Synthesis, composition and stability of anthocyanins in different berry species

V nalogi smo podrobno proučili primarne in sekundarne metabolite v plodovih različnih vrst jagodičja (Ribes rubrum L., Rubus idaeus L., Vaccinium corymbosum L. in V. myrtillus L.). Zanimalo nas je, kako raven izražanja regulatornih in strukturnih genov v povezavi z aktivnostjo encimov fenilpropanoidne/flavonoidne poti ter fenolna sestava vplivajo na različno obarvanost plodov iste vrste jagodičja. Raziskave so vključevale analizo metabolitov z metodama HPLC in HPLC-MS, izražanje genov z metodo RT-qPCR ter analizo encimske aktivnosti s pomočjo radiografskega skeniranja TLC. Na primeru belo in modro obarvanih plodov borovnice (Vaccinium myrtillus L.) in različno obarvanih sort rdečega ribeza (Ribes rubrum L.) smo ugotovili, da je vsebnost fenolnih snovi, predvsem antocianinov značilno vplivala na izraženost barve plodov, medtem ko se njihova sestava med različno obarvanimi plodovi iste vrste ni bistveno razlikovala. Precej manjša akumulacija antocianinov pri belih v primerjavi z modro obarvanimi plodovi borovnice je bila posledica nizkega izražanja genov flavonoid 3-O-glukoziltransferaze (FGT) in antocianidin sintaze (ANS) ter znižanega izražanja in aktivnosti encima flavanon 3-hidroksilaze (FHT). Manjša akumulacija skupnih fenolnih snovi v beli borovnici bi lahko bila posledica do sedaj manj poznanega dodatnega zgodnjega zmanjšanja aktivnosti encimov sintezne poti, med fenilalanin amonijak liazo (PAL) in halkon sintazo (CHS), kar bi lahko pojasnilo tudi relativno večjo vsebnost derivatov hidroksicimetnih kislin. Podobno je imela roza obarvana sorta rdečega ribeza \u27Pink Champagne\u27, v primerjavi z rdeče obarvano sorto \u27Jonkheer van Tets\u27, manjšo aktivnost encimov halkon sintaze/izomeraze (CHS/CHI), FHT in dihidroflavonol 4-reduktaze (DFR) ter znižano izražanje gena ANS, medtem ko encima FHT in DFR v sorti \u27Zitavia\u27 (beli plodovi) nista bila aktivna. Ugotovili smo, da so se vsebnosti tako primarnih kot sekundarnih metabolitov med zorenjem plodov rdečega ribeza spreminjale, kar je bilo v skladu z razlikami v aktivnosti glavnih encimov flavonoidne poti. Prav tako smo ugotovili, da smo s toplotno obdelavo vplivali na vsebnost fenolnih snovi v kaši iz ameriške borovnice (Vaccinium corymbosum L.). Višje temperature so povzročile zmanjšanje zlasti vsebnosti skupnih antocianinov. S tehnološkim ukrepom prikrajšanja enoletnih poganjkov pri dvakrat rodni malini (Rubus idaeus L.) smo vplivali na zakasnitev zorenja plodov, vendar enotnega vpliva na vsebnost antocianinov in drugih fenolnih spojin nismo potrdili.The thesis deals with the investigation of primary and secondary metabolites in fruits of various berry species (Ribes rubrum L., Rubus idaeus L., Vaccinium corymbosum L. and V. myrtillus L.). We aimed to link the expressions of regulatory and structural genes, related phenylpropanoid/flavonoid enzyme activities and phenolic composition in differently colored berry fruits of the same species. Metabolites were analyzed with HPLC and HPLC-MS, gene expression with RT-qPCR and enzyme activities with radiographic TLC scan. The results indicate that fruit color of white vs. blue colored bilberry (Vaccinium myrtillus L.) and differently colored cultivars of red currant (Ribes rubrum L.) is predominantly affected by anthocyanin content in fruit skin. Phenolic profile remained similar regardless of fruit coloration. Suppressed anthocyanin biosynthesis in white-colored bilberry compared to more common blue hues of bilberry fruit, were primarily correlated with reduced gene expression of flavonoid 3-O-glycosyltransferase (FGT) and anthocyanidin synthase (ANS) and secondly, with reduced gene expression/enzyme activity of flavanone 3-hydroxylase (FHT). Lower total phenolic content of white bilberry fruit can be associated with an additional bottleneck within the phenylpropanoid pathway at an unknown step between phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS). This could explain significant increase in relative levels of hydroxycinnamic acid derivatives in white bilberries. Similarly, pink-colored red currant cultivar \u27Pink Champagne\u27 was characterized by lower chalcone synthase/isomerase (CHS/CHI), FHT and dihydroflavonol 4-reductase (DFR) activity and ANS expression in comparison with \u27Jonkheer van Tets\u27 (standard red berries). Moreover, no FHT and DFR activities were detected in red currant \u27Zitavia\u27 cultivar, which bears white fruits. Primary and secondary metabolite contents changed during red currant fruit ripening, which coincided with different activities of predominant flavonoid enzymes. Additionally, it was established that thermal treatment of highbush blueberry (Vaccinium corymbosum L.) purees affected the stability of phenolics. Greatest effect of higher temperature treatment was detected in total anthocyanin levels, which were significantly decreased. Cultivation technique − tipping the young first-year canes of primocane raspberry (Rubus idaeus L.) significantly delayed fruit ripeninghowever, the results did not show any uniform effects on the content of anthocyanins and other phenolic groups