Size exclusion chromatography of pectic extract

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Extraction of phenolic compounds

Polyphenol extracts were prepared as described by Guyot et al. (2001). About 30 mg of freeze-dried flesh and peel were directly submitted to extraction (“crude” samples) or thioacidolysis. For thioacidolysis, 400 μL of dried methanol acidified by concentrated HCl (0.4 mol/L) and 800 μL of a toluene-α-thiol solution (50 mL/L in dried methanol) were added.
The extraction was performed by incubation of mixture for 30 min at 40°C with agitation on a vortex every 10 min. Samples were cooled in ice in order to stop thioacidolysis reaction. For crude extraction, samples were dissolved in 1200 μL of dried methanol acidified by acetic acid (10 mL/L). The reaction was carried out in an ultrasonic batch during 15 min. All samples (“thioacidolysis” and “crude” extracts) were filtered (PTFE, 0.45 μm) and injected (20 μL) into HPLC-DAD (see 2.5 section).
Cultivars Abbreviations Origins Main uses Astringency perception Abate AB Italy Dessert pears No astringent Arbi Bouficha AF Bouficha, Sousse, Tunisia Dessert pears Perceivable astringency Arbi Chiheb AC Monastir, Tunisia Dessert pears Perceivable astringency Arbi Sidi Bou Ali AS Sidi Bou Ali, Sousse, Tunisia Dessert pears Perceivable astringency Comice CO France Dessert pears No astringent Conference CF England Dessert pears No astringent De Cloche DC Sées, France Perry pears Very astringent Fausset FA Sées, France Perry pears Very astringent Jrani JR Monastir, Tunisia Dessert pears Perceivable astringency Louise Bonne LB France Dessert pears No astringent Meski Arteb MA Sousse, Tunisia Dessert pears Perceivable astringency Radsi RD Sousse, Tunisia Dessert pears Perceivable astringency Passe Crassane CR France Dessert pears No astringent Plant De Blanc PB Sées, France Perry pears Perceivable astringency Rochas RC Portugal Dessert pears No astringent Soukri SK Sousse, Tunisia Dessert pears Perceivable astringencyTourki TR Monastir, Tunisia Dessert pears Perceivable astringency William rouge WR United Kingdom Dessert pears No astringent William vert WV United Kingdom Dessert pears No astringent.

Identification of phenolic compounds by HPLC/ESI-MS2

HPLC/ESI-MS2 analysis was performed on an Acquity Ultra performance LC (UPLC) apparatus from Waters (Milford, MA, USA), equipped with a photodiode array detector (detection at 280, 320, 350 and 520 nm) coupled with a Bruker Daltonics (Bremen, Germany) HCT ultra ion trap mass spectrometer with an electrospray ionization source. Separations were achieved using a Licrospher RP-18 5μm column (Merck, Darmstadt, Germany) protected by a guard column of the same material (Licrospher RP-18 5μm column, Merck Darmstadt, Germany) operated at 30°C. The mobile phase consisted of water/formic acid (99:1, mL/mL) (eluent A) and acetonitrile (eluent B). The flow rate was 1 mL/min. The elution program was as follows: 3−9% B (0−5 min); 9−16% B (5−15 min); 16−50% B (15- 45min); 50−90% B (45−48 min); 90−90% B (48−52 min). Samples (crud extracts) were injected at a level of 10 μL. For polyphenol characterization, a capillary voltage of 2 kV was used in the negative ion mode. Nitrogen was used as drying and nebulizing gas with a flow
rate of 12 L/min. The desolvation temperature was set at 365°C and the nebulization pressure at 0.4 MPa. The ion trap was operated in the Ultrascan mode from m/z 100 to 1000. For anthocyanin characterization, a capillary voltage of 1.8 kV was used in the positive ion mode with the same previous conditions.

Quantification of polyphenols by HPLC-DAD

Phenolic compounds were quantified by Reversed-Phase High-Performance Liquid Chromatography-Diode Array Detection (RP-HPLC-DAD) (Shimadzu, Kyoto, Japan) including two pumps LC-20AD Prominence liquid chromatograph UFLC, a DGU-20A5 Prominence degasser, a SIL-20ACHT Prominence autosampler, a CTO-20AC Prominence column oven, a SPD-M20A Prominence diode array detector, a CBM-20A Prominence communication bus module and controlled by a LC Solution software.
Analyzes of polyphenols were carried out with or without thioacidolysis as described by Guyot et al. (2001) . Thioacidolysis was used to determine the content of procyanidins, their subunit composition and their number average degree of polymerization (DPn). The DPn of procyanidins was calculated as the molar ratio of all the flavan-3-ol units (thioether adducts plus terminal units, minus (-)-epicatechin and (+)-catechin from crude extract) to (-)- epicatechin and (+)-catechin corresponding to terminal units minus (-)-epicatechin and (+)- catechin from crude extract. HPLC DAD analysis of methanolic extracts (crude extracts) which were not submitted to thioacidolysis were performed to assay separately monomeric catechins. Individual compounds were quantified in mg/kg of fresh weight (FW) by comparison with external standards at 280 nm for (+)-catechin, (-)-epicatechin and (-)- epicatechin benzyl thioether (quantified as (-)-epicatechin); at 320 nm for 5’-caffeoylquinic acid; at 350 nm for flavonols (quantified as quercetin and isorhamnetin), and at 520 nm for cyanidin glycosides (quantified as cyanidin-3-O-hexosidee) and peonidin (quantified as peonidin-3-O-hexoside).

Statistical analyses

Results are presented as mean values of triplicates for each cultivar and phenolic compound. The analytical reproducibility of the results was determined as pooled standard deviations (Pooled SD). Pooled SDs were calculated for each series of replicates using the sum of individual variances weighted by individual degrees of freedom (Box et al., 1978). To respect homogeneity of value ranges, the pooled standard deviations were separately calculated for pears of high and low polyphenol concentrations. ANOVA and Principal Component Analysis (PCA) were performed on chemical data using the Excel stat package of Microsoft Excel.

Pear fruit phenolic profile from HPLC/ESI-MS2 analysis

Example of HPLC-MS² chromatogram at 280 nm for peel and flesh “crude” extracts is presented in figures 30A and 30B respectively. The chromatographic profile showed 27 peaks that included phenolic acids, flavan-3-ol monomers, flavan-3-ol polymers, flavonols, anthocyanins and others phenolic compounds such as hydroquinones (Table 10). Phenolic compounds were identified by comparison with authentic standards and by interpretation of the observed MS² spectra with those found in the literature.

Phenolic acids and derivatives

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Eight phenolic acid derivatives could be identified. Compounds 3 and 6 were identified as 3’-caffeoylquinic acid and 5’-caffeoylquinic acid respectively and were confirmed by authentic standards. Compound 9 displayed a major fragment ion at m/z 353 and fragments ion at m/z 191 and 173 typical of 4’-caffeoylquinic acid (Bujor et al., 2016; Chanforan et al., 2012). Compound 21 displayed a parent ion at m/z 515 and fragment ions at m/z 353 and 191 corresponding to caffeoylquinic acid and quinic acid respectively, and was assigned as di-O-caffeoylquinic acid as reported in Polish pear cultivars by Kolniak-Ostek (2016c) and Oszmiànski and Wojdyło (2014). Compound 11 displaying a parent ion m/z 377 and a typical fragmentation at m/z 191 corresponded to p-coumaroylquinic acid (Kolniak- Ostek, 2016c; Ncube et al., 2014). Compound 8 was identified as a derivative of pcoumaroylquinic acid (coumaroyl hexoside malic acid, m/z 441, Rt=14.6 min) as described by Abu-Reidah et al. (2015). Compound 14 was characterized as a derivative of syringic acid (syringic acid hexoside) based on its m/z 359, which fragmented at m/z 197, due to the loss of an hexose, and 153 due to the loss of CO2 (Barros et al., 2012). Compound 16 had a parent ion at m/z 163 typical for coumaric acid (Kolniak-Ostek, 2016c).

Flavan-3-ol monomers and polymers

Six flavan-3-ols were detected in pear as: catechin, epicatechin and 4 procyanidin dimers. Compounds with retention times at 12.5 min (compound 5) and 16.1 min (compound 10) were identified as (+)-catechin and (-)-epicatechin. Their retention times, m/z, UV spectrum, were consistent with the values achieved for their standards. Compounds 4, 7, 13 and 17 were characterized as dimer procyanidins, due to their m/z 577 and fragmentation ions at m/z 289 corresponding to the monomer of catechin as reported in different pear extracts (Kalisz et al., 2015; Kolniak-Ostek, 2016c).

Phenolic contents in fruit flesh vs peel

Some phenolic compounds were identified by HPLC/ESI-MS2 but not quantified by HPLC-DAD, such as arbutin, 3’-caffeoylquinic acid, 4’-caffeoylquinic acid, di-O105 caffeolyquinic acid and coumaric acid, as they were present in low concentrations. Only the concentrations of the major phenolic compounds are shown in Tables 11 and 12.
The sum of phenolic compounds ranged between 115 mg/kg fresh weight (FW) in ‘Conference’ flesh to 40400 mg/kg FW in ‘Arbi Chiheb’ peel. Thus, the polyphenol content in peel was higher than in the flesh as reported in other pear cultivars by Galvis Sánchez et al. (2003) and in apple by Guyot et al. (2002a). The phenolic profile in flesh was simple with mainly two classes, flavan-3-ols and hydroxycinnamic acids, as reported by Ferreira et al. (2002), Le Bourvellec et al. (2013) and Renard (2005) and probably some traces of arbutin.

Table of contents :

Chapitre1 : Composés phénoliques des poires (Diversité & purification)
Partie 1 : Diversité des composés Phénoliques
1. Introduction
2. Materials and methods
2.1. Chemicals compounds
2.2. Plant Material
2.3. Extraction of phenolic compounds
2.4. Identification of phenolic compounds by HPLC/ESI-MS2
2.5. Quantification of polyphenols by HPLC-DAD
3.1. Pear fruit phenolic profile from HPLC/ESI-MS2 analysis
3.1.1. Phenolic acids and derivatives
3.1.2. Flavan-3-ol monomers and polymers
3.1.3. Flavonols
3.1.4. Anthocyanins
3.1.5. Others phenolic compounds
3.2. Phenolic contents in fruit flesh vs peel
3.2.1. Flavan-3-ol monomers and polymers
3.2.3. Flavonols
3.2.3. Anthocyanins
3.3. Source of variability of pear phenolic composition
3.4. Impact of pear procyanidins on fruit and perry astringency
4. Conclusions
5. Acknowledgements
Partie 2 : Purification des procyanidines
1. Plant material
2. Procyanidin extraction and purification:
3. Polyphenol characterization
4. Phenolic composition of perry pear flesh
5. Procyanidin characterization and evolution at the overripe stage
Chapitre 2 : Préparation & caractérisation des parois de poires
1. Introduction
2. Materials and methods
2.1. Standards and Chemicals
2.3. Firmness
2.4. Cell wall isolation and extraction
2.5. Mid infrared spectroscopy (MIR)
2.6. Scanning Electron Microscopy (SEM)
2.7. Analytical
2.7.1. Neutral sugar composition
2.7.2. Uronic acids content
2.7.5. Procyanidins content
2.7.6. Acetic acid content
3.1. Texture evolution during overripening
3.2. General characteristics of cell walls
3.2.1. Mid infrared spectroscopy
3.2.2. Scanning electron microscopy (SEM)
3.3. Cell walls: Yields and compositions
3.3.1. Cell wall yields
3.3.2. Cell wall compositions
3.3.2.1. Elimination of intracellular polyphenols
3.3.2.2. Polysaccharides composition
3.4. Sequential extractions: yields and characterizations
3.4.1. Composition of pectic extracts
3.4.2. Size exclusion chromatography of pectic extract
3.4.3. Composition of hemicellulosic extracts
3.4.4. Composition of residue
4. Conclusion
5. Acknowledgements
Chapitre 3 : Interactions parois-procyanidines
1. Introduction
2. Materials and methods
2.2. Plant material
2.3. Procyanidin purification and characterization
2.4. Cell wall extraction and characterization
2.5. Procyanidin-cell wall interactions
2.5.1. Binding isotherm methodology
2.5.3. Isothermal titration microcalorimetry (ITC)
2.6. Statistical analysis
3. Results
3.1. Composition of the fractions
3.1.2. Cell walls
3.2. Quantification of binding level
3.2.2. Infrared spectroscopy (MIR)
3.3.3. Isothermal titration calorimetry (ITC)
5. Conclusion
6. Acknowledgments
Chapitre 4 : Validation des résultats sur un modèle réel (jus de poire)
1. Introduction
2. Materials and methods
2.1. Chemicals and standards
2.2. Plant material
2.3. Preparation of samples
2.4. Analysis of phenolic compounds
2.5. Cell walls preparation and characterization
2.6. Statistics
3. Results and discussion
3.1. Phenolic composition of perry pears, juice and pomace
3.2. Composition of alcohol insoluble solids (AIS)
3.3. Impact of cell walls on procyanidins transfer on juice
4. Conclusion
5. Acknowledgements
Chapitre 5 : Localisation des procyanidines in situ dans les poires à poiré à deux stades de maturité
2. Materials and methods
2.1. Solvents and reagents
2.2. Plant Materials
2.3. DMACA preparation
2.4. Transmission electron microscopy (TEM)
3. Results and discussions
3.1. Procyanidins localization by light microscopy coupled to DMACA staining
3.2. Procyanidins localization by transmission electron microscopy
4. Conclusion
5. Acknowledgments
Conclusions & perspectives
Références bibliographiques

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