experimental mass spectrum and the MS
2 data are compared
with the data available in the literature for carotenoid identification in food samples, as well as with authentic carotenoid
standards isolated from their natural sources. For carotenoid
quantification, external calibration curves are applied for individual free carotenoids, while xanthophyll esters are quantified
with the calibration curves of their corresponding free xanthophyll counterparts.
3.5 Assessment of
Lipolysis Efficiency
As the carotenoids belong to the lipid profile of foods, they should
incorporate into mixed micelles to be accessible to intestinal cells
for absorption. Micellarization is the outcome of a complex process
where several parameters are key players, but particularly the action
of digestive secretions that include enzymes and bile salts impacts
the efficiency of this stage. Therefore, to obtain reliable results is
critical to measure the efficiency of the lipolysis in the chyme
obtained after the in vitro digestion protocol.
1. Add 1 g of chyme to a 15 mL centrifuge tube and mix with
5 mL of the Folch solution, cap and mix for 10 min. Mixture
could be stored at À20
C at this point if required.
2. Add 1 mL of 1 M NaCl to the tube and mix for 10 min.
3. Centrifuge for 10 min at 1000 g at 4
C.
4. Discard the aqueous upper layer and transfer the solvent phase
(see Note 14) to a rotatory flask and evaporate to dryness. Add
0.5 mL of chloroform to the dry lipid extract and vortex to
dissolve.
5. Spot 200 μL of the lipid extract onto a TLC plate and develop
the TLC plate with the elution solvent in the TLC chamber
until solvent reaches 4/5 up plate.
6. Remove the TLC plate from the chamber and allow solvents to
evaporate at room temperature.
7. Spray the TLC plate with H 2 O–H 2 SO 4 (1:1, v/v) solution and
place it in a plate heater at 180
C for 15 min. Subsequently, let
the TLC plate to reach ambient temperature and scan it. Use an
image processing software to analyze one-dimensional images
and obtain the grey scale intensity values (see Note 15).
3.6 Calculations and
Statistical Analysis
1. The micellarization index is expressed as percentage value
regarding the total initial carotenoid content in the meal and
the total carotenoid content in the micellar fraction.
2. A minimum of three replicates per in vitro digestion process is
required to determine whether the observed effect (if it is so) is
statistically significant.
Carotenoid In Vitro Digestion
383
2 data are compared
with the data available in the literature for carotenoid identification in food samples, as well as with authentic carotenoid
standards isolated from their natural sources. For carotenoid
quantification, external calibration curves are applied for individual free carotenoids, while xanthophyll esters are quantified
with the calibration curves of their corresponding free xanthophyll counterparts.
3.5 Assessment of
Lipolysis Efficiency
As the carotenoids belong to the lipid profile of foods, they should
incorporate into mixed micelles to be accessible to intestinal cells
for absorption. Micellarization is the outcome of a complex process
where several parameters are key players, but particularly the action
of digestive secretions that include enzymes and bile salts impacts
the efficiency of this stage. Therefore, to obtain reliable results is
critical to measure the efficiency of the lipolysis in the chyme
obtained after the in vitro digestion protocol.
1. Add 1 g of chyme to a 15 mL centrifuge tube and mix with
5 mL of the Folch solution, cap and mix for 10 min. Mixture
could be stored at À20
C at this point if required.
2. Add 1 mL of 1 M NaCl to the tube and mix for 10 min.
3. Centrifuge for 10 min at 1000 g at 4
C.
4. Discard the aqueous upper layer and transfer the solvent phase
(see Note 14) to a rotatory flask and evaporate to dryness. Add
0.5 mL of chloroform to the dry lipid extract and vortex to
dissolve.
5. Spot 200 μL of the lipid extract onto a TLC plate and develop
the TLC plate with the elution solvent in the TLC chamber
until solvent reaches 4/5 up plate.
6. Remove the TLC plate from the chamber and allow solvents to
evaporate at room temperature.
7. Spray the TLC plate with H 2 O–H 2 SO 4 (1:1, v/v) solution and
place it in a plate heater at 180
C for 15 min. Subsequently, let
the TLC plate to reach ambient temperature and scan it. Use an
image processing software to analyze one-dimensional images
and obtain the grey scale intensity values (see Note 15).
3.6 Calculations and
Statistical Analysis
1. The micellarization index is expressed as percentage value
regarding the total initial carotenoid content in the meal and
the total carotenoid content in the micellar fraction.
2. A minimum of three replicates per in vitro digestion process is
required to determine whether the observed effect (if it is so) is
statistically significant.
Carotenoid In Vitro Digestion
383