1. LIFE13 ENV IT 000461 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

2. EU project LIFE13 ENV IT 000461 / “EVERGREEN” “Environmentally friendly biomolecules from agricultural wastes as substitutes of pesticides for plant diseases control” Accademia dei Georgofili, Firenze – 13 novembre 2015

3. TIMETABLETIMETABLE

4. 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.

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

7. 1 2 3 4 5 6 7 8 9 WATER EXTRACTION REVERSE OSMOSIS & NANO- FILTRATION STANDARDIZED EXTRACTS PRE- TREATMENTS FEED FIBER (1% TANNINS) THE PROCESS MULCHING TRACED SOURCE

8. Accademia dei Georgofili, Firenze – 13 novembre 2015

9. 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 142.674.327.363.957.342.727.2 274.486.410.675.886.113.915.7 324.369.440.562.335.065.082.0 438.379.033.164.148.451.675.3 562.283.617.166.474.325.743.2 66.0017.933.850.733.366.7121 7100 0.00-1000.02.68 863.084.015.160.174.925.1138 931.859.631.166.753.346.763.6 1021.448.424.443.944.455.6372 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

10. peakRtcompoundλ max MWnegative ions m/z 16.8vescalin230, 280 sh632631 [M-H] - 28.9castalin230, 280 sh632631 [M-H] - 311.3pedunculagin I222, 282 sh784783 [M-H] -, 481 413.9monogalloyl glucose I274332331 [M-H] -, 271, 169 514.6gallic acid272170169 [M-H] -, 125 615.2monogalloyl glucose II274332331 [M-H] -, 271, 169 717.2roburin D228, 286 sh1850933, 924, 915, 683, 301 818.3vescalagin224, 276 sh934933 [M-H] -, 466, 301 920.5dehydrated tergallagic-C-glucoside250, 374614613 [M-H] -, 301 1021.1castalagin224, 276 sh934933 [M-H] -, 466, 301 1123.4digalloyl glucose274484483 [M-H] -, 377, 271, 169 1224.2O-galloyl-castalagin isomer220, 280 sh10861085, 520, 542 1331.8trigalloyl glucose276636635 [M-H] -, 465, 241, 169 1437.7tetragalloyl glucose276788787 [M-H] -, 356, 169 1538.9ellagic acid254, 370302301 [M-H] - 1640.1pentagalloyl glucose274940939 [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

11. 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 11.012.3820.6360.695 21.010.6400.4132.007 31.069.1481.1820.444 41.047.9050.9381.000 51.032.3700.5651.429 61.2530.991.3191.516450.4 71.000.0670.2502.399 81.077.3670.6161.510 91.080.4930.0520.545 1056.991.2100.5863050 Accademia dei Georgofili, Firenze – 13 novembre 2015

12. ADWB in YPD - 25°C Diameter of inhibition area (mm) ABC concentrations (mg/mL) corresponding to 5.00 mM of total polyphenols 6.419.85.26 SpeciesStrain C. albicans6133 16 C. albicans6157 18 C. glabrata7212332521 C. glabrata3828302320 P. guilliermondii6140 C. parapsilosis6150 17 C. tropicalis3982 C. zeylanoides6163 Cl. lusitaniae6142 Cl. lusitaniae6148 14 I. orientalis678220 K. marxianus6141 16 S. cerevisiae6173192219 S. cerevisiae649728 R24 R21 R S. cerevisiae6500182112 Y. lipolitica6053 Cr. laurentii388325 R Cr. laurentii4272 3630 Cr. laurentii6265 Fil. neoformans342825 R18 R Fil. neoformans601024 R19 R18 R Fil. neoformans6225252421 Fil. neoformans6981333425 Fil. neoformans6982292425 P. wickerhamii887919 R24 R A.Pomegranate peel (Punica granatum L.); B.Sweet Chestnut frac. 6(Castanea sativa Mill.); C. Myrtle leaves (Myrtus communis L.).

13. 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

15. 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

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

17. 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 ± 0.104.69 ± 0.676.18 ± 0.583.63 ± 0.64279.89 ± 18.2424.69 ± 3.4725.21 ± 1.5623.55 ± 0.0315.98 ± 0.96 SI der. 2.74 ± 1.7525.13 ± 8.8826.62 ± 8.142.44 ± 1.74 nd 164.19 ± 1.4711.09 ± 0.4578.18 ± 16.70 25.41 ± 11.20 EA der. 0.82 ± 0.284.05 ± 1.334.15 ± 0.451.05 ± 0.370.51 ± 0.0428.34 ± 0.437.54 ± 0.4016.98 ± 1.179.30 ± 4.46 HC der 0.03 ± 0.020.24 ± 0.130.30 ± 0.670.21 ± 0.127.83 ± 0.251.42 ± 0.064.30 ± 0.311.26 ± 0.881.49 ± 0.61 Flavonoids 0.15 ± 0.090.56 ± 0.180.83 ± 0.130.29 ± 0.21 nd 1.27 ± 0.011.00 ± 0.414.38 ± 1.633.08 ± 1.05 Verb 0.09 ± 0.030.99 ± 0.310.83 ± 0.230.71 ± 0.491.69 ± 0.176.76 ± 0.105.85 ± 1.054.13 ± 0.442.27 ± 0.37 Lignansnd 3.18 ± 1.16 nd 17.48 ± 0.012.65 ± 0.23 nd Total PF4.12 ± 2.1238.84 ± 10.3138.91 ± 8.248.33 ± 2.51289.93 ± 18.70244.15 ± 5.5457.63 ± 4.42128.5 ± 20.84 57.53 ± 18.66 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).

18. 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. 267-268.

19. 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 ± 0.5865.19 ± 13.286.61± 1.3414.23± 0.48 DCC 2.81 ± 1.193.96 ± 5.607.64± 0.697.63± 0.20 Chlorogenic acid 2.04 ± 0.4734.00 ± 7.3811.93± 1.7212.36± 0.03 Cynarin 0.50 ± 0.4328.94 ± 14.891.62± 0.014.41± 0.34 Flavonols 0.23 ± 0.0610.11 ± 5.391.09± 0.273.48± 0.56 Total Polyphenols6.57 ± 1.92142.21 ± 9.5828.90± 4.0242.10± 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.

20. 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)6.76 10 -3 Olea DL soft extract (dried leaves)5.44 10 -3 Olea OH-TYR soft extract (pitted olive pulps)1.22 10 -2 Cynara GL Soft extract3.25 10 -3

21. Accademia dei Georgofili, Firenze – 13 novembre 2015 w/w % MW (Da) Gallic acidtraces170 Catechin dimer B30.222578 Catechin1.107290 Catechin trimer0.321866 Catechin dimer B60.261578 Catechin dimer B20.537578 Epicatechin1.362290 Catechin trimer0.371866 Epicatechin gallate dimer0.665730 Epicatechin gallate0.610442 Oligomers quantified as tetramers5.488 Epicatechin gallate dimer18.06882 ECG oligomers quantified as trimers38.30 ECG oligomers quantified as trimers14.97 TOT82.27 Grape seeds extract: quali-quantitative characterization

22.   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 (EC50 7.75  M) Tannic acid (EC50 2.5  M) Grape seeds (EC50 5.75  M) Pine bark extract (EC50 5.25  M) Green tea extract (EC50 5.75  M) Myrtle aqueous extract (EC50 4.5  M) Myrtle hydroalcoholic extact (EC50 6.75  M) Catechin (EC50 12.5  M) Myricitrin (EC50 11.7  M)

23. EGCG w/w %MW (Da) Gallic acidtraces170 Catechin dimer B30.222578 Catechin1.107290 Catechin trimer0.321866 Catechin dimer B60.261578 Catechin dimer B20.537578 Epicatechin1.362290 Catechin trimer0.371866 Epicatechin gallate dimer0.665730 Epicatechin gallate0.610442 Oligomers quantified as tetramers5.488 Epicatechin gallate dimer18.06882 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

24. 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

27. 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

28. 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

29. Polyphenols Waste water Energy Anaerobic fermetnationOrganic fertilizerVegetal water ZERO waste

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

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

32. LIFE13 ENV IT 000461 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