Employment of state-of-the-art technologies towards to metabolomic approaches on sweet cherry postharvest performance
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1 Employment of state-of-the-art technologies towards to metabolomic approaches on sweet cherry postharvest performance V. Goulas 1,2, P.M. Kourdoulas 1, I. Minas 3, A. Lazaridou 3, A.N Molassiotis 3,I. Gerothanasis 2, G.A. Manganaris 1 1 Department of Agricultural Sciences, Biotechnology & Food Science, Cyprus University of Technology, 3603 Lemesos, Cyprus 2 University of Ioannina, Department of Chemistry, NMR Center, Ioannina, Greece 3 School of Agriculture, Aristotle University of Thessaloniki, Thessaloniki, Greece
2 Source: Montanari
3 Background information on sweet cherry fruit physiology Non climacteric fruit with moderate respiration rate Highly perishable commodity with reduced potential storage life Parameters affecting fruit quality: Loss of acidity, Softening, Fruit cracking, Extended weight loss, Stem desiccation Physical/physiological disorders: pitting, bruising, cracking Pathological disorders: brown rot, gray mold, blue mold, Rhizopus rot Numerous cultivars with different qualitative/phytochemical profile and ripening properties and storage potential The extreme perishability of cherries creates challenges for everyone in the supply chain, from grower to retailer
4 Sweet cherry metabolites Sugars Taste: sweetness Organic acids Phenolic compounds Amino acids Taste: acidity Color: anthocyanins Health-promoting properties Nutritional aspects
5 Research on cherry fruit ripening physiology 24 documents [7 Conference Proceedings]. Edible coatings (Aloe vera based, alginate coatings, Semperfresh TM ) CA/MA/Modified atmosphere packaging Application of active molecules (salicylic acid, acetylsalicylic acid, oxalic acid) Heat treatment Removal of field heat with forced-air cooling and hydrocooling Hydro-cooling with CaCl 2 (OptiCAL TM ) for 5 min
6 Research on cherry phytochemical status 73 documents. Phytochemical profile has been mainly monitored at harvest No available information during postharvest ripening at different time points
7 Phytochemicals as an extra criterion that defines fruit quality Phytochemical affects colour and taste of fruits AR Vicente, GA Manganaris, GO Sozzi, CH Crisosto
8 Cherry phenolic composition Phenolic acids Anthocyanins Chlorogenic Acid Derivatives (mg 100 g -1 ) Hydroxycinnamic derivative Neochlorogenic acid p-coumaroylquinic acid Chlorogenic acid Hydroxybenzoic acid Caffeic acid Ferulic acid Individual Anthocyanin (mg 100 g -1 ) Cyanidin 3-glucoside Cyanidin 3-rutinoside Pelargonidin 3-rutinoside Myricetin 3-rutinoside Kaemfperol 3-rutinoside Quercetin 3-rutinoside a Sweet Sweet Sweet Sweet Sweet Heart Late Heart Late Heart. Total phenolic content: (1) Major classes represented by anthocyanins and hydroxycinnamic acids, (2) Neoclorogenic acid, p-coumarolquinic acid, chlorogenic acid are regarded as the dominant polyphenols in sweet cherry fruit S Anthocyanins: (1) Varietal differences in content ( mg g -1 FW among 11 cultivars, Diaz-Mula et al., 2009), (2) cyanidin 3-rutinoside and cyanidin 3-glucoside are the most represented/predominant anthocyanins in cherry cultivars (Gao & Mazza, 1995). Martínez-Esplá et al J. Agric. Food Chem. 62:
9 Analytical approach The estimated phytochemical composition of fruits is strongly affected by sample preparation and extraction methods and instrumental analysis.
10 Aim of the study Define sweet cherry fruit postharvest performance with a complete set of indicators related to: qualitative attributes mechanical properties antioxidant potency phytochemical profile - spectrophotometry - HPLC analysis -Nuclear magnetic resonance (NMR) study Establishment/optimization of a protocol adapted to sweet cherry fruits
11 Fruit material and experimental design Fruit were harvested at commercial maturity stage and were randomly divided into 6 treatment units per cultivar Each treatment unit included 60 sweet cherries and were analyzed at harvest (Day 0) and after maintenance at 20 o C for 1, 2, 4, 6 and 8 days, respectively
12 Qualitative [Objective] analyses Weight loss (%) Respiration rate Soluble solids content (%) Titratable acidity (% malic acid) Color (L*,a*, b*) Stem removal force [A tensile grip was used to remove the stem] Tissue firmness (15% deformation force) Skin penetration force
13 Qualitative [Subjective] analyses Stem browning index 0 Green, fresh appearance, less than 30% brown 1 Substantially green with 30-50% brown 2 Substantially brown with less than 50% green Surface pitting 0 None or less than 0,3 cm 2 pitting 1 Pitting affecting 0,3-0,5 cm 2 2 Pitting affecting greater than 0.5 cm 2 Overall acceptance 0 Good commercial quality 1 Some damage, but still commercially salable 2 Not commercially salable
14 Instrumental analysis (colorimetric assays) Fruit material + Methanol:Water (8/2) Homogenization Total phenolic content (Folin Ciocalteu) Totals anthocyanin content (ph differential method) Total antioxidant capacity - DPPH (2,2-diphenyl-2-picrylhydrazyl) - FRAP (Ferric Reducing Antioxidant Power) - ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] Centrifugation Useful & rapid tool for screening of phytochemical content and giving an estimation of the concentration of a group of compounds
15 Instrumental analysis (HPLC analysis) Fruit material + Methanol:Water (8/2) Centrifugation Homogenization Column: Sherisorb, phase ODS 2, particle 5 μm, l x i.d 15 cm x 4.6 mm Solvent A: 1% Acetic acid in Water Solvent B: Acetonitrile Elution: 0 5 min, 3 9% B; 5 15 min, 9 16% B; min, 16 50%B; min, 50% isocratic Flow rate: 1 ml/min Injection volume: 20 μl
16 Instrumental analysis [Nuclear Magnetic Resonance (NMR) spectroscopy] Why? Powerful instrumental techniques for structural elucidation of phytochemicals in crude fruit extracts. Allows the simultaneous detection and quantification of individual compounds (phytochemicals, amino acids, sugars, organic acids) in complex fruit extracts at the same time in a single running, thus avoiding timeconsuming and tedious chromatographic techniques for their separation. A simple extraction protocol is needed using deuterated solvents in order to obtain 1 H-NMR spectra.
17 Instrumental analysis (NMR) Fruit material + Methanol:Water (8/2) Homogenization +D 2 O (ph=3.0) Centrifugation Freeze drying
18 0d 0d 8d 8d Canada Giant Ferrovia
19 TA (malic acid, %) 15% deformation force (N mm -1 ) Overall acceptability index SSC (%) Penetration force (N) Surface pitting index Weight loss (%) Traction force (N) Stem browning index (A) (A) (A) (B) (B) (B) (C) (C) (C) Days at 20 o C Days at 20 o C Days at 20 o C
20 Respiration rate (CO 2 ml kg -1 h -1 ) Respiration rate Canada Giant Ferrovia Days after harvest (20 o C)
21 Total phenols (GAE mg 100 g -1 FW) FRAP (Trolox μmol 100 g -1 FW) ABTS (Trolox μmol 100 g -1 FW) Total anthocyanins C3GE mg 100 g -1 FW DPPH (Trolox μmol 100 g -1 FW) (A) (A) (B) (B) (C) Days at 20 o C Days at 20 o C
22 HPLC data Hydroxycinnamic acids Flavonols Neochlorogenic acid p-coumaroylquinic acid Chlorogenic acid Catechin Epicatechin Rutin
23 HPLC data Hydroxycinnamic acids Flavonols
24 NMR metabolic profile of fruits NMR spectroscopy alone or in combination with LC-MS and GC-MS has been used to develop metabolomic platforms for: Table grapes Sea buckthorn Melon Black raspberry Citrumelo Apple Mandarin Mango Papaya
25
26 Optimization of an NMR protocol for sweet cherry fruit Extraction: Recovery of metabolites in a single step Freeze-drying: Remove protonated solvents Redissolve in D 2 O and adjust ph at 3 Acquiring NMR Spectra
27 Classify resonances in NMR spectra Aliphatic region (sugars, organic acids) Aromatic region???
28 Classify resonances in NMR spectra Aromatic region Hydroxycinnamic acids, Flavonols, Anthocyanins X 512
29 Discrimination of sugars Sorbitol Fructose Glucose Aliphatic region
30 Discrimination of organic acids Fumaric acid Malic acid Citric acid Aliphatic region
31 Discrimination of hydroxycinnamic acids Neochlorogenic acid Chlorogenic acid Aromatic region
32 Discrimination of anthocyanins A characteristic peak (H-4) for anthocyanins were found at ppm The chemical shift of characteristic peak is ph dependent ph has to be adjusted ph=5.30 ph=3.41 ph=2.43 ph=1.80 ph=1.01 Cyanidin Aromatic region
33 1 H-NMR spectra of the examined cultivars [at harvest] Canada Giant Ferrovia Aliphatic region
34 1 H-NMR spectra of the examined cultivars [at harvest] Canada Giant Ferrovia Aromatic region
35 1 H-NMR spectra of Canada Giant fruit Glucose Malic acid Fructose Sorbitol Citric acid Postharvest ripening Aliphatic region
36 1 H-NMR spectra of Canada Giant fruit Cy-3-rut Chlorogenic acid Neochlorogenic acid Postharvest ripening Aromatic region
37 Monitoring biochemical changes on sweet cherry fruit Traditional approach Sugars Organic acids Phenolic acids Flavonols Anthocyanins 210 nm 280 nm 320 nm 520 nm HPLC-RI HPLC-UV HPLC-UV HPLC-Vis HPLC-Vis Need for standard compounds Different runnings 3 columns -2 detector
38 Monitoring biochemical changes on sweet cherry fruit Novel approach Sugars Organic acids Phenolic acids 210 nm 280 nm HPLC-RI HPLC-UV HPLC-UV Flavonols Anthocyanins 320 nm 520 nm HPLC-Vis HPLC-Vis Extraction with 600 μl D 2 O ~20 min NMR
39 The identification of amino acids at region 0,8-1,5 ppm Aliphatic region
40 What s next in metabolic profiling of sweet cherry? Employment of state-of-the-art technologies towards to metabolomic approaches on sweet cherry postharvest performance 1 H-NMR fingerprinting to determine phytochemicals and nutrients GC-MS fingerprinting to determine volatile compounds
41 Contributors & Acknowledgements V. Goulas P.M. Kourdoulas G.A. Manganaris I.S. Minas A. Lazaridou A.N. Molassiotis I. Gerothanasis
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