Isolation of fucoxanthin chlorophyll protein complexes of the centric diatom Thalassiosira pseudonana associated with the xanthophyll cycle enzyme diadinoxanthin de-epoxidase.

In the present study, low concentrations of the very mild detergent n-dodecyl-α-d-maltoside in conjunction with sucrose gradient ultracentrifugation were used to prepare fucoxanthin chlorophyll protein (FCP) complexes of the centric diatom Thalassiosira pseudonana. Two main FCP fractions were observed in the sucrose gradients, one in the upper part and one at high sucrose concentrations in the lower part of the gradient. The first fraction was dominated by the 18 kDa FCP protein band in SDS-gels. Since this fraction also contained other protein bands, it was designated as fraction enriched in FCP-A complex. The second fraction contained mainly the 21 kDa FCP band, which is typical for the FCP-B complex. Determination of the lipid composition showed that both FCP fractions contained monogalactosyl diacylglycerol as the main lipid followed by the second galactolipid of the thylakoid membrane, namely digalactosyl diacylglycerol. The negatively charged lipids sulfoquinovosyl diacylglycerol and phosphatidyl glycerol were also present in both fractions in pronounced concentrations. With respect to the pigment composition, the fraction enriched in FCP-A contained a higher amount of the xanthophyll cycle pigments diadinoxanthin (DD) and diatoxanthin (Dt), whereas the FCP-B fraction was characterized by a lower ratio of xanthophyll cycle pigments to the light-harvesting pigment fucoxanthin. Protein analysis by mass spectrometry revealed that in both FCP fractions the xanthophyll cycle enzyme diadinoxanthin de-epoxidase (DDE) was present. In addition, the analysis showed an enrichment of DDE in the fraction enriched in FCP-A but only a very low amount of DDE in the FCP-B fraction. In-vitro de-epoxidation assays, employing the isolated FCP complexes, were characterized by an inefficient conversion of DD to Dt. However, in line with the heterogeneous DDE distribution, the fraction enriched in FCP-A showed a more pronounced DD de-epoxidation compared with the FCP-B.

Discrimination of Green Coffee (Coffea arabica and Coffea canephora) of Different Geographical Origin Based on Antioxidant Activity, High-Throughput Metabolomics, and DNA RFLP Fingerprinting.

The genus Coffea is known for the two species C. arabica (CA) and C. canephora (CC), which are used to prepare the beverage coffee. Proper identification of green beans of coffee varieties is based on phenotypic and phytochemical/molecular characteristics. In this work, a combination of chemical (UV/Vis, HPLC-DAD-MS/MS, GC-MS, and GC-FID) and molecular (PCR-RFLP) fingerprinting was used to discriminate commercial green coffee accessions from different geographical origin. The highest content of polyphenols and flavonoids was always found in CC accessions, whereas CA showed lower values. ABTS and FRAP assays showed a significant correlation between phenolic content and antioxidant activity in most CC accessions. We identified 32 different compounds, including 28 flavonoids and four N-containing compounds. The highest contents of caffeine and melatonin were detected in CC accessions, whereas the highest levels of quercetin and kaempferol derivatives were found in CA accessions. Fatty acids of CC accessions were characterized by low levels of linoleic and cis octadecenoic acid and high amounts of elaidic acid and myristic acid. Discrimination of species according to their geographical origin was achieved using high-throughput data analysis, combining all measured parameters. Lastly, PCR-RFLP analysis was instrumental for the identification of recognition markers for the majority of accessions. Using the restriction enzyme AluI on the trnL-trnF region, we clearly discriminated C. canephora from C. arabica, whereas the cleavage performed by the restriction enzymes MseI and XholI on the 5S-rRNA-NTS region produced specific discrimination patterns useful for the correct identification of the different coffee accessions. This work extends our previous studies and provides new information on the complete flavonoid profile, combining high-throughput data with DNA fingerprinting to assess the geographical discrimination of green coffee.