10 Analytical Protocols in the Measurement …
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Most studies on microalgae focus on the production and application of biomass
and pigments extracted from biomass. Determination of the bioaccessibility and
bioavailability of microalgal pigments has been the focus of very few researches in
the last years. However, some aspects are more exploited than others. From the data
shown in the literature, the most studied pigments from microalgae are carotenoids.
Researches about the behavior of chlorophylls and phycobiliproteins are very rare.
In addition, even for carotenoids, there is no unified protocol specific for microalgal
sources, although, for food in general, a harmonized method called INFOGEST was
developed and tested for the in vitro digestion method (Egger et al. 2016).
Just as the different pigments classes present distinct ways to be produced, they
also behave differently in the gastrointestinal system and, hence, perform differently
against bioavailability. So, it is mandatory to answer the main questions around the
bioavailability of pigments evaluated by different protocols, regardless of the work
applies in vitro models or in vivo (animal and/or human) protocols.
Several methods have been proposed to assess bioavailability, including in vitro,
ex vivo, and in vivo protocols, and the found articles are summarized in Table 10.1. A
discussion remains concerning the quality of the data produced from in vitro and/or
in vivo methods. Some authors consider in vivo approaches more trustworthy since
the reactions and components occur in the human body. However, several studies
have shown a correlation between the results obtained for the same compound using
in vivo and in vitro systems (Brown et al. 2014).
Bioavailability assessment is grounded on three main steps: digestibility and solubility of the component in the gastrointestinal tract; absorption of the component by
the intestinal cells and transport into the circulation; and incorporation from the
circulation to the functional entity or target (Mackie and Rigby 2015).
10.3 In Vitro Bioavailability Methods
All methods to assess the bioavailability of microalgal pigments applied and
described in the articles follow almost the same steps and sequence of experiments as
outlined in Fig. 10.1. Most of the studies about bioavailability of microalgal pigments
used in vitro methods.
Particularly regarding in vitro methods, they range from simple enzyme reactions,
such as hydrolysis of proteins by pepsin under rather non-physiological conditions
in a beaker, to the complex sophistication of the TNO gastrointestinal model and
other similar dynamic models (Mackie and Rigby 2015).
Static digestion models are widely used as the first step to determine bioavailability
of pigments from microalgae. Gille and collaborators (2017) evaluated the in vitro
digestion process for carotenoids’ extraction from Phaeodactylum tricornutum. The
authors performed in vitro digestion using porcine pepsin (40 mg/mL in 0.1 N HCl),
then the pH was adjusted to a value between 2.2 and 2.4 and this step was followed
by shaking at 37 °C in a water bath for 1 h. Subsequently, porcine bile extract
(12 mg/mL in 0.1 M Na 2 CO 3 ) and pancreatin (0.2 mg/ml in 0.1 M Na 2 CO 3 ) were
231
Most studies on microalgae focus on the production and application of biomass
and pigments extracted from biomass. Determination of the bioaccessibility and
bioavailability of microalgal pigments has been the focus of very few researches in
the last years. However, some aspects are more exploited than others. From the data
shown in the literature, the most studied pigments from microalgae are carotenoids.
Researches about the behavior of chlorophylls and phycobiliproteins are very rare.
In addition, even for carotenoids, there is no unified protocol specific for microalgal
sources, although, for food in general, a harmonized method called INFOGEST was
developed and tested for the in vitro digestion method (Egger et al. 2016).
Just as the different pigments classes present distinct ways to be produced, they
also behave differently in the gastrointestinal system and, hence, perform differently
against bioavailability. So, it is mandatory to answer the main questions around the
bioavailability of pigments evaluated by different protocols, regardless of the work
applies in vitro models or in vivo (animal and/or human) protocols.
Several methods have been proposed to assess bioavailability, including in vitro,
ex vivo, and in vivo protocols, and the found articles are summarized in Table 10.1. A
discussion remains concerning the quality of the data produced from in vitro and/or
in vivo methods. Some authors consider in vivo approaches more trustworthy since
the reactions and components occur in the human body. However, several studies
have shown a correlation between the results obtained for the same compound using
in vivo and in vitro systems (Brown et al. 2014).
Bioavailability assessment is grounded on three main steps: digestibility and solubility of the component in the gastrointestinal tract; absorption of the component by
the intestinal cells and transport into the circulation; and incorporation from the
circulation to the functional entity or target (Mackie and Rigby 2015).
10.3 In Vitro Bioavailability Methods
All methods to assess the bioavailability of microalgal pigments applied and
described in the articles follow almost the same steps and sequence of experiments as
outlined in Fig. 10.1. Most of the studies about bioavailability of microalgal pigments
used in vitro methods.
Particularly regarding in vitro methods, they range from simple enzyme reactions,
such as hydrolysis of proteins by pepsin under rather non-physiological conditions
in a beaker, to the complex sophistication of the TNO gastrointestinal model and
other similar dynamic models (Mackie and Rigby 2015).
Static digestion models are widely used as the first step to determine bioavailability
of pigments from microalgae. Gille and collaborators (2017) evaluated the in vitro
digestion process for carotenoids’ extraction from Phaeodactylum tricornutum. The
authors performed in vitro digestion using porcine pepsin (40 mg/mL in 0.1 N HCl),
then the pH was adjusted to a value between 2.2 and 2.4 and this step was followed
by shaking at 37 °C in a water bath for 1 h. Subsequently, porcine bile extract
(12 mg/mL in 0.1 M Na 2 CO 3 ) and pancreatin (0.2 mg/ml in 0.1 M Na 2 CO 3 ) were