it has been recently discussed [1]. However, these protocols have
been applied to single carotenoid food sources (green vegetables
and oils, carrots, tomatoes, peppers, oranges, grains, dairy products, food emulsions, etc.) with the objective of gaining information of the factor(s) playing a role in the micellarization of
carotenoids. This reductionist approach has been necessary to
clearly correlate the observed effect(s) in the efficiency of the
micellarization with the examined factor(s), or whether the latter
has no influence in the former process. With this strategy a substantial amount of information has been obtained [9] to figure out
several weighting factors in the micellarization of carotenoids,
including food processing, technological treatments and cooking
procedures, application of novel techniques, deposition in the food
microstructures, texture, fiber networks and particle size, and
amount and kind of lipids (preferably polar lipids). The next move
forward could include a holistic assessment of the micellarization of
carotenoids in the context of a standard meal or dietary pattern,
according to the FDA guidelines for bioavailability and bioequivalence studies [10] and to the harmonized INFOGEST in vitro
digestion protocol [2]. Hence, this protocol describes the measurement of the food effect in the micellarization of carotenoids considering normo-, hypo-, and hypercaloric meals to tabulate
predictive data of the potential bioaccessibility of carotenoids
from dietary food sources, a protocol that could be extended to
other lipophilic bioactives and vitamins.
2 Materials
2.1 Standard Meals
1. The standard meals are designed following the guidelines of the
report for Human Energy Requirements, which are based on
sex BMI and PAL [11] (see Note 1). For a single meal with a
caloric intake of 650–750 kcal (see Note 2), the macronutrient
ratio is established according to the following reference intake
ranges: >50% total carbohydrates, 15–30% total fat, and
10–20% total protein (percentage of total energy intake per
meal). In addition, the goal of 400 g of fruits and vegetables
a day is followed [12]. The design of high or low caloric meals
is made applying the same reference intake ranges for macronutrients, and the goal of fruits and vegetables daily intake is
followed but increasing or decreasing ca. 30% the total caloric
intake in the meal, respectively. Accordingly, the design of high
or low-fat meals is made increasing or decreasing the mean
reference intake of total fat a 60%, keeping the reference intake
of protein in both cases, and decreasing or increasing the
reference intake of total carbohydrates, respectively [10]. The
food composition tables published by Mataix [13] are taken as
reference to calculate the caloric content and macronutrient
Carotenoid In Vitro Digestion
377
been applied to single carotenoid food sources (green vegetables
and oils, carrots, tomatoes, peppers, oranges, grains, dairy products, food emulsions, etc.) with the objective of gaining information of the factor(s) playing a role in the micellarization of
carotenoids. This reductionist approach has been necessary to
clearly correlate the observed effect(s) in the efficiency of the
micellarization with the examined factor(s), or whether the latter
has no influence in the former process. With this strategy a substantial amount of information has been obtained [9] to figure out
several weighting factors in the micellarization of carotenoids,
including food processing, technological treatments and cooking
procedures, application of novel techniques, deposition in the food
microstructures, texture, fiber networks and particle size, and
amount and kind of lipids (preferably polar lipids). The next move
forward could include a holistic assessment of the micellarization of
carotenoids in the context of a standard meal or dietary pattern,
according to the FDA guidelines for bioavailability and bioequivalence studies [10] and to the harmonized INFOGEST in vitro
digestion protocol [2]. Hence, this protocol describes the measurement of the food effect in the micellarization of carotenoids considering normo-, hypo-, and hypercaloric meals to tabulate
predictive data of the potential bioaccessibility of carotenoids
from dietary food sources, a protocol that could be extended to
other lipophilic bioactives and vitamins.
2 Materials
2.1 Standard Meals
1. The standard meals are designed following the guidelines of the
report for Human Energy Requirements, which are based on
sex BMI and PAL [11] (see Note 1). For a single meal with a
caloric intake of 650–750 kcal (see Note 2), the macronutrient
ratio is established according to the following reference intake
ranges: >50% total carbohydrates, 15–30% total fat, and
10–20% total protein (percentage of total energy intake per
meal). In addition, the goal of 400 g of fruits and vegetables
a day is followed [12]. The design of high or low caloric meals
is made applying the same reference intake ranges for macronutrients, and the goal of fruits and vegetables daily intake is
followed but increasing or decreasing ca. 30% the total caloric
intake in the meal, respectively. Accordingly, the design of high
or low-fat meals is made increasing or decreasing the mean
reference intake of total fat a 60%, keeping the reference intake
of protein in both cases, and decreasing or increasing the
reference intake of total carbohydrates, respectively [10]. The
food composition tables published by Mataix [13] are taken as
reference to calculate the caloric content and macronutrient
Carotenoid In Vitro Digestion
377