Polyphenols, including flavonoids and stilbenes, are an essential part of human diet and constitute
one of the most abundant and ubiquitous group of plant secondary metabolites. The level of these
compounds is inducible by stress or fungal attack, so attempts are being made to identify likely
biotic and abiotic elicitors and to better understand the underlying mechanism.
Resveratrol (3,5,4’-trihydroxystilbene), which belongs to the stilbene family, is a naturally
occurring polyphenol, found in several fruits, vegetables and beverages including red wine. It is one
of the most important plant polyphenols with proved benefic activity on animal health. In the last
two decades, the potential protective effects of resveratrol against cardiovascular and
neurodegenerative diseases, as well as the chemopreventive properties against cancer, have been
largely investigated. The most important source of polyphenols and in particular resveratrol for
human diet is grape (Vitis vinifera).
Since stilbenes and flavonoids play a very important role in plant defence responses and
enviromental interactions, and their effects on human health seem promising, the aim of the
research of this Thesis was to study at different levels the activation and the regulation of their
biosynthetic pathways after chitosan treatment. Moreover, the polyphenol production in grape cells
and the optimisation of cultural conditions bioreactor scale-up, were also investigated.
Cell suspensions were obtained from cv. Barbera (Vitis vinifera L.) petioles and were treated with a
biotic elicitor, chitosan (50 μg/mL, dissolved in acetic acid) to promote phenylpropanoid
metabolism. Chitosan is a D-glucosamine polymer from fungi cell wall and therefore mimes fungal
pathogen attack.
Liquid cultures have been monitored for 15 days, measuring cell number, cell viability, pH and
grams of fresh weight. The endogenous and released amounts of 7 stilbenes (trans and cis isomers
of resveratrol, piceid and resveratroloside, and piceatannol), gallic acid, 6 hydroxycinnamic acids
(trans-cinnamic, p-coumaric, caffeic, ferulic, sinapic and chlorogenic acids), 5 catechines (catechin,
epicatechin, epigallocatechin-gallate (EGCG), epigallocatechin and epicatechin-gallate) and other 5
flavonoids (chalcon, naringenin, kaempferol, quercetin and rutin) in cells and cultural medium,
were measured by HPLC-DAD analysis and total anthocyanins were quantified by
spectrophotometric analysis.
Chitosan was effective in stimulating trans-resveratrol endogenous accumulation with a sharp peak
at day 4 (exceeding acetic acid and water controls by 36% and 63%, respectively), while it did not influence the production of the cis-isomer. Compared to both water and acetic acid controls,
chitosan decreased the release of both trans- and cis-resveratrol respect to controls.
No effect was shown on the accumulation of single resveratrol mono-glucoside isomers, but
considering their total amount, normalized for the relative water control, it was possible to evidence
an increase in both accumulation and release of those compounds, in chitosan-treated cells,
throughout the culture period and particularly during the second week.
Many of the analysed flavonoids and hydroxycinnamic acids were not present or detectable in trace
amounts. Catechin, epicatechin and epigallocatechin-gallate (EGCG) were detectable both inside
the cells and in the culture media, but chitosan did not affect their amounts. On the contrary, total
anthocyanins have been stimulated by chitosan and their level, from day 4 to 14, was about 2-fold
higher than in both controls, confirming macroscopic observations that treated suspensions showed
an intense brown-red color, from day 3 onwards.
These elicitation results suggest that chitosan selectively up-regulates specific biosynthetic
pathways, without modifying the general accumulation pattern of other flavonoids.
Proteins have been extracted from cells at day 4 of culture (corresponding to the production peak of
trans-resveratrol) and separated by bidimensional electrophoresis. The 73 proteins that showed a
consistently changed amount between untreated, chitosan and acetic acid (chitosan solvent) treated
cells, have been identified by mass spectrometry. Chitosan induced an increase in stilbene synthase
(STS, the resveratrol biosynthetic enzyme), chalcone-flavanone isomerase (CHI, that switches the
pathway from chalcones to flavones and anthocyanins), pathogenesis-related proteins 10 (PRs10, a
large family of defence proteins), and a decrease in many proteins belonging to primary
metabolisms. A train of six distinct spots of STS encoded by the same gene and increased by
chitosan, was detected on the 2-D gels, and related to the different phosphorylation degree of STS
spots.
Northern blot analyses have been performed on RNA extracted from cells treated with chitosan and
relative controls, using probes for STS, PAL (phenylalanine ammonia lyase, the first enzyme of the
biosynthetic pathway), CHS (chalcone synthase, that shares with STS the same precursors), CHI
and PR-10. The up-regulation of PAL, CHS and CHI transcript expression levels correlated with the
accumulation of anthocyanins. The strong increase of different molecular weight PR-10 mRNAs,
correlated with the 11 PR-10 protein spots identified in proteomic analyses.
The sudden decrease in trans-resveratrol endogenous accumulation after day 4 of culture, could be
simply explained by the diminished resveratrol biosynthetic activity due to the lower amount of
biosynthetic enzymes. This might be indirectly demonstrated by northern blot expression analyses,
that showed lower levels of phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) mRNAs starting from day 4. Other possible explanations could be a resveratrol oxidation process
and/or the formation of other different mono-, di-glucosides and resveratrol oligomers such as
viniferins.
Immunolocalisation experiments performed on grape protoplasts and the subsequent analyses by
confocal microscope, showed that STS, and therefore the resveratrol synthetic site, is mostly
associated to intracellular membranes close to the cytosolic side of plasma membrane and in a
smaller amount is localized in the cytosol. STS seemed not to be present inside vacuole and
nucleus. There were no differences in the STS intracellular localisation between the different
treatments. Since it was shown that stilbenes are largely released in the culture medium and that
STS is a soluble protein, a possible interaction of STS with a plasma membrane transporter
responsible for the extrusion of stilbenes in the culture medium, might be hypothesized. Proteomic
analyses performed on subcellular fractions identified in the microsomial fraction 5 proteins taking
part in channel complexes or associated with channels, that significantly changed their amount after
chitosan treatment. In soluble and membrane fractions respectively 3 and 4 STS and 6 and 3 PR-10
have been identified. Proteomic results obtained from subcellular fractions substantially confirmed
previous result obtained from total cell protein extracts and added more information about protein
localisation and co-localisation.
The interesting results obtained on Barbera cell cultures with the aim to increase polyphenol
(especially stilbenes) production, have encouraged scale up tests in 1 litre bioreactors. The first trial
fermentation was performed in parallel with a normal time-course in 20 mL flasks, showing that the
scale-up (bigger volume and different conditions) process influenced in a very relevant way
stilbenes production.
In order to optimise culture parameters such as medium sucrose amount, fermentation length and
inoculum cell concentration, few other fermentations were performed. Chitosan treatments were
also performed. The modification of each parameter brought relevant variations in stilbenes and
catechins levels, so that the production of a certain compound (or class of compounds) could be
hypothetically promoted by modulating one or more culture parameters. For example the catechin
yield could be improved by increasing sucrose content and the time of fermentation.
The best results in stilbene yield were obtained in a 800 mL fermentation inoculated with 10.8
grams of cells and supplemented with chitosan. The culture was fed with MS medium added with
30 g/L sucrose, 25 μg/mL rifampicin and 50 μg/mL of chitosan, and was maintained at 24°C, stirred
by marine impeller at 100 rpm and supplied of air at 0.16 L/min rate. Resveratroloside was the
stilbene present in the larger amount, 3-5 times more than resveratrol. Because resveratrol
glucosides are similarly active and more stable than free resveratrol, their production using a bioreactor could be a great advantage in an hypothetical industrial process. In my bioreactor tests,
stilbenes were mainly released in the culture medium (60-80% of the total) and this fact could be
another advantage for industrial applications, because it allows recovering the products directly
from the culture medium without stopping the fermentation and/or killing the cells. In my best
cultural conditions, it was possible to obtain 3.95 mg/L of stilbenes at day 4 (maximum resveratrol
accumulation) and 5.13 mg/L at day 14 (maximum resveratroloside production).
In conclusion, chitosan effect in inducing Vitis vinifera defense mechanisms can be related to its
ability to increase the intracellular content of a large spectrum of antioxidants, and in particular of
resveratrol, its derivates and anthocyanins. Its effect can be observed at transcriptional, proteomic
(variation of soluble and membrane protein amounts) and metabolic (polyphenols production) level.
The chitosan ability to elicit specific plant matabolisms can be useful to produce large quantities of
antioxidant compounds from cell culture in bioreactor.
| Identifer | oai:union.ndltd.org:unibo.it/oai:amsdottorato.cib.unibo.it:678 |
| Date | 03 April 2008 |
| Creators | Ferri, Maura <1979> |
| Contributors | Bagni, Nello |
| Publisher | Alma Mater Studiorum - Università di Bologna |
| Source Sets | Università di Bologna |
| Language | Italian |
| Detected Language | English |
| Type | Doctoral Thesis, PeerReviewed |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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