Page 386 - IJB-10-6

International Journal of Bioprinting Innovative manufacturing of ω-3-enriched chocolate

compression testing machine (EZ-LX; Shimadzu, Japan), spectrophotometer 6 min after initial mixing. To conduct
the mechanical strength of the 3D-printed chocolates the CUPRAC assay, 1 mL portions of CuCl (0.01 M),
2
was determined. ammonium acetate buffer (1 M, pH 7.0), and neocuproine
(7.5 mM) were mixed. After adding 0.1 mL of the extract and
2.10. Differential scanning calorimetry 1 mL of distilled water, the mixture was incubated at room
Differential scanning calorimetry (DSC) was performed temperature for 1 h in the dark. Absorbance measurements
using DSC-6O Plus (Shimadzu, Japan) to examine were obtained at 450 nm using a spectrophotometer. The
the thermal properties of the chocolates (as described results are expressed as μmol Trolox equivalent (TE)/g dw.
by Aydin et al. ); a heating rate of 10°C/min over a
29
temperature range of 25–350°C in an N stream was used 2.13. In vitro simulation of gastrointestinal digestion
2
in the study. The parameters representing the melting In vitro simulated digestion studies were performed as
profile associated with the samples, i.e., onset temperature described by Tolve et al. and Ozkan et al. Briefly, 1 g
31
32
(T onset ; the temperature at which the samples begin to of chocolate samples was mixed with 0.7 mL of simulated
melt), peak temperature (T peak ; the temperature at which salivary fluid (SSF), 5 µL of 3 M CaCl , 195 µL of water,
2
the highest melting rate was noted), end temperature and 0.1 mL of α-amylase. After 3 min, 1.5 mL of simulated
(T ; the temperature at which the samples completely gastric fluid (SGF), 139 µL of water, 1 µL of 0.3 M CaCl ,
end
2
melted), and ΔH (the energy that is required for complete and 0.32 mL of pepsin were added. The pH of the solution
sample melting) were determined from thermograms was subsequently adjusted to 3.0 with 40 µL of HCl (1
obtained from the measurements. M); the sample was incubated at 37°C for 2 h. After 2 h
2.11. Determination of total polyphenol content of incubation at 37°C, 1.3 mL of simulated intestinal fluid
The chocolate samples were defatted prior to phenolic (SIF), 1 mL of lipase, 1 mL of pancreatin, 0.5 mL of bile
extraction using the approach described by Hu et al. salts, 8 µL of 0.3 M CaCl , and 162 µL of water were added.
30
2
with some modifications. Briefly, the samples were sliced The pH was adjusted to a pH value of 7 in this phase using
into small pieces; 0.5 g of each piece was extracted using 30 µL of NaOH (1 M). The sample was incubated at 37°C for
2.5 mL of n-hexane twice via shaking for 45 min at 25 ± 3 h. The aliquots collected at the end of the salivary, gastric,
0.03°C at 150 ×g. After each extraction step, the mixtures and intestinal phases were added to 9 mL of methanol/
were centrifuged at 2000 ×g for 10 min; the supernatant HCl/water (79.9:0.1:20, v/v/v) and brought to −20°C in
was subsequently discarded. After the second extraction order to inhibit the enzyme activity prior to polyphenol
step, the samples were dried in a hot-air dryer for 24 h at determination. A blank test tube without a chocolate
25°C. The defatted samples were subsequently weighed; sample but with all digestion fluids was also analyzed. The
the phenolics were extracted using 5 mL of 0.1% HCl bioaccessibility index (BI) value was determined as follows:
and 80% aqueous methanol from the defatted samples.
After vortexing for 2 min, the solution was sonicated for
20 min. Next, the solution was centrifuged for 10 min BI (%) = A × 100 % (I)
31
at 2000 ×g. The supernatant was subsequently recovered, B
filtered through a 0.45 μm filter, and then stored at −20°C
until further extraction. The total phenolic content (TPC) where A is the TPC in the intestinal phase after in
was finally measured. The TPC results are presented as vitro digestion and B is the TPC in the samples before in
32
mg gallic acid equivalent (GAE)/100 g dw (dw: dry weight). vitro digestion.
2.12. Antioxidant capacity assessment 2.14. In vitro ω-3 release study
The 2,2-Diphenyl-1-picrylhydrazylradical (DPPH) radical Standard ω-3 solutions were prepared using an ultraviolet
scavenging activity, 2,2-azino-bis-3-ethylbenzothiazoline- (UV) spectrometer (Shimadzu UV-3600; Shimadzu,
33
6-sulphonic acid (ABTS) radical scavenging activity, and Japan) as described by Baykara et al. A calibration graph
copper-reducing antioxidant capacity (CUPRAC) were (0.2, 0.4, 0.6, 0.8, and 1 µL/mL) was plotted accordingly.
32
measured accordingly. To conduct the DPPH assay, 0.1 mL In vitro release studies of ω-3-mixed chocolate and ω-3-
of the extract was mixed with 4.9 mL of DPPH solution SA MP-coated chocolates were performed. Briefly, 5 mg of
(0.1 mM in methanol). The mixture was subsequently chocolate samples were placed in Eppendorf tubes; 1 mL of
incubated at room temperature in the dark for 20 min. phosphate-buffered saline (PBS; pH 4 and 7.4) was added
The absorbance was then measured at 517 nm using a to each sample. The release of ω-3 was determined in PBS
spectrophotometer. To conduct the ABTS assay, 0.1 mL of in a shaking incubator (37°C; 1.5 ×g). Thereafter, 1 mL of
the extract was mixed with 2 mL of diluted ABTS solution; the solution was removed from the tubes at the specified
the absorbance values were measured at 734 nm using a intervals, and a UV-visible (Vis) spectrophotometer

Volume 10 Issue 6 (2024) 378 doi: 10.36922/ijb.3969