## 3 Characterization of Food-Grade Systems
### Technological Characterization
#### 3.1.4 In Vitro Release Testing  
In the context of DDS and its corresponding food applications (dietary supplements and fortified food), in vitro release testing aims to evaluate over time the amounts of loaded substance that are released from the system under conditions simulating GIT. The extensive literature available proposes different protocols (direct beaker dissolution, dissolver dissolution, dialysis method, etc.) for colon-targeted DDS [65; 113; 114; 115]. Therefore, in order to properly compare release profiles different variables must be considered including, intrinsic properties of the system (shape, size, rheology/viscosity, nature of components and their interactions, if any, production procedures, %EE and/or %DL and release mechanism, etc. ), number and type of simulated environments (mouth, stomach, intestine, colon and any other specific sections) and their specific reference parameters (simulated environment in empty or fed conditions, pH, travel time, volumes and fluid composition, thus presence or absence of specific enzymes, surfactants, salts, etc.) [7; 46; 104; 116; 117; 118; 119; 120]. Added to this is the common research effort to develop in vitro dissolution/release equipment/protocols with increasing significance and predictivity of the in vivo process [121; 122; 123]. During the test, aliquots are taken and appropriately treated and are subjected to quantitative analysis of the released active substance (e.g., HPLC and UV). From the data obtained, profiles and release kinetics are derived. All these data allow us to assess the proportion of the active substance available for uptake, i.e., bioavailable, or in situ action. Referring to recent scientific literature, we propose an overview of this stage of the characterization of total food-grade systems for targeted delivery to the colon. Curcio et al., studying polymeric systems based on EUGB and EUGC for colon-targeted delivery of RSV, carried out an in vitro release test according to the pH-varying model provided by the Eur. Pharm. X Edn. (Method A, Apparatus 2, Delayed-release solid dosage forms) with some minor modifications. Briefly, 750 mL of a 0.1 N HCl solution to simulate the gastric environment at pH 1.2 (SGF) and the sample to be analyzed were placed in the beaker of the paddle dissolver (thermostated to 37 \(\pm\) 0.5 \({}^{\circ}\)C). After 2 h to simulate the intestinal environment, the pH was raised to 6.8 (SIF) by adding 250 mL of a 0.20 M Na\({}_{3}\)PO\({}_{4}\) solution. Finally, the colon environment was simulated after 8 h by bringing the pH to 7.4 (SCF) with a few drops of a 3N solution of NaOH. The test had a total duration of 24 h. Aliquots of the medium replaced with equal volume of specific fresh buffer were taken at predetermined intervals. By UV analysis, the corresponding release rates were derived. All systems prepared with EUGB exhibited low releases under simulated gastric conditions (less than 30%) except for the one with the RSV:EUGB weight ratio of 1:5 in which 40% release was exceeded. This is probably due to the presence of unencapsulated RSV as confirmed by the low value of %EE = 75.2% and FTIR and DSC analysis. In addition, it was shown that the release rate gradually increased in SIF to reach the plateau in simulated colonic environment. The test showed that the minimum RSV:EUGB weight ratio required to control RSV release was 1:10; whereas systems with weight ratios of 1:20 and 1:50 had nearly overlapping release profiles. Systems formed from EUGC alone had low sustained RSV release. Mixed matrices allowed the release plateau to shift (about 80%) in the interval 4-8 h after the start of the test (SIF-SCF) with release rates in the gastric environment varying from 20 to 60% [65]. The in vitro release of RSV from pectin-zinc-chitosan nanoparticles (10:1:3) with or without polyethylene glycol (PEG) prepared (%EE = 63% with PEG) by Andishmand et al. was studied by dialysis technique.