Hydrolytic efficiency and isomerization during de-esterification of natural astaxanthin esters by saponification and enzymolysis

Background: Astaxanthin from natural sources is typically esterified with fatty acids; hence, it must be hydrolyzed to remove esters before identification and quantification by conventional HPLC. Alkaline-catalyzed saponification and enzyme-catalyzed enzymolysis are the most commonly used de-esterification methods. However, information on the efficiency and isomerization during de-esterification of natural astaxanthin esters by these two methods remains scarce. Therefore, we conducted two HPLC-based experiments to determine which method is better for hydrolyzing astaxanthin esters. Results: To assess the effect of enzymolysis (0.67 U/mL cholesterol esterase, at 37°C) and saponification (0.021 M NaOH, at 5°C) conditions on free astaxanthin recovery and destruction or structural transformation of astaxanthin, we varied the total treatment time across a range of 195 min. The results showed that enzymolysis and saponification were complete in 60 min and 90 min, respectively. After complete hydrolysis, the maximum free astaxanthin recovery obtained by enzymolysis was 42.6% more than that obtained by saponification. The identification of by-products, semi-astacene and astacene, during the process of saponification also indicated that a more severe degradation of astaxanthin occurred during saponification. Moreover, the composition of astaxanthin isomers during saponification was similar to that of the isomers during enzymolysis between 30 min and 75 min (all-trans:9-cis:13-cis = 21:3:1, approximately) but dramatically changed after 90 min, whereas the composition in the enzymolysis treatment remained relatively stable throughout. Conclusion: Compared with saponification, enzymolysis with cholesterol esterase was recommended as a more accurate method for de-esterification of natural astaxanthin esters for further qualitative and quantitative HPLC analysis.

Carotenoid genes transcriptional regulation for astaxanthin accumulation in fresh water unicellular alga Haematococcus pluvialis by gibberellin A3 (GA3).

The fresh water unicellular alga Haematococcus pluvialis is a promising natural source of astaxanthin. The present study investigated the transcriptional expression of carotenoid genes for astaxanthin accumulation in H. pluvialis using real-time fluorescence quantitative PCR (qRT-PCR). With treatments of 20 and 40 mg/L of gibberllin A3 (GA3), five genes ipi-1, ipi-2, psy, pds and bkt2 were up-regulated with different expression profiles. GA20 (20 mg/L of GA3) treatment had a greater effect on transcriptional expression of bkt2 than on ipi-1 ipi-2, psy and pds (>4-fold up-regulation). However, GA40 (40 mg/L of GA3) induced more transcriptional expression of ipi-2, psy and bkt2 than both ipi-1 and pds. The expression of lyc, crtR-B and crtO for astaxanthin biosynthesis was not affected by GA3 in H. piuvialis. In the presence of GA3, astaxanthin biosynthesis genes of ipi-1, pds and bkt2 were up-regulated at transcriptional level, psy at post-transcriptional level, whereas ipi-2 was up-regulated at both levels. The study could potentially lead to a scale application of exogenous GA3 in astaxanthin production with H. pluvialis just like GAs perform in increasing crops production and it would provide new insight about the multifunctional roles of carotenogenesis in response to GA3.

Photoinhibition and photosynthetic acclimation of rice (Oryza sativa L. cv Jyothi) plants grown under different light intensities and photoinhibited under field conditions.

Thirty-days old rice (Oryza sativa L. cv. Jyothi) plants grown under the greenhouse (150-200 µmol m-2 s-1) or shade (600-800 µmol m-2s-1) were exposed to 7 days of full sunlight and compared with plants grown under direct sunlight (1200-2200 µmol m-2s-1).Transfer of greenhouse and shade plants to full sunlight for a day resulted in a decline in their photosynthetic efficiency (Fv/Fm) and an increase in non-photochemical quenching (qN). The decline in Fv/Fm was much greater in transferred greenhouse plants (33%) as compared to transferred shade-plants (20%). Sun-plants did not show much variation in the Fv/Fm ratio (4%) from their predawn measurements (control). The sun-grown plants showed a higher pool of xanthophyll pigments (violaxanthin + antheraxanthin + zeaxanthin). Transfer of greenhouse and shade-plants to full sunlight resulted in an increase in lutein, Chl a/b ratio, antheraxanthin (A) and zeaxanthin (Z) content. Increase in A and Z was correlated with the increase in the qN. The increase in the A and Z content was due to increase in the activity of violaxanthin de-epoxidase. Greenhouse and shade plants on exposure to sunlight showed an increase in lipid peroxidation (LPO). Prolonged exposure of greenhouse and shade plants up to 7 days resulted in recovery of the Fv/Fm, an increase in Z and A and a decline in the LPO. The study demonstrated that rice plants grown at lower light intensities initially underwent photoinhibitory damage on exposure to full sunlight, but were able to acclimate to the high irradiance by dissipating the excess light through various mechanisms such as an increase in lutein, high Chl a/b ratio and xanthophyll cycle, suggesting use of energy dissipation as a mechanism of protection against high irradiance, but to different extent and to some extent by different processes. The study was unique, as plants were grown and photoinhibited under natural conditions rather than the artificial light, as was the case in most of the studies so far. Results showed better adaptation of high-light grown plants and suggested role for chl a/b ratio and lutein, in addition to xanthophylls cycle in shade plants. Low-light grown plants could also completely adapt to full level of sunlight within 3 days of the treatment and xanthophylls cycle (measured as V, A and Z) and activity of de-epoxidase seemed to be important in this adaptation.

Morphology and cultural behavior of Botryococcus protuberans with notes on the genus.

The green alga Botryococcus protuberans was isolated from its natural environment and its morphology under different cultural conditions was examined. The alga was characterized by a high starch content and reddish oil drops as the assimilatory products. Photosynthetic pigments, Chl a, Chl b, carotenoids and xanthophylls are present. Modification of environmental conditions in modified Chu-10 medium resulted in optimum growth of the alga. Fatty acid composition revealed palmitic acid being the major component, while lauric acid, myristic acid and stearic acid were found in less quantity.