LIFE13 ENV IT DIFESA INNOVATIVA DELLE PIANTE DAI LORO PATOGENI, NEL RISPETTO DELL’AMBIENTE E NELL’AMBITO DELL’ECONOMIA CICLICA Accademia dei Georgofili, Firenze – 13 novembre 2015 Polifenoli estratti dai sottoprodotti e reflui agricoli come biostimolanti e prodotti per la difesa: un’importante opportunità per ridurre i rifiuti e stimolare l’economia circolare Prof.ssa Annalisa Romani – Phytolab-DiSIA, Università di Firenze Polifenoli estratti dai sottoprodotti e reflui agricoli come biostimolanti e prodotti per la difesa: un’importante opportunità per ridurre i rifiuti e stimolare l’economia circolare Prof.ssa Annalisa Romani – Phytolab-DiSIA, Università di Firenze

EU project LIFE13 ENV IT / “EVERGREEN” “Environmentally friendly biomolecules from agricultural wastes as substitutes of pesticides for plant diseases control” Accademia dei Georgofili, Firenze – 13 novembre 2015

TIMETABLETIMETABLE

CIRCULAR ECONOMY A circular economy is one that is restorative and regenerative by design, and which aims to keep products, components and materials at their highest utility and value at all times, distinguishing between technical and biological cycles.

CIRCULAR ECONOMY TRADITIONAL USE DiSCARD DISPOSAL OR COMBUSTION INNOVATIVE GREEN REUSE OR EXTRACTIONS NATURAL ACTIVE COMPOUNDS AND ENERGY BIOMASS AND WASTE

WATER EXTRACTION REVERSE OSMOSIS & NANO- FILTRATION STANDARDIZED EXTRACTS PRE- TREATMENTS FEED FIBER (1% TANNINS) THE PROCESS MULCHING TRACED SOURCE

Accademia dei Georgofili, Firenze – 13 novembre 2015

Quantitative analysis by subclasses (% calculated from their contents expressed in mM) for all the fractions from the productive process. A. % gallic acid on total tannins; B. % gallic acid on total GTs; C. % castalagin + vescalagin on total tannins; D. % castalagin + vescalagin on total ETs; E. % GTs on total tannins; F. % ETs on total tannins. ABCDEFTOT a a Total tannins content expressed as mM for the liquid fractions and as µmol/g for the fraction 10 (spray-dried). Sweet Chestnut fractions: quali-quantitative characterization Accademia dei Georgofili, Firenze – 13 novembre 2015

peakRtcompoundλ max MWnegative ions m/z 16.8vescalin230, 280 sh [M-H] castalin230, 280 sh [M-H] pedunculagin I222, 282 sh [M-H] -, monogalloyl glucose I [M-H] -, 271, gallic acid [M-H] -, monogalloyl glucose II [M-H] -, 271, roburin D228, 286 sh , 924, 915, 683, vescalagin224, 276 sh [M-H] -, 466, dehydrated tergallagic-C-glucoside250, [M-H] -, castalagin224, 276 sh [M-H] -, 466, digalloyl glucose [M-H] -, 377, 271, O-galloyl-castalagin isomer220, 280 sh , 520, trigalloyl glucose [M-H] -, 465, 241, tetragalloyl glucose [M-H] -, 356, ellagic acid254, [M-H] pentagalloyl glucose [M-H] -, 469, 169 Single compounds qualitative analysis of the Sweet Chestnut liquid commercial fraction (fraction 6). Rt=retention time (min); λ max =maximum absorbance wavelength (nm); sh = shoulder. m/z of the most abundant ESI-MS ion is underlined. Sweet Chestnut fractions: quali-quantitative characterization Accademia dei Georgofili, Firenze – 13 novembre 2015

Sweet Chestnut fractions: antioxidant and antiradical activities Measured values of GAE, EC 50 and TE. d: density (g/mL); GAE1: Gallic Acid Equivalents (g gallic acid/100g sample); GAE2: Gallic Acid Equivalents (g gallic acid/mmol total tannins by HPLC/DAD); EC 50 : polyphenolic concentration inhibiting DPPH· activity to 50% (μM); TE: Trolox Equivalents (µmol/g sample). dGAE1GAE2EC 50 µmolTE/g Accademia dei Georgofili, Firenze – 13 novembre 2015

ADWB in YPD - 25°C Diameter of inhibition area (mm) ABC concentrations (mg/mL) corresponding to 5.00 mM of total polyphenols SpeciesStrain C. albicans C. albicans C. glabrata C. glabrata P. guilliermondii6140 C. parapsilosis C. tropicalis3982 C. zeylanoides6163 Cl. lusitaniae6142 Cl. lusitaniae I. orientalis K. marxianus S. cerevisiae S. cerevisiae R24 R21 R S. cerevisiae Y. lipolitica6053 Cr. laurentii R Cr. laurentii Cr. laurentii6265 Fil. neoformans R18 R Fil. neoformans R19 R18 R Fil. neoformans Fil. neoformans Fil. neoformans P. wickerhamii R24 R A.Pomegranate peel (Punica granatum L.); B.Sweet Chestnut frac. 6(Castanea sativa Mill.); C. Myrtle leaves (Myrtus communis L.).

P HYTOTERAPICS, COSMETICS AND NUTRACEUTICALS P HYTOTERAPICS, COSMETICS AND NUTRACEUTICALS The antioxidant, antiradical and antimicrobial activities suggest the use of individual fractions from aqueous extracts of sweet chestnut, olea and cynara for phytoterapics and cosmetics, both in order to prolong the shelf life of products in combination or in place of the traditional chemicals, and as active ingredients. Accademia dei Georgofili, Firenze – 13 novembre 2015

Vegetable biomass and waste from olive-oil process VEGETAL WATER SOLID OLIVE WASTE VEGETAL DISCARD TISSUES WASTE WATER COMBUSTION GREEN ENERGY NOVEL FOODS FUNCTIONAL FOODS NATURAL ACTIVE COMPOUNDS EXTRACTION PROCESS PURE WATER LEAVES OLIVE PIT REUSE CIRCULAR ECONOMY

Treatment plant for leaves/branches/pulp and dry/wet husks diluted with waste water

Accademia dei Georgofili, Firenze – 13 novembre 2015 Olea fractions: quali-quantitative characterization Plant Fractions (g/L)Concentrated Fractions (mg/g)Spray Dried (mg/g) GL Olea CMF GL Olea CNF GL Olea CRO DL Olea CRO Soft Extract Olea OH-Tyr Soft Extract Olea GL Soft Extract Olea DL Olea GLOlea DL OH T der 0.29 ± ± ± ± ± ± ± ± ± 0.96 SI der ± ± ± ± 1.74 nd ± ± ± ± EA der ± ± ± ± ± ± ± ± ± 4.46 HC der 0.03 ± ± ± ± ± ± ± ± ± 0.61 Flavonoids 0.15 ± ± ± ± 0.21 nd 1.27 ± ± ± ± 1.05 Verb 0.09 ± ± ± ± ± ± ± ± ± 0.37 Lignansnd 3.18 ± 1.16 nd ± ± 0.23 nd Total PF4.12 ± ± ± ± ± ± ± ± ± OH T der: Hydroxytyrosol derivatives; SI der: Secoiridoids derivatives; EA der: Elenolic Acid derivatives; HC der: Hydroxycynnamic derivatives; Verb: Verbascoside. HPLC/DAD quantitative analyses of Olea plant fractions, concentrate fractions and powders (spray dried). Data are mean values of triplicate analyses (±SD).

Cynara Active R, nutraceutical standardized extracts from Cynara scolymus L. Scardigli A., Vignolini P., Pinelli P., Romani A. XXVI International Conference on Polyphenols, Polyphenols Communication 2012, Vol. I, pp

Accademia dei Georgofili, Firenze – 13 novembre 2015 Cynara fractions: quali-quantitative characterization CUF Cynara GL mg/L CRO Cynara GL mg/L Cynara GL Soft extract mg/g Cynara GL Spray Dried mg/g MCC 1.07 ± ± ± ± 0.48 DCC 2.81 ± ± ± ± 0.20 Chlorogenic acid 2.04 ± ± ± ± 0.03 Cynarin 0.50 ± ± ± ± 0.34 Flavonols 0.23 ± ± ± ± 0.56 Total Polyphenols6.57 ± ± ± ± 0.42 HPLC/DAD quantitative analyses of different plant fractions from Cynara leaves (CUF and CRO), and two concentrated fractions from CRO: soft extract and spray dried from green leaves. Data are mean values of triplicate analyses (±SD). CUF = Concentrate of Ultrafiltration; CRO = Concentrate of Reverse Osmosis; GL = Green Leaves.

Olea and Cynara fractions: antiradical activity with DPPH assay Accademia dei Georgofili, Firenze – 13 novembre 2015 FractionEC 50 (mM) Olea GL soft extract (green leaves) Olea DL soft extract (dried leaves) Olea OH-TYR soft extract (pitted olive pulps) Cynara GL Soft extract

Accademia dei Georgofili, Firenze – 13 novembre 2015 w/w % MW (Da) Gallic acidtraces170 Catechin dimer B Catechin Catechin trimer Catechin dimer B Catechin dimer B Epicatechin Catechin trimer Epicatechin gallate dimer Epicatechin gallate Oligomers quantified as tetramers5.488 Epicatechin gallate dimer ECG oligomers quantified as trimers38.30 ECG oligomers quantified as trimers14.97 TOT82.27 Grape seeds extract: quali-quantitative characterization

  Antiradical Activity vs DPPH  (50,7 μM) at 5 concentrations: 3,3- 8,3- 16,7- 25,1- 33,3 μM Antiradical Activity vs DPPH  (50,7 μM) at 5 concentrations: 3,3- 8,3- 16,7- 25,1- 33,3 μM Gallic acid (EC  M) Tannic acid (EC  M) Grape seeds (EC  M) Pine bark extract (EC  M) Green tea extract (EC  M) Myrtle aqueous extract (EC  M) Myrtle hydroalcoholic extact (EC  M) Catechin (EC  M) Myricitrin (EC  M)

EGCG w/w %MW (Da) Gallic acidtraces170 Catechin dimer B Catechin Catechin trimer Catechin dimer B Catechin dimer B Epicatechin Catechin trimer Epicatechin gallate dimer Epicatechin gallate Oligomers quantified as tetramers5.488 Epicatechin gallate dimer ECG oligomers quantified as trimers38.30 ECG oligomers quantified as trimers14.97 TOTALE82.27 Tannins content and single compounds molecular weights in grape seeds extract Accademia dei Georgofili, Firenze – 13 novembre 2015

Liquid and gel ormulations for biostatic/biocide and repelling products  Tests on Olea crops.  Repelling activity  Repelling, biostatic/biocide activity, plant biostimulant activity: field tests on tobacco nematodes Accademia dei Georgofili, Firenze – 13 novembre 2015  Antimicrobial activity toward selected pathogenic bacterial strains  Tests on soil

FOOD ANTIMICROBIAL AND ANTIOXIDANT ACTIVITIES Use of tannins mixed with extracts from Olea Europaea and Cynara replacing chemical additives FOOD ANTIMICROBIAL AND ANTIOXIDANT ACTIVITIES Use of tannins mixed with extracts from Olea Europaea and Cynara replacing chemical additives

Natural and quality foods Use of tannins mixed with extracts from Olea Europaea and Cynara replacing chemical additives Natural and quality foods Use of tannins mixed with extracts from Olea Europaea and Cynara replacing chemical additives

Polyphenols Waste water Energy Anaerobic fermetnationOrganic fertilizerVegetal water ZERO waste

A circular economy seeks to rebuild capital, whether this is financial, manufactured, human, social or natural. This ensures enhanced flows of goods and services.

Annalisa Romani Margherita Campo Elisa Fierini Francesca Ieri Stefano Mulas Patrizia Pinelli Arianna Scardigli Pamela Vignolini Chiara Vita

LIFE13 ENV IT DIFESA INNOVATIVA DELLE PIANTE DAI LORO PATOGENI, NEL RISPETTO DELL’AMBIENTE E NELL’AMBITO DELL’ECONOMIA CICLICA Accademia dei Georgofili, Firenze – 13 novembre 2015 Polifenoli estratti dai sottoprodotti e reflui agricoli come biostimolanti e prodotti per la difesa: un’importante opportunità per ridurre i rifiuti e stimolare l’economia circolare Prof.ssa Annalisa Romani – Phytolab-DiSIA, Università di Firenze Polifenoli estratti dai sottoprodotti e reflui agricoli come biostimolanti e prodotti per la difesa: un’importante opportunità per ridurre i rifiuti e stimolare l’economia circolare Prof.ssa Annalisa Romani – Phytolab-DiSIA, Università di Firenze