**5. Production Procedures**

The major objective of the encapsulation is creating coating-sensitive compounds or reducing side effects of some useful compounds applied in high concentrations; these compounds are located in the core and coated by suitable wall materials. Encapsulation techniques protect nutraceuticals or bioactive compounds from unbalanced and unfavorable conditions, including pH, light, moisture, heat, chemical and biological degradation, and oxygen during storage, processing and utilization. Wall materials, including lipids and surfactants, have a critical role in the encapsulation technique because of their important effects on target delivery, bioavailability, biocompatibility and protection of bioactive compounds. Additionally, these materials should be safe and do not have an impact on flavor, color, texture or other properties of foods. The most important properties of suitable wall materials include a low cost, low viscosity, film-forming capacity, high solubility, low hydroscope, high stability in the media of the target, high protection, abundance, nontoxicity and compatibility in food or drug formulations. Several techniques are used for encapsulating bioactive agents; the preferred encapsulation technique depends on the bioactive compound structure and its end use. The most common encapsulation techniques for lipid NPs include emulsification, homogenization at high pressure, microemulsion and emulsion–evaporation of the solvent, sometimes combined with sonication. The main production procedures are discussed below for their use in the food industry.

#### *5.1. Homogenization at High Pressure (HPH)*

The melted lipid is emulsified in an aqueous solution containing the surfactant at the same temperature by agitation at high speed or ultrasounds. The pre-emulsion is then subjected to high-pressure homogenization. As typical production conditions, 500 bar pressure and between 3 and 5 homogenization cycles are repeated. Finally, the nanoemulsion is cooled, the lipid phase solidifies, and the suspension of lipid nanoparticles is formed. It must be highlighted that increasing homogenization cycles may lead to particle coalescence, resulting in a bigger particle size. This technique is especially aimed at the encapsulation of lipophilic molecules since the hydrophilic ones diffuse in a large proportion to the aqueous phase during the homogenization phase, giving rise to a low encapsulation efficiency. One of the drawbacks of this technology is the exposure of the active ingredients to high temperatures, although for a very short time, this allows sensitive compounds to resist the

process. Additionally, the high temperatures used in hot HPH may reduce the emulsifying capacity of most surfactants, therefore causing nanocarriers' instability [64,66,67,74].

For the encapsulation of thermosensitive compounds, a cold homogenization method was designed in which the molten lipid is rapidly cooled in dry ice, the solid form of carbon dioxide, or in liquid nitrogen. In this way, the fragility of the lipid is increased to facilitate the grinding process for obtaining microparticles. These are dispersed in the cold solution of the surfactant, and finally, the suspension is subjected to high-pressure homogenization at or below room temperature [87]. HPH is the most used production technique for nanocarriers encapsulating food ingredients due to the advantages that it has compared to other methods, including large-scale production, disuse of organic solvents and shorter production time.

#### *5.2. Preparation Technique via Microemulsion*

This method requires low energy and is based on the basic mechanism of microemulsions, which can be transformed into an ultrafine nanoemulsion after their rupture by adding a certain volume of water.

In the microemulsion formation, the lipid melts and the active substance or a drug is dissolved in it. Next, the surfactant, cosurfactant and water are added at a high temperature to form the microemulsion, which is poured over cold water, breaking into nanoparticles of emulsion, which crystallize to form lipid nanoparticles. As drawbacks of this process, we can point out the high concentration of the surfactant and cosurfactant which is required, the use of solvents to form the emulsion and the high dilution to which the particles are subjected, which leads to the final content in particles being below 1%. The temperature difference between the chilled water and the microemulsion extremely influences the particle size in this method. The faster the solidification, the smaller the particle sizes. Although this method is operated under mild conditions, it requires abundant surfactant and cosurfactant, which could be a disadvantage for its use in the food industry [63].

#### *5.3. Solvent Emulsification–Evaporation Technique*

In this method, very low or no energy is required, and it is widely used for the preparation of polymeric micro- and nanoparticles. The lipid material in this case is dissolved in a water-immiscible organic solvent, in which the active ingredient is also dissolved. This organic phase is emulsified with the aqueous phase containing the surfactant agen<sup>t</sup> by means of mechanical agitation or an ultrasound probe. After evaporation of the solvent at reduced pressure, the dispersion of nanoparticles occurs after the precipitation of the lipid. The preparation of double emulsion in this technique allows the encapsulation of numerous compounds. As there is no heat involved, this method is suitable for heat-sensitive active compounds. The low energy required is another grea<sup>t</sup> advantage of this method. The main disadvantages of this technique are solvent-residue-associated toxicity and diluted particles. These disadvantages can be reduced by the selection of a food-grade solvent such as ethanol or ethyl acetate, making this method a good option for the encapsulation of food ingredients [63,77].

#### *5.4. Solvent Emulsification–Diffusion Technique*

This technique is similar to the previous one, differing only in the method of precipitation of the lipid from the emulsion. In this case, it is achieved by adding extra water to the aqueous phase, which causes immediate diffusion of the organic solvent, with the consequent precipitation of the lipid.

In the solvent emulsification–evaporation process, the lipid is dissolved in the waterimmiscible solvent, and then it is emulsified in an aqueous phase containing the surfactant, followed by evaporation of the solvent under reduced pressure. Lipid precipitation occurs upon solvent evaporation, leading to nanocarriers' formation. Merits of this method include its lab-scale acceptability, higher stability and ability to obtain the smallest particle size, but

its demerits are the use of toxic solvents, the increase in lipid content, which leads to an increase in the polydispersity index, and particle size distribution [31,78].

#### **6. Conclusions and Future Prospects**

Lipid nanoparticles are especially interesting for oral administration, for different reasons, including, in the first place, the mucoadhesive properties that they present due to their colloidal nature, and to which their ability to facilitate the release in the area of the intestine to which they adhere is attributed. On the other hand, there is the possibility that they are internalized by the intestinal cells, and the promoting effect of the absorption of the constituent lipid components must also be considered. Nanotechnology has wide applications in nutrition, food supplements, nutraceuticals and medical science [88–90]. The recent literature suggests that nanotechnology will overcome the current main challenges that bioactive compounds and nutraceuticals must face, such as their stability, low solubility, targeted delivery and prolonged release. Additionally, with regard to the food industry, new products must avoid problems related to their color, flavor or nutrient content. Accommodation of each health need could be achieved with the aid of pharmaceutical nanotechnology. In fact, it seems like a promising technology approach to reduce the dose levels and to achieve better and longer stability of the nutraceuticals. The formulations of the bioactives as nanostructured products will help in their superior characterization, improved patient acceptability and, above all, high reproducibility of their therapeutic effectiveness. Thus, a lot of nutraceuticals in nanosized forms have been developed in many works regarding the optimum production procedure or the most adequate wall materials for each nanoparticle, considering lipid types and surfactants. Additionally, many nutraceutical products containing NPs are commercially available on the market. Therefore, it can be concluded that nano-based carrier systems provide better means for enhancing the efficacy and availability of nutraceuticals having issues with solubility, stability and bioavailability.

Nevertheless, components of lipid NPs should be carefully selected since they will directly influence product stability and effectiveness. For future prospects, it should be remarked that studies on orally administered NPs are still very limited, and the molecular mechanisms by which they are absorbed through the intestinal lumen into the circulation should be better clarified by studying each lipid component. Although NPs possess grea<sup>t</sup> potential as delivery carriers, more preclinical and clinical studies are needed to better understand their behavior. Additionally, NPs have some related challenges, such as the need to improve their colloidal stability under harsh conditions, including food processing (heating, high pressure, drying, etc.) and the gastrointestinal environment (low pH, bile salt and digestive enzymes); studying interactions between bioactive compounds and nanoparticles for optimal encapsulation; and accepting the biological fate of these nanoparticles upon oral administration. Thus, further investigation on food nanotechnology is needed with regard to the in vivo and food processing stages.

**Author Contributions:** Methodology, formal analysis, investigation, resources and data curation: C.B.-L., J.F., A.D., M.L. and A.S. Writing of the original manuscript: C.B.-L., F.J.S. and E.B.S. Conceptualization, review and editing of the manuscript, project administration, supervision and funding acquisition: C.B.-L., A.S., F.J.S., A.S. and E.B.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work received the funding support from Santander/Autonoma University of Madrid, granted to the first author.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
