## 3 Characterization of Food-Grade Systems
### Technological Characterization
#### 3.1.4 In Vitro Release Testing
developed hollow/QUE-filled microspheres of alginate (AL-E/Q), alginate/inulin (ALIN-E/Q), and alginate/inulin/chitosan (ALINCH-E/Q), with particle sizes ranging from 25.1 \(\pm\) 1.8 to 79.4 \(\pm\) 4.5 \(\upmu\)m.  
The presence of inulin allowed the enlarged pores of the alginate lattice to be occluded in an alkaline environment constituting an additional factor controlling release. In vitro gastrointestinal digestion was performed with a protocol (INFOGEST) simulating the progression in saliva (SSF), gastric fluid (SGF, pH = 3) and intestinal fluid (SIF, pH = 7). The combination of inulin as the pore-filling material of the alginate network and chitosan as the coating material in loaded ALINCH-Q (%EE = 53.2 \(\pm\) 1.2%) outperformed the other formulations with a QUE retention rate of 80.3 \(\pm\) 4.4% after in vitro gastrointestinal digestive treatment. Therefore, ALINCH-Q was subjected to colonic fermentation (Figure 14) using pig fecal material as the source of microbiota. Fecal inoculum was prepared by homogenizing 20 g of freshly collected porcine feces (from four healthy females) in 80 mL of sterilized 0.1 M phosphate buffer (pH = 7.0) and filtered through a sterile muslin cloth. Fermentation was conducted by adding 100 mg of microspheres in 5 mL of sterilized basic culture medium to which 5 mL of fecal inoculum was added, and the mixture was washed with nitrogen, vortexed and incubated in anaerbiosis under positive nitrogen pressure for 24 h. Free QUE was gradually metabolized by the porcine fecal microbiome, disappearing over the 24 h of fermentation, but encapsulated QUE (ALINCH-Q) delayed metabolism. In fact, at time zero, the free form had the highest level (0.39 mg/mL), and then gradually decreased with time, while for the (ALINCH-Q) system, a minimum amount of encapsulated quercetin was detected and then reached 0.33 mg/mL concentration in the fermentation fluid at 3 h. The level of quercetin in both cases decreased substantially within 6 h, and by 12 h most was degraded to phenolic metabolites by the action of the porcine microbiome. Since chitosan is poorly fermentable by intestinal bacteria, it would appear that the release of quercetin is mainly due to ionization of the carboxyl groups of alginate at neutral pH (enlargement of the hydrogel network) and its degradation by excreted microbial polysaccharide hydrolases. From the results obtained, ALINCH-Q microspheres have been proposed as a valid system for the sustained release of QUE by prolonging its retention time during colonic fermentation [79]. A more recent study published proposed an interesting site-specific delivery system of nutraceuticals for ulcerative colitis (UC). The natural anti-inflammatory products berberine (BBR) and epigallocatechin gallate (EGCG) were assembled (BBR/MPN NPs) and encapsulated in yeast microcapsules (YM), generating the BBR/MPN@YM system (%EE = 62.45 \(\pm\) 1.16% for BBR and %EE = 95.27 \(\pm\) 0.33% for EGCG). The system was designed for site-specific delivery to inflammation-prone areas of the colon for the treatment of ulcerative colitis. Indeed, due to the rigid structure of \(\beta\)-glucan on the yeast microcapsules, decomposition of the system by gastric acid and pepsin could be reduced to a certain extent. Upon reaching the colon, the system could be phagocytosed by M cells, transported into the payer's patch and internalized into macrophages. Then, the degradation of \(\beta\)-glucan and nanoparticle structure could allow the release of BBR and EGCG by transforming M1 macrophages into anti-inflammatory M2  
Figure 14: Quercetin degradation during 24 h of in vitro colonic fermentation. Blank: no substrate addition (10 mL fecal inoculum), ALINCH-E: empty microspheres (100 mg/10 mL fecal inoculum), UQ: unencapsulated quercetin (5.46 mg quercetin in 10 mL fecal inoculum, equivalent to the amount encapsulated in 100 mg ALINCH-Q), ALINCH-Q: quercetin loaded microspheres (100 mg containing 5.46 mg encapsulated quercetin/10 mL fecal inoculum).