*2.3. Polysaccharide Matrices*

Polysaccharides that have various enzymatic susceptibilities to ensure specific degradation in the small or large intestine when used as a NP coating can efficiently retard the nonspecific release of encapsulated bioactive compounds until the coating is exposed to its intended environment of release, and such coated NPs can be potentially targeted to different GI tract organs and taken up by the enterocytes, providing improved oral bioavailability [89].

High amylose corn and potato starches nanocarriers with granular structure and particle sizes ranging from 32.04 to 99.2 nm were used to encapsulate vitamin D3, and their EE ranged from 22.34 to 94.8%. By using ultrasonic treatment, an increase of the hydrocarbon chain length was observed resulting in van der Waals and *H*-bonds of vitamin D3 with the potato starch and greater thermal stability [90]. Low-molecular-weight octenyl succinic anhydride modified starches were reported to be suitable to form stable vitamin E nanocapsules for potential application in beverages [91].

Recent findings concerning the use of cellulosic nanomaterials for food and nutraceutical needs were summarized by Khan et al. [92]. The addition of cellulose nanocrystals and lecithin into alginate microbeads improved the viability of encapsulated probiotic (*Lactobacillus rhamnosus* ATCC 9595) during gastric passage and storage, and at 25 and 4 ◦C storage conditions, a decrease in the viability of *L. rhamnosus* by 1.23 and 1.08 log, respectively, was estimated, while at encapsulation of the probiotic with alginate microbeads, a 3.17 and 1.93 log reduction, respectively, was observed [93]. The oligo-hyalurosomes nanoscale DES based on oligo-hyaluronic acid-CUR polymer co-loaded with both CUR and resveratrol (RES) showing the average particle size of 134.5 ± 5.1 nm, spherical shape, and zeta potential of −29.4 ± 1.2 mV at pH 7.4 phosphate buffer conditions exhibited excellent stability and sustained release character and higher radical scavenging activity compared to the single formulations and liposomes suggesting that this system could be considered as a promising nanofood DES applicable in juice, yoghourt and nutritional supplements [94].

CS/tripolyphosphate-nanoliposomes core-shell nanocomplexes as vitamin E carriers showed vitamin E retention rate >80% during the 30-day storage and 92% and 97% after heating at 65 ◦C for 30 min and at 80 ◦C for 16 s, respectively, and based on the enhanced stability of liposomes against temperature stress reflected in reduced particle aggregation, zeta potential inversion, and membrane fluidity, this formulation could be considered as appropriate for commercial use in the food industry [95]. CS hydrochloride/carboxymethyl CS nanocomplexes loaded with anthocyanins with particle size 178.1 nm, zeta potential +25.6 mV, and polydispersity index 0.315 showed a higher stability when placed at different conventional storage temperatures, various L-ascorbic acid concentrations, varying pH, or white fluorescent light, suggesting that such nanocomplexes could be applied in food ingredients associated with stable anthocyanins in functional foods and nutraceutical applications [96].

In food-grade alginate/CS nanolaminates obtained by the layer-by-layer technique, in which folic acid (FA) was incorporated by post-diffusion, a higher stability of FA under ultraviolet light exposure compared to free FA was estimated, and the higher rate and concentration of FA released from nanolaminates at pH 7 in comparison with that at pH 3 suggested that nanolaminates containing hydrophilic active compounds can be used for food applications [97].

Insulin encapsulated in antacid-loaded calcium alginate microgels (diameter 280 μm) had higher biological activity in simulated gastric conditions than free insulin, and considerably increased Akt phosphorylation at Thr308 and Ser473 in L6 myotubes was observed [98].

Papagiannopoulos and Vlassi [99] reported preparation of multi-functional stimuli-responsive NPs for food and biomedical applications by combining electrostatic complexation between proteins and polysaccharides with following thermal protein denaturation for the production of chondroitin sulfate/bovine serum albumin NPs. The irreversible protein–protein contacts upon temperature treatment provide the complexes with properties of nanogels, and the surface charge of the prepared NPs reversed at pH 5.3, while their size depended on the solution ionic strength and pH. Protein-polysaccharide-surfactant ternary complex particles prepared by anti-solvent co-precipitation using zein, propylene glycol alginate, and either rhamnolipid or lecithin pronouncedly improved the photostability and bioaccessibility of CUR suggesting that they could be used to deliver hydrophobic nutraceuticals for applications in foods, supplements, and pharmaceuticals [100].

#### *2.4. Protein-Based Carriers*

The state of the art of protein-based nanoencapsulation approaches as well as protein modification approaches in order to extend their functionality in nanocarrier systems to achieve an improvement in encapsulation, retention, protection, and release of bioactive agents was summarized by Fathi et al. [101]. A review paper discussing the latest findings concerning the nanoscale phenomena of whey protein denaturation and aggregation, which could contribute to the design of protein nanostructures with new or improved properties for the incorporation of nutraceuticals in food matrices and their release was presented by Ramos et al. [102]. Using whey protein isolate as an encapsulating agent, Parthasarathi and Anandharamakrishnan [103] presented a spray freeze-drying based microencapsulation technique as a promising strategy to enhance the oral bioavailability of poorly water-soluble bioactive compounds like vitamin E.

Significant aggregation and sedimentation of zein NPs encapsulating lutein (ZLNPs) with hydrodynamic radius approx. 75 nm were observed at gastric digestion conditions, and the ZLNPs that were not fully digested by gastric enzymes adhered to lipid droplets; however, the aggregation was reduced and digestion was stimulated when salt (i.e., high ion concentration) was left out. On the other hand, thanks to the encapsulation of lutein into NPs, its digestive stability was increased [104].

The size of egg albumin (Alb)-FA nanocomplexes prepared by mixing egg Alb NPs with FA did not change after adjusting the pH from 3 to 4, but showed considerable increase after adjusting pH to 5, 6, or 7; however, the bioavailability of FA in the form of digested nanocomplexes for *Lactobacillus rhamnosus* was improved [105].

The degree of FA binding to β-lactoglobulin (β-Lglb) and type A gelatin carriers was affected by their pH-dependent zeta-potential, which indicated the occurrence of ionic bonds, and the binding of FA reached 100% at pH 3. At pH 3, particle size considerably increased at increasing the molar FA/protein ratio; however, shifting back the pH to 7 totally reversed it, which means that these formulations could protect FA at pH 3 prevailing in the stomach, but they are strongly favorable for its delivery to the duodenum (pH 7) [106]. β-Lglb nanostructures were reported to be suitable carriers for riboflavin and its controlled release in an in vitro GI system: approx. 11% was released during their passage through the stomach, while 35%, 38%, and 5% of the total riboflavin were released during their passage through duodenum, jejunum, and ileum, respectively. At food simulant conditions (yoghurt simulant, 3% acetic acid), β-Lglb nanostructures were stable for more than 14 days and had protective impact on riboflavin activity, releasing it in a 7-day period [107].

Isolated 7S and 11S globulins (Glbs) obtained from defeated soy flour, which were complexed with FA and included in culture media, showed higher bacterial growth of *Lactobacillus casei* BL23. Therefore, Glbs-FA based nanocomplexes have potential to be used in nutraceutical, pharmaceutical, and food industries [108]. *Lactobacillus casei* BL23 produces microvesicles carrying proteins that have been connected with its probiotic effect, and, using a proteomic approach, Rubio et al. [109] identified

proteins described as mediators of *Lactobacillus*' probiotic effects, namely p40, p75, and the product of LCABL\_31160, which was annotated as an adhesion protein. The expression and subsequent encapsulation of proteins into microvesicles of bacteria generally considered as safe could be also used in applications of foods and nutraceuticals.

Negatively charged ( −41 mV) sophorolipid-coated CUR NPs with the particle size of 61 nm showing relatively high EE and loading capacity for CUR that was present in an amorphous state exhibited 2.7–3.6-fold higher bioavailability than free CUR crystals, which was connected primarily with their higher bioaccessibility [110].

Protein–lipid composite NPs having a three-layered structure (barley protein layer, α-tocopherol layer, and phospholipid layer) and an inner aqueous compartment to load the hydrophilic nutraceutical vitamin B12 exhibited controlled release behavior in simulated GI media, and in an in vivo experiment, the NPs loaded with vitamin B12 increased serum vitamin B12 levels in rats upon their oral administration and reduced the level of methylmalonic acid more efficiently than the free vitamin B12 form without any toxicity of the formulation observed during 14 days. These NPs could be used for increasing vitamin B12 absorption upon oral administration [111].

The enhanced physicochemical stability and in vitro bioaccessibility of vitamin D3 in corn protein hydrolysate-based vitamin D3 nanocomplexes showing spherical structure with sizes 102–121 nm was reported by Lin et al. [112]. In vitamin D–potato protein co-assemblies, the nanocomplexation provided pronounced protection and reduced vitamin D losses during pasteurization and also under several different sets of storage conditions, suggesting that potato protein could be used as a protective carrier for hydrophobic nutraceuticals suitable for enrichment of clear beverages and other food or drink products with beneficial impact on human health [113].

After drying and reconstitution, vitamin D-loaded re-assembled casein micelles (r-CMs) were found to improve the in vitro bioavailability of vitamin D in a Caco-2 cell model and showed strong protective effect against its gastric degradation, providing 4-fold higher bioavailability compared to free vitamin D [114]. Ghayour et al. [115] encapsulated CUR and Q using a hierarchical approach (binding of ligand to SCas with subsequent re-assembling of micellar nanostructures or formation of casein NPs). r-CMs had smaller mean particle size than casein NPs, and the entrapment efficiency of both ligands was >90%. An incorporated phenolic compound showed notably improved chemical stability during an accelerated shelf-life test. The aqueous solubility of CUR and Q after loading in r-CMs was higher than that of free polyphenol molecules, and the viability of treated MCF-7 human breast cancer cells decreased as follows: free polyphenol molecules >> non-digested polyphenol-loaded carriers > digested polyphenol-loaded r-CMs. Based on the investigation of the stability and bioavailability of CUR in mixed SCas and pea protein isolate NEs, Yerramilli et al. [116] reported that pea proteins could be used to partially replace SCas as an emulsion stabilizer for the protection and delivery of oil-soluble bioactive compounds. Based on in vitro proteolysis, it was found that in low-fat yogur<sup>t</sup> supplemented with the spray- and freeze-dried casein micelles loaded with vitamin D2, 90% of the vitamin remained active compared to 67% estimated with free vitamin [117].

FA–loaded casein NPs of 150 nm fabricated with the use of a coacervation process, stabilized with lysine or arginine, and finally dried by spray-drying were administered to laboratory animals p.o. at dose 1 mg FA/kg and ensured considerably higher serum levels of the vitamin than an aqueous solution of FA administered to animals, and the release profile and oral bioavailability of FA were not affected by the treatment of casein NPs by high hydrostatic pressure [118].
