7 Analytical Protocols in Carotenoid Analysis
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a rivet-locking function, while the secondary pigments accumulate in cytosolic lipid
droplets, so that the method applied for exhaustive extraction would require different
strategies. Analysis of carotenoids in microalgae encompasses several aims including
microalgae chemotaxonomy—some pigments are characteristic of a strain, species,
class or taxon-assessment of ocean productivity and changes in marine ecosystem,
control of the targeted metabolic or genetic engineering approach for enhancing the
production of a pursued bioactive compound, monitoring of the pigment metabolism
and biosynthetic responses to different cultivation conditions, and high-throughput
screening to unravel unknown or unprecedent carotenoid structures in microalgae
species (Serive et al. 2017). Therefore, the comprehensive description of the analytical procedures applied in analysis of carotenoids in microalgae is a key issue with
multidisciplinary objectives and the present chapter has been outlined according to
this characteristic.
7.2 Analysis
7.2.1 Extraction
An efficient extraction of carotenoids from microalgae is achieved by considering
the different problems that the isolation of the pigment (lipophilic) fraction from
aquatic environments must face. Usually, a protocol based in just one single strategy
for extraction is not successful because of the wide range polarity that carotenoid
pigments display and the different opposition of the cellular environments to the
force that drives extraction. Indeed, carotenoids are prone to oxidation mediated
by temperature, light, and oxygen, as well as those degradative processes that may
occur when the cellular content, grouped in subcellular structures, is mixed and
homogenized in a single environment. Thus, acids, enzymes, and other compounds
originally inert in the cells may promote transformation and/or degradation of the
initial carotenoid content. Therefore, minimization of these processes and situations
is advised to reduce the generation of artifacts and lack of correspondence with the
natural carotenoid profile. To achieve this objective, the following good laboratory
practices are recommended. Extraction time should be short, and the temperature
should be monitored, controlling unnecessary excessive heating. Direct exposure of
samples and extracts to sunlight must be avoided by working in a lab with diminished
light or illuminated with yellow-light bulbs. Effect of cellular acids is neutralized
by adding calcium carbonate to the extracting solvents, which might be supplemented with synthetic antioxidants (butylated hydroxytoluene, pyrogallol, ascorbyl
palmitate) as well, at the 0.1–0.2% (w/v) range.
Another issue to consider in the extraction protocol is the application of a pretreatment step that may considerably facilitate the subsequent extraction activities.
153
a rivet-locking function, while the secondary pigments accumulate in cytosolic lipid
droplets, so that the method applied for exhaustive extraction would require different
strategies. Analysis of carotenoids in microalgae encompasses several aims including
microalgae chemotaxonomy—some pigments are characteristic of a strain, species,
class or taxon-assessment of ocean productivity and changes in marine ecosystem,
control of the targeted metabolic or genetic engineering approach for enhancing the
production of a pursued bioactive compound, monitoring of the pigment metabolism
and biosynthetic responses to different cultivation conditions, and high-throughput
screening to unravel unknown or unprecedent carotenoid structures in microalgae
species (Serive et al. 2017). Therefore, the comprehensive description of the analytical procedures applied in analysis of carotenoids in microalgae is a key issue with
multidisciplinary objectives and the present chapter has been outlined according to
this characteristic.
7.2 Analysis
7.2.1 Extraction
An efficient extraction of carotenoids from microalgae is achieved by considering
the different problems that the isolation of the pigment (lipophilic) fraction from
aquatic environments must face. Usually, a protocol based in just one single strategy
for extraction is not successful because of the wide range polarity that carotenoid
pigments display and the different opposition of the cellular environments to the
force that drives extraction. Indeed, carotenoids are prone to oxidation mediated
by temperature, light, and oxygen, as well as those degradative processes that may
occur when the cellular content, grouped in subcellular structures, is mixed and
homogenized in a single environment. Thus, acids, enzymes, and other compounds
originally inert in the cells may promote transformation and/or degradation of the
initial carotenoid content. Therefore, minimization of these processes and situations
is advised to reduce the generation of artifacts and lack of correspondence with the
natural carotenoid profile. To achieve this objective, the following good laboratory
practices are recommended. Extraction time should be short, and the temperature
should be monitored, controlling unnecessary excessive heating. Direct exposure of
samples and extracts to sunlight must be avoided by working in a lab with diminished
light or illuminated with yellow-light bulbs. Effect of cellular acids is neutralized
by adding calcium carbonate to the extracting solvents, which might be supplemented with synthetic antioxidants (butylated hydroxytoluene, pyrogallol, ascorbyl
palmitate) as well, at the 0.1–0.2% (w/v) range.
Another issue to consider in the extraction protocol is the application of a pretreatment step that may considerably facilitate the subsequent extraction activities.