Diapositiva 1 - Life

LIFE13 ENV IT 000461
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
DIFESA INNOVATIVA DELLE PIANTE DAI LORO PATOGENI, NEL RISPETTO DELL’AMBIENTE E
NELL’AMBITO DELL’ECONOMIA CICLICA
Accademia dei Georgofili, Firenze – 13 novembre 2015
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
TIMETABLE
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.
Accademia dei Georgofili, Firenze – 13 novembre 2015
BIOMASS AND WASTE
TRADITIONAL
USE
CIRCULAR
ECONOMY
DiSCARD
DISPOSAL OR
COMBUSTION
INNOVATIVE
GREEN REUSE OR
EXTRACTIONS
NATURAL ACTIVE
COMPOUNDS AND
ENERGY
THE PROCESS
1
TRACED
SOURCE
8
2
STANDARDIZED
EXTRACTS
REVERSE
OSMOSIS &
NANOFILTRATION
MULCHING
9
FEED FIBER (1%
TANNINS)
PRETREATMENTS
5
7
4
6
WATER
EXTRACTION
3
Accademia dei Georgofili, Firenze – 13 novembre 2015
Sweet Chestnut fractions: quali-quantitative characterization
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.
A
B
C
D
E
F
TOTa
1
42.6
74.3
27.3
63.9
57.3
42.7
27.2
2
74.4
86.4
10.6
75.8
86.1
13.9
15.7
3
24.3
69.4
40.5
62.3
35.0
65.0
82.0
4
38.3
79.0
33.1
64.1
48.4
51.6
75.3
5
62.2
83.6
17.1
66.4
74.3
25.7
43.2
6
6.00
17.9
33.8
50.7
33.3
66.7
121
7
100
100
0.00
100
0.0
2.68
8
63.0
84.0
15.1
60.1
74.9
25.1
138
9
31.8
59.6
31.1
66.7
53.3
46.7
63.6
10
21.4
48.4
24.4
43.9
44.4
55.6
372
a Total tannins content expressed as mM for the liquid fractions and as µmol/g for the
fraction 10 (spray-dried).
Accademia dei Georgofili, Firenze – 13 novembre 2015
Sweet Chestnut fractions: quali-quantitative characterization
Single compounds qualitative analysis of the Sweet Chestnut liquid commercial fraction (fraction 6).
Rt=retention time (min); λmax=maximum absorbance wavelength (nm); sh = shoulder. m/z of the
most abundant ESI-MS ion is underlined.
peak
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Rt
6.8
8.9
11.3
13.9
14.6
15.2
17.2
18.3
20.5
21.1
23.4
24.2
31.8
37.7
38.9
40.1
compound
vescalin
castalin
pedunculagin I
monogalloyl glucose I
gallic acid
monogalloyl glucose II
roburin D
vescalagin
dehydrated tergallagic-C-glucoside
castalagin
digalloyl glucose
O-galloyl-castalagin isomer
trigalloyl glucose
tetragalloyl glucose
ellagic acid
pentagalloyl glucose
λmax
230, 280 sh
230, 280 sh
222, 282 sh
274
272
274
228, 286 sh
224, 276 sh
250, 374
224, 276 sh
274
220, 280 sh
276
276
254, 370
274
MW
632
632
784
332
170
332
1850
934
614
934
484
1086
636
788
302
940
negative ions m/z
631 [M-H]631 [M-H]783 [M-H]-, 481
331 [M-H]-, 271, 169
169 [M-H]-, 125
331 [M-H]-, 271, 169
933, 924, 915, 683, 301
933 [M-H]-, 466, 301
613 [M-H]-, 301
933 [M-H]-, 466, 301
483 [M-H]-, 377, 271, 169
1085, 520, 542
635 [M-H]-, 465, 241, 169
787 [M-H]-, 356, 169
301 [M-H]939 [M-H]-, 469, 169
Accademia dei Georgofili, Firenze – 13 novembre 2015
Sweet Chestnut fractions: antioxidant and antiradical activities
Measured values of GAE, EC50 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); EC50: polyphenolic concentration
inhibiting DPPH· activity to 50% (μM); TE: Trolox Equivalents (µmol/g sample).
d
GAE1
GAE2
EC50
1
1.01
2.382
0.636
0.695
2
1.01
0.640
0.413
2.007
3
1.06
9.148
1.182
0.444
4
1.04
7.905
0.938
1.000
5
1.03
2.370
0.565
1.429
6
1.25
30.99
1.319
1.516
7
1.00
0.067
0.250
2.399
8
1.07
7.367
0.616
1.510
9
1.08
0.493
0.052
0.545
56.99
1.210
0.586
10
µmolTE/g
450.4
3050
Accademia dei Georgofili, Firenze – 13 novembre 2015
ADWB in YPD - 25°C
A.
Pomegranate peel (Punica granatum L.);
B.
Sweet Chestnut frac. 6(Castanea sativa Mill.);
C.
Myrtle leaves (Myrtus communis L.).
concentrations (mg/mL)
corresponding to 5.00 mM of total
polyphenols
Species
Strain
C. albicans
6133
C. albicans
6157
C. glabrata
7212
C. glabrata
3828
P. guilliermondii
6140
C. parapsilosis
6150
C. tropicalis
3982
C. zeylanoides
6163
Cl. lusitaniae
6142
Cl. lusitaniae
6148
I. orientalis
6782
K. marxianus
6141
S. cerevisiae
6173
S. cerevisiae
6497
S. cerevisiae
6500
Y. lipolitica
6053
Cr. laurentii
3883
Cr. laurentii
4272
Cr. laurentii
6265
Fil. neoformans
3428
Fil. neoformans
6010
Fil. neoformans
6225
Fil. neoformans
6981
Fil. neoformans
6982
P. wickerhamii
8879
Diameter of inhibition area
(mm)
A
B
C
6.41
9.8
5.26
33
30
16
18
25
23
21
20
17
14
20
19
28 R
18
16
22
24 R
21
19
21 R
12
36
30
18 R
19 R
24
34
24
24 R
18 R
21
25
25
25 R
25 R
24 R
25
33
29
19 R
PHYTOTERAPICS, 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
Accademia dei Georgofili, Firenze – 13 novembre 2015
Vegetable biomass and waste from olive-oil process
CIRCULAR ECONOMY
VEGETAL DISCARD TISSUES
SOLID
OLIVE WASTE
COMBUSTION
GREEN ENERGY
OLIVE PIT
EXTRACTION
PROCESS
VEGETAL WATER
NOVEL FOODS
LEAVES
PURE WATER
WASTE WATER
REUSE
NATURAL ACTIVE
COMPOUNDS
FUNCTIONAL
FOODS
Diafiltration
Diafiltrated water
Leaves/branches
Washing waters
Leaves/branches
process
Hot water
extraction
MF
Extracted vegetal material
Pre-filtration
60 L
Dosing
Tanks
Chopped olives,
wet/dry pomaces
diluted with waste
water
NF
OI
Pulps/husks
process
Waste
Solid and semi-solid waste intended
for the storage tank for subsequent
use eg. fertilizers and biogas
2 thermostated reactors
Pre-treatment section
UF
Ultrapure
water
Marketed products
PRODUCTS REFINING with RESINS
Treatment plant for leaves/branches/pulp and dry/wet husks diluted with waste water
Olea fractions: quali-quantitative characterization
HPLC/DAD quantitative analyses of Olea plant fractions, concentrate fractions and powders (spray dried). Data are
mean values of triplicate analyses (±SD).
Plant Fractions (g/L)
Concentrated Fractions (mg/g)
GL Olea
CMF
GL Olea
CNF
GL Olea
CRO
OH T der
0.29 ± 0.10
4.69 ± 0.67
6.18 ± 0.58
SI der.
2.74 ± 1.75
25.13 ± 8.88 26.62 ± 8.14 2.44 ± 1.74
EA der.
0.82 ± 0.28
4.05 ± 1.33
4.15 ± 0.45
1.05 ± 0.37
0.51 ± 0.04
28.34 ± 0.43
7.54 ± 0.40 16.98 ± 1.17 9.30 ± 4.46
HC der
0.03 ± 0.02
0.24 ± 0.13
0.30 ± 0.67
0.21 ± 0.12
7.83 ± 0.25
1.42 ± 0.06
4.30 ± 0.31
1.26 ± 0.88
1.49 ± 0.61
Flavonoids
0.15 ± 0.09
0.56 ± 0.18
0.83 ± 0.13
0.29 ± 0.21
nd
1.27 ± 0.01
1.00 ± 0.41
4.38 ± 1.63
3.08 ± 1.05
Verb
0.09 ± 0.03
0.99 ± 0.31
0.83 ± 0.23
0.71 ± 0.49
1.69 ± 0.17
6.76 ± 0.10
5.85 ± 1.05
4.13 ± 0.44
2.27 ± 0.37
nd
3.18 ± 1.16
nd
nd
nd
17.48 ± 0.01
2.65 ± 0.23
nd
nd
Lignans
Total PF
4.12 ± 2.12
DL Olea
CRO
Soft Extract
Olea OH-Tyr
Spray Dried (mg/g)
3.63 ± 0.64 279.89 ± 18.24
nd
38.84 ± 10.31 38.91 ± 8.24 8.33 ± 2.51 289.93 ± 18.70
Soft Extract
Olea GL
Soft Extract
Olea DL
24.69 ± 3.47
25.21 ± 1.56 23.55 ± 0.03 15.98 ± 0.96
Olea GL
164.19 ± 1.47 11.09 ± 0.45 78.18 ± 16.70
244.15 ± 5.54 57.63 ± 4.42 128.5 ± 20.84
OH T der: Hydroxytyrosol derivatives;
SI der: Secoiridoids derivatives;
EA der: Elenolic Acid derivatives;
HC der: Hydroxycynnamic derivatives;
Verb: Verbascoside.
Accademia dei Georgofili, Firenze – 13 novembre 2015
Olea DL
25.41 ±
11.20
57.53 ±
18.66
Cynara ActiveR , 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.
MF
UF
OI
pre-filtration
Aqueous
Extract from
TIMATIC
animal feeding,
agricultural
products
Spray-dryer
Pure
Water
Natural Extracts
(phytotherapy/functional food)
pura
Cynara fractions: quali-quantitative characterization
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 Cynara GL
mg/L
CRO
Cynara GL mg/L
Cynara GL Soft
extract
mg/g
Cynara GL Spray
Dried mg/g
MCC
1.07 ± 0.58
65.19 ± 13.28
6.61± 1.34
14.23± 0.48
DCC
2.81 ± 1.19
3.96 ± 5.60
7.64± 0.69
7.63± 0.20
Chlorogenic acid
2.04 ± 0.47
34.00 ± 7.38
11.93± 1.72
12.36± 0.03
Cynarin
0.50 ± 0.43
28.94 ± 14.89
1.62± 0.01
4.41± 0.34
Flavonols
0.23 ± 0.06
10.11 ± 5.39
1.09± 0.27
3.48± 0.56
Total Polyphenols
6.57 ± 1.92
142.21 ± 9.58
28.90± 4.02
42.10± 0.42
CUF = Concentrate of Ultrafiltration; CRO = Concentrate of Reverse Osmosis; GL = Green Leaves.
Accademia dei Georgofili, Firenze – 13 novembre 2015
Olea and Cynara fractions: antiradical activity with DPPH assay
Fraction
Olea GL soft extract (green leaves)
Olea DL soft extract (dried leaves)
Olea OH-TYR soft extract (pitted olive pulps)
Cynara GL Soft extract
EC50 (mM)
6.76 10-3
5.44 10-3
1.22 10-2
3.25 10-3
Accademia dei Georgofili, Firenze – 13 novembre 2015
Grape seeds extract: quali-quantitative characterization
Gallic acid
traces
MW
(Da)
170
Catechin dimer B3
0.222
578
Catechin
1.107
290
Catechin trimer
0.321
866
Catechin dimer B6
0.261
578
Catechin dimer B2
0.537
578
Epicatechin
1.362
290
Catechin trimer
0.371
866
Epicatechin gallate dimer
0.665
730
Epicatechin gallate
0.610
442
Oligomers quantified as tetramers
5.488
Epicatechin gallate dimer
18.06
ECG oligomers quantified as trimers
38.30
ECG oligomers quantified as trimers
14.97
TOT
82.27
w/w %
882
Accademia dei Georgofili, Firenze – 13 novembre 2015
gallico
quercetina
0,3000
clorogenico
catechina
grape seeds
pine bark
0,2500
3,4diidrossibenz
cinarina
mirto idroalc
abs (550nm) DPPH
0,2000
carciofo
teavigo
Antiradical Activity vs DPPH
(50,7 μM)
at 5 concentrations:
3,3- 8,3- 16,7- 25,1- 33,3 μM
ac. caffeico
0,1500
esculina
miricitrina
oleuropeina
foglie olea
0,1000
rami olea
verbascoside
pinoresinolo
0,0500
apigenina 7-O-gluc
estr.olio
luteolina 7-O-gluc
0,0000
mirto decotto
0,0
5,0
10,0
15,0
20,0
25,0
30,0
35,0
μM
40,0
0,3000
45,0
AV1
gallico
AV2
catechina
ac. tannico
abs (550nm) DPPH
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)
0,2500
grape seeds
pine bark
0,2000
3,4diidrossibenz
mirto idroalc
0,1500
teavigo
0,1000
miricitrina
mirto decotto
0,0500
ac. Tannico mw 940
ripesato
0,0000
0,0
10,0
20,0
30,0
uM
40,0
50,0
EGCG
Gallic acid
w/w %
traces
MW (Da)
170
Catechin dimer B3
0.222
578
Catechin
1.107
290
Catechin trimer
0.321
866
Catechin dimer B6
0.261
578
Catechin dimer B2
0.537
578
Epicatechin
1.362
290
Catechin trimer
0.371
866
Epicatechin gallate dimer
0.665
730
Epicatechin gallate
0.610
442
Oligomers quantified as tetramers
5.488
Epicatechin gallate dimer
18.06
ECG oligomers quantified as trimers
38.30
ECG oligomers quantified as trimers
14.97
TOTALE
82.27
882
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
 Antimicrobial activity toward selected pathogenic bacterial strains
 Tests on soil
Accademia dei Georgofili, Firenze – 13 novembre 2015
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
Energy
Waste
water
Anaerobic fermetnation
Organic fertilizer
Polyphenols
Vegetal water
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 000461
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
DIFESA INNOVATIVA DELLE PIANTE DAI LORO PATOGENI, NEL RISPETTO DELL’AMBIENTE E
NELL’AMBITO DELL’ECONOMIA CICLICA
Accademia dei Georgofili, Firenze – 13 novembre 2015