated fats according to the latest report by H. Li, et al, “Nutritional values, beneficial effects, and food applications of broccoli (Brassica oleracea var. italica Plenck)”, Trends in Food Science & Technology, Volume 119, 2022, Pages 288-308, ISSN 0924-2244, https: / / doi.org / 10.1016 / j.tifs.2021.12.015, which indicates 71.39% of polyunsaturated fatty acids in the broccoli seed, which would explain the presence of fat droplets, see Figure 2A, and the difference in their sizes, one being inside the other. Furthermore, in the mixture of SFN extract with petrolatum, it can be observed how the petrolatum drops are less agglomerated (Figure 2G) than when the petrolatum is present alone (Figure 2F), and then the SFN extract could contain surfactant in the mixture which would reduce the interfacial tension between the petrolatum drops.Example 3: Characterization of SFN microcapsules Optical microscope observation The dried microcapsules, which were dissolved in distilled water, were observed using an Optical Microscope / Carl Zeiss, WT 6V / 10W, Germany and AxioVision Rel.4.8 software. Spherical-shaped microcapsules are shown in Figures 1A and 1B. Wang, G., & Sukumar, S. (2020). Characteristics and antitumor activity of polysorbate 80 curcumin micelles preparation by cloud point cooling. Journal of Drug Delivery Science and Technology, 59(February), 101871. Amphiphilic surfactant molecules such as Tween 80 form micelles above the critical micellar concentration (CMC), and water-insoluble compounds can be solubilized in the core. The micelles form spontaneously and are similar to the nanoemulsions produced by the emulsification method, which is achieved by first mixing the oil and the surfactant and then pouring the mixture into the aqueous medium, so micelles could exist in this complex. The mixture of the components was observed separately, and they were stirred in an ultraturrax at the same speed and time, to determine the moment of formation and distribution of the drops. The images obtained from the optical microscope can be seen in Figures 2A-2G, where the similarity in shape and appearance of the drops is evident. Figure 3C shows an FTIR spectrum with bands at: 1044 cm -1 , corresponding to the sulfoxide functional group found in the SFN structure; 1254 cm -1, corresponding to group CN and 1747 cm -1 corresponding to the carbonyl ester functional group of triglycerides, which corroborates the presence of fat in the SFN extract. Figure 3B shows an FTIR spectrum with bands at: 1635 cm -1 corresponding to the C=O functional group. In Figure 3A it can be observed that the typical bands of each of the components disappear and the SFN band is not evident, indicating an interaction between the components that keeps the SFN associated and stabilized.

Claims

CLAIMS 1. Method for microencapsulating sulforaphane (SFN) to increase thermal stability, making it useful for the food and pharmaceutical industries, characterized in that it comprises the steps of: a) obtaining an extract rich in SFN from broccoli seeds from: a.1) grinding broccoli seeds until obtaining a flour which is then dissolved in ultrapure water, the proportion by weight (mg) of broccoli seed flour to the volume (ml) of water being from 8:7 to 3:2, and obtaining an SFN homogenate which is incubated at a temperature in the range of 40-50ºC; a.2) the double organic extraction of the incubated SFN homogenate with methylene chloride in a sonication bath and vacuum filtration of the sonicated SFN organic extract to separate the SFN-rich organic phase which is then vacuum dried at a temperature in the range of 25-35ºC by rotary evaporation, and a.3) resuspending the SFN-rich organic phase dried by rotary evaporation in ethanol (50% v / v) and obtaining an SFN-rich ethanolic extract, and b) preparing stabilized SFN microcapsules from: b.1) preparing an oil-in-water emulsion by mixing the SFN-rich ethanolic extract with petrolatum, preferably oral liquid petrolatum, as the oily phase of the emulsion, under stirring and in a water bath at a temperature in the range of 15 to 25°C, and then adding 20% ​​gum arabic (GA) as the aqueous phase, and in the presence of a surfactant, obtaining an O / W emulsion homogenate of SFN in petrolatum and GA, where the O / W emulsion ratio of SFN in petrolatum and GA (μg / mg) is 0.7, and the oil phase and surfactant ratio (SOR) is 1; and b.2) drying by dehydrating the O / W emulsion of SFN in petroleum jelly and GA, in layers no greater than 1 mm, under vacuum and at 37°C, preferably in an oven, and grinding the O / W emulsion of SFN in petroleum jelly and GA to obtain powdered microcapsules. 2.The method of claim 1 characterized in that in step a.1), the ratio by weight (mg) of broccoli seed flour to the volume (ml) of water is 5:

4.

3. The method of claim 1 characterized in that in step a.1), the incubation temperature is 45 ° C.

4. The method of claim 1 characterized in that in step a.1), the incubation is conducted in a water bath.

5. The method of claim 1 characterized in that in step a.2), the drying temperature is 30 ° C.

6. The method of claim 1 characterized in that in step b.1), the temperature of the water bath is 15 ° C.

7. The method of claim 1, characterized in that in step b.1), the surfactant has a concentration of 7% (mg surfactant / mg emulsion).

8. The method of claim 1 or 7, characterized in that in step b.1), the surfactant is Tween 80.

9. An oil-in-water emulsion of sulforaphane (SFN) characterized in that it comprises 20% w / w gum arabic (GA) as a wall component, containing an oil-in-water, O / W, emulsion, comprising petrolatum as the oil phase and an extract of SFN in 50% v / v ethanolic solution as the aqueous phase and Tween 80. TMas surfactant, where the ratio of SFN extract to GA is 0.7 and the concentration of the surfactant is 7%.

10. Microcapsules with stabilized SFN characterized in that they comprise an oil-in-water emulsion of sulforaphane (SFN) comprising 20% ​​w / w gum arabic (GA) as a wall component, containing an oil-in-water emulsion, O / W, comprising petrolatum as the oil phase and an SFN extract in 50% v / v ethanolic solution as the aqueous phase and Tween 80 TM as surfactant, where the ratio of SFN extract to GA is 0.7 and the concentration of the surfactant is 7%.

11. Food additive characterized in that it comprises microcapsules with SFN stabilized from an oil-in-water emulsion of sulforaphane (SFN) comprising gum arabic (GA) at 20% w / w as a wall component, containing an oil-in-water emulsion, O / W, comprising petrolatum as the oil phase and an SFN extract in 50% v / v ethanolic solution as the aqueous phase and Tween 80 TMas surfactant, where the ratio of SFN extract to GA is 0.7 and the concentration of the surfactant is 7%, and one or more food agents.

12. Pharmacological additive characterized in that it comprises microcapsules with SFN stabilized from an oil-in-water emulsion of sulforaphane (SFN) comprising gum arabic (GA) at 20% w / w as a wall component, containing an oil-in-water emulsion, O / W, comprising petrolatum as the oil phase and an SFN extract in 50% v / v ethanolic solution as the aqueous phase and Tween 80 TMas surfactant, where the ratio of SFN extract to GA is 0.7 and the concentration of the surfactant is 7%, and one or more pharmacological agents.

13. Cosmetic additive characterized in that it comprises microcapsules with SFN previously stabilized from an oil-in-water emulsion of sulforaphane (SFN) comprising gum arabic (GA) at 20% w / w as a wall component, containing an oil-in-water emulsion, O / W, comprising petrolatum as the oil phase and an extract of SFN in 50% v / v ethanolic solution as the aqueous phase and Tween 80 TM as a surfactant, where the ratio of SFN extract to GA is 0.7 and the concentration of the surfactant is 7%, and one or more cosmetic agents.

14. Nutraceutical additive characterized in that it comprises microcapsules with SFN stabilized from an oil-in-water emulsion of sulforaphane (SFN) comprising Gum arabic (GA) at 20% w / w as a wall component, containing an oil-in-water emulsion, O / W, comprising petrolatum as the oil phase and an SFN extract in 50% v / v ethanolic solution as the aqueous phase and Tween 80 TM as a surfactant, where the ratio of SFN extract to GA is 0.7 and the concentration of the surfactant is 7%, and one or more nutraceutical agents.

15. Use of the food additive, pharmacological additive, cosmetic additive or nutraceutical additive of any of claims 11 to 14, characterized in that it is useful for preparing food formulations, pharmacological formulations, cosmetic formulations or nutraceutical formulations.

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