Biotechnological Production of Microalgal Carotenoids with reference to Astaxanthin and Evaluation of its Biological Activity

Microalgal biotechnology has gained importance due to its potential to produce
bioactive compounds. Green alga Haematococcus pluvialis, being a potent source
for ketocarotenoid astaxanthin, has been an attractive species for commercial
exploitation. The present work focused on production of astaxanthin from
H. pluvialis and evaluation of its biological activity. Modified medium was
developed for autotrophic cultivation of H. pluvialis in open and closed system.
Haematococcus was grown in different prototype bioreactors under optimized
culture condition. The high biomass yield in closed tubular bioreactors suggested
that maintenance of the constant carbon dioxide level in the airspace is essential
for effective gas-liquid mass transfer. Maximum biomass yield of
0.89 g/L with a growth rate of 0.13 d-1 and astaxanthin content of 1.8% (w/w) was
obtained in closed tubular bioreactor. H. pluvialis culture of 60 L prototype
raceway tank, after 9 to 12 days growth period and exposed to sunlight and
salinity stress for 5 days, produced a biomass yield of 0.5 g/L and astaxanthin
content of 1.4 % (w/w). Digital image processing based method was developed for
estimation of carotenoid content in H. pluvialis cells, a good correlation of
R²=0.967 was observed between carotenoid content as estimated by analytical
method.
H. pluvialis mutants were isolated using chemical and physical mutagen
treatment and were characterized for growth, astaxanthin production,
photosynthetic property and carotenoid gene expression. Mutants obtained with 1-
methyl 3-nitro 1-nitrosoguanidine (NTG) have shown significant enhancement in
total carotenoid and astaxanthin content (23-59% w/w) in comparison with parent
culture. The mutant obtained by UV irradiation showed highest lycopene cyclase
activity (458 nmole of β-carotene formed/mg of protein/hr) followed by NTG
mutant (315 nmole of β-carotene formed/mg of protein/hr) when compared to that
of parent strain (105 nmole of β-carotene formed/mg of protein/hr). Expression
analysis of carotenoid biosynthetic genes in the mutants exhibited increase in
transcript levels compared to wild type.
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Astaxanthin esters and free astaxanthin from H. pluvialis were evaluated for
their biological activity. Results indicated that free astaxanthin from H. pluvialis
has 4.4 fold higher free radical scavenging activity (IC50 value of 8.1μg/ml) when
compared to that of astaxanthin esters. Free astaxanthin also showed maximum
reducing power of 59.6U/g equivalents to that of tannic acid (48.5 U/g). The above
data showing better antioxidant activity of free astaxanthin is substantiated by
comparing with the activity of standard astaxanthin. Free astaxanthin exhibited 5
fold higher soybean lipoxygenase inhibitory activity (IC50 ∼3.4 μg/ml) when
compared to total carotenoid fraction. Further, astaxanthin esters effectively
inhibited the gastric proton potassium ATPase enzyme that is involved in the acid
secretion during gastric conditions. Free astaxanthin was potent inhibitor of gastric
H+ K+ ATPase with IC50 -6.2μg/ml than astaxanthin esters (IC50 – 18.2 μg/ml).
Results of in vivo studies revealed that astaxanthin esters at 500μg/kg b.w.,
protected ulcerous condition in rats by ∼67% equivalent to that of known antiulcer
drug- omeprazole which offered ∼72% protection at 20 mg/kg b.w. Attractive skin
colouration in ornamental fish was achieved by feeding astaxanthin rich
H. pluvialis biomass. Poultry birds fed with astaxanthin rich H. pluvialis showed
an increase in yolk colour intensity as indicated by Roche Yolk colour fan (Yolk
colour score-11.00) and improved egg quality as per FAO standards (Haugh unit
score -76 and USDA grade AA). A maximum of 44μg of carotenoid content per
gram of yolk was observed in experimental birds, which is 2-3 fold higher
compared to control (15μg/g of egg yolk). The findings of this study have
substantiated biological activity of astaxanthin such as antioxidant, pigmentation
efficiency and established its antiulcer properties. It has also provided insight on
autotrophic cultivation of Haematococcus pluvialis for production of astaxanthin.