CV - ASL Teramo

Transcript

CV - ASL Teramo

ASL TERAMO PROTOCOLLO UNICO
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Prot. nr. 0008009113 del 21/02/2013
Al Direttore Generale dell r\L''-'' ·~-
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64100 Teramo
Il sottoscritto Federico Di Fabio chiede di essere ammesso a partecipare all'avviso
di pubblica selezione, per titoli e colloquio, finalizzato alla formulazione di una
graduatoria di merito per il conferimento della borsa di studio per N. 2 laureato in
SCIENZE E TECNOLOGIE ALIMENTARI - NELL'AMBITO DEL PROGETTO LETTERA "A"
MIGLIORAMENTO DELLE ACQUE DESTINATE AL CONSUMO UMANO;
A tal fine dichiara, sotto la propria responsabilità , anche agli effetti previsti dal
D.P.R. n. 445 del 28.12.2000:
o di chiamarsi: Federico Di Fabio;
o
o
o
di essere nato a
di essere residente in
telefonico n.
il
;
alla via
n.
cap. 6
recapito
;
di essere in possesso della cittadinanza
o di non aver riportato condanne penali, non avere carichi penali pendenti e non
essere sottoposto a procedimento penale
o
di possedere un'adeguata conoscenza della lingua italiana (solo per i cittadini
degli altri Paesi dell'Unione Europea).
o di essere in possesso dei seguenti requisiti di ammissione:
o Laurea in _S_cie!Jle e Tecnologie Alimentari conseguita in data 23/03/2000
presso l'Università degli Studi di Bologna sede di Cesena (FC);
o Competenze professionali ed esperienza richiesti per la partecipazione alla
suindicata borsa di studio;
o
di aver prestato i seguenti servizi presso Pubbliche amministrazioni:
- dal 1 dicembre 2012 ad oggi presso Istituto Zooprofilattico Sperimentale
dell'Abruzzo e Molise "G.Caporale" via Campo Boario 64100 Teramo;
- dal 1 ottobre 2011 al 31 novembre 2012 presso Istituto Zooprofilattico
Sperimentale dell'Abruzzo e Molise " G.Caporale" via Campo Boario 64100
Teramo;
- dal 07 agosto 2006 al 31 dicembre 2009 presso Agenzia Regionale Tutela
Ambiente Strada Prov.le per Monticchio, Caselle di Bazzana 67100 L'Aquila~
·
- dal 21 gennaio 201 O al 20 aprile 201 O presso Agenzia Regionale Tutela
Ambiente Strada Prov.le per Monticchio, Caselle di Bazzana 67100 L'Aquila;
·
- anno accademico 2005/2006 presso Università degli Studi di Teramo
dipartimento Scienze degli Alimenti Piazza Aldo Moro 4 64100 Teramo;
Domanda di selezione
112
- dal 19 aprile 2004 al 18 ottobre 2004 presso
Sperimentale dell'Abruzzo e Molise "G.Caporale" via
Teramo;
- dal 01 novembre 2003 al 17 marzo 2004 presso
Teramo;
- dal Ol novembre 2002 al 31 ottobre 2003 presso
Teramo;
- dal 19 agosto 2002 al 31 ottobre 2002 presso
Teramo.
Istituto Zooprofilattico
Campo Boario 64100
Campo Boario 64100
Campo Boario 64100
Campo Boario 64100
D di eleggere il seguente domicilio ove inviare ogni comunicazione relativa al
presente avviso:
Sig. Federico Di Fabio Via
Comune
Prov.
Cap
n.
Telefono
D di dare il proprio consenso al trattamento dei dati personali ai sensi del O.Lgs. 30
giugno 2003, n. 196;
Allega la documentazione indicata nell'unito elenco descrittivo:
Elenco dei documenti presentati
CV vitae
Dichiarazione sostitutiva di certificazione
Dichiarazione sostitutiva dell'atto di notorietà
Fotocopia del documento d'identità
Data
.Àc:{l\)L/10,(;:,
Domanda di selezione
212
DICHIARAZIONE SOSTITUTIVA DELL'ATTO DI NOTORIETÀ
(Art. 47 del D.P.R. 28 dicembre 2000, n. 445)
Il sottoscritto Di Fabio Federico nato a
il
residente in
Via
con riferimento all'istanza di partecipazione
all'avviso di pubblica selezione, per titoli e colloquio, finalizzato alla formulazione di
una graduatoria di merito per il conferimento della borsa di studio per N. 2
laureato in SCIENZE E TECNOLOGIE ALIMENTARI NELL'AMBITO DEL PROGETTO
LETTERA "A" MIGLIORAMENTO DELLE ACQUE DESTINATE AL CONSUMO UMANO; ai
sensi e per gli effetti dell'art. 47 del D.P.R. n. 445 del 28 dicembre 2000, sotto la
propria responsabilità e consapevole delle sanzioni penali richiamate dall'art. 76 in
caso di dichiarazioni mendaci e della decadenza dei benefici eventualmente
conseguenti al provvedimento emanato sulla base di dichiarazioni non veritiere di
cui all'art. 75 del succitato D.P.R., informato/a su quanto previsto dal D.Lgs. 30
giugno 2003, n. 196
DICHIARA
D di aver prestato i seguenti servizi:
- dal 23 aprile 2010 al 31 dicembre 2011 in qualità di Collaboratore Tecnico
Prof.le Tecnologo Alimentare
presso Agenzia per il Lavoro "OBIETIIVO
LAVORO" filiale di Pescara via Trilussa 79 /81 sede attività lavoro Agenzia
Regionale Tutela Ambiente Strada Prov.le per Monticchio, Caselle di Bazzane
67100 L'Aquila
- dal 04 dicembre 2006 al 28 febbraio 2007 in qualità di Collaboratore Tecnico )
Prof.le Tecnologo Alimentare presso Agenzia per il Lavoro "ITALIA LAVORO
Guidobaldo Del Monte 60 Roma sede attività di lavoro Ente Gran Sasso e
Monti della Lago, Polo Agroalimentare di Amatrice (RI) P.zza San Francesco ;
- dal 04 dicembre 2006 al 28 febbraio 2007 in qualità di Collaboratore Tecnico
Prof.le Tecnologo Alimentare presso Agenzia per il Lavoro "ITALIA LAVORO
Guidobaldo Del Monte 60 Roma sede attività di lavoro Ente Gran Sasso e
Monti della Lago, Polo Agroalimentare di Amatrice (RI) P.zza San Francesco ;
- settembre 2000 al gennaio 2001 in qualità di Collaboratore Tecnico Prof.le
Tecnologo Alimentare presso Cantina Frentana via Perazza 32 66020 Rocca
san Giovanni (Ch) ;
D dichiara le seguenti pubblicazioni:
- Rosanna Tofalo, Clemencia Chaves-Lopez, Federico Di Fabio, Maria Schirone,
Giovanna E. Felis, Sandra Torrioni, Antonello Paparella, Giovanna Suzzi
Molecular identification and osmotolerant profile of wine yeasts that ferment
a high sugar grape must. Jnternational Journal of Food Microbiology
2009, voi. 130, n°3, pp. 179-187.
Dichiarazione sostitutiva atto di notorietà
113
(}
~
11
- Di Fabio F. {2008). Characterization Saccharomyces cerevisiae biodiversity
and dynamics during Montepulciano d'Abruzzo". Oral Communication. 13th
Workshop on the Deve/opments in the Jtalian PhD Research on Food Science
Technology and Biotechnology, University of Turin, 10-12 September 2008.
- Suzzi G., Tofalo R., Chaves-Lopez C., Ramazzotti S., Stagnari F., Di Fabio F.,
Pisante M., Barca E., Castrignanò A. {2008). Microclimatic and territorial
Montepulciano d'Abruzzo" Colline Teramane tor
characterization of
isolation, characteritation and selection of Saccharomyces cerevisiae:
preliminary results. 31 st World Congress of Vine and Wine - June 15-20, 2008
Verona, {ltaly):308.
- Tofalo R., Arfelli G., Chaves-Lopez C., Angrisani R. Teiera G., Di Fabio F., Pisante
M., Suzzi G .. (2008). Dominance and oenological properties of Saccharomyces
cerevisiae starters tor Montepulciano d' Abruzzo"vinification. 31 st World
Congress of Vine and Wine - June 15-20, 2008 Verona, {ltaly):31 O.
- Di Fabio F. {2007). Characterization of yeasts for Montepulciano d'Abruzzo,
Colline Teramane DOCG vinification". Poster Communication. 12th Workshop
on the Developments in the ltalian PhD Research on Food Science and
Technology. Reggio Calabria, ltaly, 12-14 September 2007.
- Tofalo R., Di Fabio F., Chaves-Lopez C., Piva A., Torrioni S., Suzzi G. (2007).
"Characteristics of osmotolerant yeasts from Vino Cotto", a traditional wine
produced in the Abruzzo region (ltaly) ". Proceedings 8th lnternational
symposium of Enology of Bordeaux. Bordeaux, France, 25-27 giugno 2007.
- Di Fabio F. (2006). Wine from Abruzzo Region Ecology and Microbiology of
Production". Poster Communication. 11 th Workshop on the Developments in
the ltalian PhD Research on Food Science and Technology. Mosciano
Sant' Angelo, ltaly, 27-29 September 2006.
- Tofalo R., Di Fabio, F., Chaves-Lopez, C., Piva A., Suzzi G. (2006). Yeasts from
traditional ltalian Cooked Wine". Proceedings 20th lnternational ICFMH
Symposium - Food Micro 2006 - Bologna, ltaly, August 29 - September 2, p.285
- Tofalo R., Chaves-Lopez C., Di Fabio F., Angrisani R., Suzzi G. (2006).
"Determinazione di Dekkera\Brettanomyces nel vino con metodi colturaindipendenti. Atti IV Conv. Naz. AISSA, -Qualità e sostenibilità delle produzioni
agrarie, alimentari e forestali-, 5-6 dicembre 2006, Mosciano S. Angelo (TE),
p.227-228.
- V. Langella, P. Semprini, F. Di Fabio, B.Pasini, M.T. Falda, F. Panello, S. Calvarese
(2003): Indagine preliminare sull'efficacia dell'estratto di pompelmo nella
lotta contro la peste americana. Vet. lt. Anno XXXIX-47. Gennaio-Marzo 2003,
pp 49-54.
11
11
11
11
11
11
D delle pubblicazioni indicate il sottoscritto allega fotocopia semplice conforme
agli originali in suo possesso.
D Attività di docenza:
- dal O1 dicembre 2007 al 21 dicembre 2007 docente fino agli aventi diritto, 9
ore settimanali in Scienze degli Alimenti presso Istituto professionale per i Servizi
Commerciali Turistici e Alberghieri L. Di Poppa" via F. Barnabei 2 64100
Teramo;
11
2/3
- dal 03 ottobre 2006 al 09 novembre 2006 docente fino agli aventi diritto, 3 ore
settimanali in Scienze degli Alimenti presso Istituto professionale per i Servizi
Commerciali Turistici e Alberghieri "L. Di Poppa" via F. Barnabei 2 64100
Teramo;
- dal 26 gennaio 2008 al 15 marzo 2008, 25 ore professore a contratto presso
Istituto professionale per i Servizi Commerciali Turistici e Alberghieri "L. Di
Poppa" via F. Barnabei 2 64100 Teramo;
- dal 02 febbraio 2008 al 29 marzo 2008, 25 ore professore a contratto presso
- dal 31 gennaio 2007 al 18 marzo 2007, 18 ore professore a contratto presso
ata
k(
l ;) L 'Lo,(3
3/3
j
- Corso di Formazione: "La validazione del metodo di prova: Quaderno di
laboratorio; Piano di validazione, piano di controllo qualità". ARTA Pescara 15
gennaio 2009
- Corso di Formazione: "La validazione dei metodi analitici: Metodi biologici, /.
chimici e fisici". Organizzato da Agenzia Regionale Tutela Ambiente-Abruzzo L{
(A.R.T.A.) presso L'Università degli Studi di Teramo 3-4-10-11 dicembre 2008.
- Corso di Formazione: Eusoft®.Lab-LIMS End User. Presso ARTA Abruzzo- Dip.Prov.
7
di L'Aquila 7 ottobre 2008
r
- Corso di Formazione: Implementazione procedure del S.G.Q. "Modulistica di )
sistema_ procedure Gestionali".
- Corso di Formazione: Norma ISO/IEC 17025:2005 laboratori di prova.
Organizzata da ANGQ Associazione nazionale garanzia di qualità e SINAL
SistemA Nazionale per l'Accreditamento di Laboratori. ARTA Teramo 20-21
ottobre 2008.
- Xlii-Workshop on the Developments in the ltalian PhD Research in Food
Science and Technology-Alba (TO) 9-11 settembre 2008.
- Xli-Workshop on the Developments in the ltalian PhD Research in Food
Science and Technology-Reggio Calabria 12-14 settembre 2007.
- Convegno: "Legionella un Killer Ambientale" presso A.R.T.A. Abruzzo. Pescara r
19/10/2006.
- Xl-Workshop on the Developments in th e ltalian PhD Research in Food Science
and Technology-Mosciano Sant' Angelo 27-29 settembre 2006.
- Food Micro 2006 Bologna 29 agosto - 1 settembre 2006.
- Corso di Formazione: "Validazione e valutazione dei metodi di calcolo e
dell'incertezza di misura in campo biologico". IZSAM Istituto Zooprofilattico
24-26
Sperimentale dell 'Abruzzo e del Molise "G.Caporale" Teramo,
settembre 2003.
- Corso di Formazione "Formazione Formatori". IZSAM "G.Caporale" Teramo, 2629 luglio 2004.
- Corso di Formazione "Il Sistema Qualità". IZSAM "G.Caporale" Teramo, 13-15
settembre 2004 .
- Corso di Lingua inglese Il livello. Centro Territoriale Permanente per L'Istruzione
e la Formazione in età adulta, Teramo gennaio-maggio 2002.
7
7
7
o di possedere altra idonea documentazione da cui sia possibile dedurre
attitudini professionali in relazione alle mansioni da svolgere:
')( - Partecipazione al gruppo di lavoro_relativo accreditamento SINAL_:'NORMA )
UNI CEI EN ISO/IEC 17025" dei metodi analitici di acque destinate al consumo ,
uman9 ISTISADI 2007 /31 presso ARTA- A5r0zzo dipartimento provinciale-diL' Aquila e di Teramo;
- Accreditamento e Responsabile delle prove in base alla norma ISO 17025
presso ARTA Abruzzo dip. L' Aquila
PP I AQ/CH/02/01 determinazione del pH ISTISAN 2007 /31 Met ISS BCA 023
PP I AQ/CH/02/02 determinazione della conduttività ISTISAN 2007 /31 Met ISS
BOA 022;
PP/AQ/CH/02/03 determinazione degli anioni ISTISAN 2007/31 Met ISS CBB
037
PP I AQ/CH/02/04 determinazione dei cationi ISTISAN 2007 /31 Met ISS CBB 038
Dichiarazione sostitu tiva di certific azione
2/3
yv.J
- Partecipazione al gruppo di lavoro relativo al "Monitoraggio Ambientale
Integrato del Fiume Sagittario in Valle Peligno" presso ARTA Abruzzo dip.
L'Aquila per L'Università degli Studi dell'Aquila Dip. Scienze Ambientali;
- Frequenza volontaria presso ARTA Abruzzo dipartimento provinciale di
Teramo: partecipazione al gruppo di lavoro nell'elaborazione del progetto
"Creazione di una rete territoriale per la diffusione di Sistemi di Gestione
Ambientale EMAS/SGA";
- Frequenza volontaria a scopo di studio e istruzione presso il Settore Chimico
Ambientale P.M.l.P., P.zza Martiri Pennesi 64100 Teramo;
- Frequenza volontaria a scopo di studio e istruzione presso il Settore di Igiene e
Tecnologie Alimentari e dell'Alimentazione Animale presso Istituto
Zooprofilattico Sperimentale dell 'Abruzzo e Molise "G .Caporale" via Campo
Boario 64100 Teramo.
data
I
4/ ò'I../
Dichiarazione sostitutiva di c ertific azione
'I.O,(::,
3/3
Curriculum Vitae
Europass
Informazioni personali
Nome/ Cognome
Federico Di Fabio
Indirizzo
Cellulare
E-mail
Cittadinanza
Data di nascita
Sesso
Esperienza professionale
Maschile
/I
Date _ 12/2012- 11/2013
Lavoro o posizione ricop erti
Principali attività e
responsabilità
Nome e indirizzo del datore
di lavoro
Tipo di attività o settore
Date
Lavoro o posizione ricoperti
responsabilità
di lavoro
Date
Principali a ttività e
responsabilità
Pagina 1/9- Curriculum vitae di
Federico Di Fabio
Contratto di collaborazione coordinata e continuativa in qualità di
Tecnologo Alimentare
Valutazione
delle
dinamiche
di
contaminazione
di
agenti
patogeni.dell'efficacia dei controlli e del rischio finale per il consumatore per
alcune categorie di prodotti:
Messa a punto di tecniche per la conservazione dei batteri;
•
Estrazione di materiale genico dai batteri.
" IZSA&M" Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise
"G.Caporale", reparto di Batteriologia via Campo Boario 64 100 Teramo
r/'
/
Ricerca sperimentale sull'origine e lo sviluppo delle malattie infettive e
diffusive degli animali, nella d iagnosi delle malattie animali e di quelle che si
possono trasmettere all'uomo (zoonosi). Nel settore degli alimenti di origine
animale destina ti agli uomini e agli animali vengono effettuate indagini
rricrobiologiche, chimiche e radiometriche; inoltre viene mantenuta alta la
sorveglianza epidemiologica sullo stato sanitario delle popolazioni animali,
sull'igiene delle produzioni zootecniche e sui prodotti di origine animale
yfJ
10/2011 - 11/2012
Contratto di collaborazione coordina ta e continuativa in qualità di
Determinazione dell'equivalenza tra la legislazione dell'Unione Europea e
degli Sta ti Uniti d'America in materia di produzione e trasformazione delle
carni e dei prodotti a base di carne:
•
Attività di produzione Materiali di Riferimento per prove
microbiologiche.
" IZSA&M" Istituto Zooprofilattico Sperimentale dell 'Abruzzo e del Molise ,
"G.Caporale", reparto di Batteriologia via Campo Boario 64 100 Teramo
diffusive degli animali. nella diagnosi delle malattie animali e di quelle che si
animale destinati agli uomini e agli animali vengono effettuate indagini
rricrobiologiche, chimiche e radiometriche; inoltre viene mantenuta alta la
sorveglia nza epidemiologica sullo stato sanitario delle popolazioni animali,
08/2006- 12/201 1
Collaborat0re Tecnico professionale Tecnologo Alimentare
Monitora ggio del Territorio Regionale per la rilevazione e prevenzione
dall'inquinamento ambienta le e alimentare:
•
Ac~reditamento e responsabile delle prove:
PP/AQ/CH/02/01 determinazione del pH ISTISAN 2007/31 Met ISS BCA
•
•
•
•
•
•
•
•
•
•
•
•
•
di lavoro
Date
responsabilità
di lavoro
Date
responsabilità
di lavoro
Date
responsabilità
Pagina 219- Curriculum vitae di
Federico Di Fabio
023
PP/AQ/CH/02/02 determinazione della conduttività ISTISAN 2007/31
Met ISS BOA 022
PP/AQ/CH/02/03 determinazione degli anioni ISTISAN 2007/31 Met
ISS CBB037
PP/AQ/CH/02/04 determinazione dei cationi ISTISAN 2007/31 Met ISS
CBB038
Partecipazione al gruppo di lavoro relativo al 11 Monitoraggio
Ambientale Integrato del Fiume Sagittario in Valle Peligno" per
Università degli Studi de!I' Aquila Dip. Scienze Ambientali.
Partecipazione al gruppo di lavoro relativo all'accreditamento SINAL
11
NORMA UNI CEI EN ISO/IEC 17025" dei metodi analitici di acque
destinate al consumo umano ISTISAN 2007/31.
Partecipazione a circuiti interlaboratorio UNICHIM. FAPAS, LGC
Standard.
Analisi chimiche e Controllo Sicurezza Alimenti (vino Reg. CEE
2676/90; olio Reg. CEE 2568/'2008 ; conserve vegetali, succhi, prodotti
pronti per alimentazione, prodotti da forno)
Analisi chimiche acque destinate al consumo umano (D.Lgs. 31 /01,
D.Lgs. 27 /02)
Analisi chimiche acque superficiali destinate al consumo umano
(D.Lgs. 152/06)
Analisi chimiche acque minerali (D.Lgs. 105/92, D.M. 452/92)
Analisi chimiche acque sotterranee (D.Lgs. 152/06)
Analisi chimiche siti contaminati e rifiuti (D.Lgs. 152/06)
Analisi chimiche Monitoraggio Acque Marino Costiere
Supporto all'attività di campionamento e analisi di laboratorio
relativa al Programma di Monitoraggio Acque Sotterranee.
Supporto all'attività di campionamento e analisi di laboratorio
relativa al Programma di Monitoraggio Nitrati.
Vigilanza rimozione macerie post-sisma 2009
ARTA Abruzzo Agenzia Regionale Tutela Ambiente
Distretto di L'Aquila, strada prov. Per Monticchio, caselle di Bazzana 67100
L'Aquila
Attività di prevenzione, protezione e tutela ambientale, supporto analitico
strumentale azienda SIAN Servizio di Igiene degli Alimenti e della Nutrizione
Anno accademico '2008/2009
Attività d'insegnamento, Contratto di Prestazione d'opera
"Analisi Sensoriale"
Istituto Professionale di Stato "L. DI POPPA" per i servizi Commerciali Turistici e
Alberghieri, via Bamabei n°2 ,64100 Teramo
Istituto di Istruzione Superiore
Anno accademico 2007/'2008
"Igiene degli Alimenti"
Istituto Professionale di Stato "L. DI POPPA" per i servizi Commerciali Turistici e
Alberghieri, via Bamabei n°2 ,64100 Teramo
Anno accademico 2007/2fXJ8
"Analisi Sensoriale"
,
r
di lavoro
Dote
Principali a ttività e
responsabilità
di lavoro
Istituto Professionale di Stato per i servizi Commerciali Turistici e Alberghieri, L.
DI POPPA, via Bornobei n°2 ,64 l 00 Teramo
l 2/'2ffJ6 - 02/'2ffJ7
Collaboratore Tecnico professionale Tecnologo Alimentare
Marchi d'Areo- Strumenti per lo sviluppo dell 'occupazione nel settore
agroalimentare:
• Supporto ed assistenza tecnica. per l'adeguamento di un impianto
piloto per lo produzione di mozzarella. nel rispetto delle tradizioni
tecnologiche di trasformazione in condizioni igieniche adeguate.
Progettazione e realizzazione dell'impianto. ottimizzazione e
standardizzazione del processo di produzione, validazione del
processo di produzione e redazione del manuale interno di
produzione.
Italia Lavoro Spa, via Guidoboldo Del Monte 60, Roma
Società di Lavoro
Dote
l 2/'2ffJ6 - 02/'2ffJ7
responsabilità
di lavoro
Dote
responsabilità
di lavoro
Dote
responsabilità
di lavoro
Tipo di a ttività o settore
Dote
responsabilità
di lavoro
Federico Di Fabio
IV' ,/
Collaboratore Tecnico professionale Tecnologo Alimentare
Marchi d'Areo- Strumenti per lo sviluppo dell'occupazione nel settore
agroalimentare:
•
Redazione di un protocollo operativo per lo gestione dell'igiene
delle strutture. delle tecniche di allevamento e dei trattamenti
farmacologici delle api allevate: localizzazione dell'allevamento.
nutrizione artificiale. qualità e gestione dell'alveare
Obiettivo Lavoro Spa, via Guidoboldo Del Monte 60, Roma
Società di Lavoro
Anno accademico '2ffJ7/'XJJ8
Professore fino agli aventi diritto
Insegnamento in Scienze degli Alimenti
Istituto Professionale di Stato per i servizi Commerciali Turistici e Alberghieri. L.
DI POPPA. via Bornobei n°2Teromo.
Anno accademico '2ffJ6/'2ffJ7
Professore fino agli aventi diritto
Insegnamento in Scienze degli Alimenti
Istituto Professionale di Stato per i servizi Commerciali Turistici e Alberghieri, L.
DI POPPA. via Bornobei n°2 Teramo.
Anno accademico '2ffJ6/'2ffJ7
Attività di insegnamento. Contratto di Prestazione d'opero
"Laboratorio del gusto"
Istituto Professionale di Stato per i servizi Commerciali Turistici e Alberghieri. L.
DI POPPA, via Bornobei n°2 ,64100 Teramo
1
Date
responsabilità
12/2005 - 11 /2006
Compensi per l'esercizio di arti e professioni Cod. Min 1040
Campionamento di matrici agroalimentari per le attività di ricerca
microbiologica.
Isolamento e caratterizzazione fenotipica e genotipica dei lieviti autoctoni
responsabili della fermentazione alcolica nei mosti di uve Montepulciano
d'Abruzzo e Trebbiano d'Abruzzo.
Caratteristiche osmotolleranti dei lieviti vinari isolati durante la fermentazione
alcolica del mosto cotto utilizzato per la produzione di "Vino Cotto"; Prodotto
tipico tradizionale Abruzzese, inserito nella lista dei prodotti tipici italiani
(Repubblica Italiana 2000; 2003).
di lavoro
Dipartimento di Scienze degli Alimenti- Sede di Agraria- Università degli Studi
di Teramo P.zza Aldo Moro, 45 64100 Teramo
Il Dipartimento di Scienze degli Alimenti ha tre obbiettivi principali,
l'educazione di nuove generazioni di scienziati nel settore alimentare, la
ricerca e l'innovazione dei processi alimentari, il miglioramento della
sicurezza e funzionalità degli alimenti
Date
responsabilità
19/08/2002 - 31 Il 0/2002
01/11/2002- 31/10/2003
ol I 11 /2003 - 17/03/2004
19/04/2004 - 18/ l 0/2004
Contratto di collaborazione coordinata e continuativa in qualità di
•
•
•
•
•
di lavoro
Date
responsabilità
Federico Di Fabio
Armonizzazione e validazione dei metodi di prova per la ricerca
qualitativa e quantitativa dei microrganismi negli alimenti di origine
animale.
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e nutrizione, Chimica degli alimenti e bromatologia, Commercializzazione e
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produttivi e di trasformazione, Microbiologia e igiene applicata agli alimenti
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ISCED5A
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utente
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Bl
Utente
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sociali
Capacità di lavorare in gruppo attraverso l'esperienza maturata nel corso
delle varie esperienze professionali e lavorative
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Autorizzo il trattamento dei miei dati personali ai sensi del Decreto
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•
Firma
Federico Di Fabio
12 febbraio 2013
Federico Di Fabio
Allegato 1
Pubblicazioni e Poster
1.
2.
3.
4.
5.
6.
7.
8.
Rosanna Tofalo, Clemencia Chaves-Lopez, Federioo Di Fabio, Maria Schirone. Giovanna E. Felis. Sandra Torriani, Antonello
Paparella. Giovanna Suzzi Molecular identification and osmotolerant profile of wne yeasts that ferment a high sugar grape must.
/ntemational Joumal of Food Microbiology 2009. voi. 130, n<>3, pp. 179-187.
Di Fabio F. (2008). "Characterization Saccharomyces cerevisiae biodiversity and dynamics during Montepulciano d'Abruzzon.
Oral Communication. 13th Workshop on the Deve/opments in the lta/ian PhD Research on Food Science Technology and
Biotechnology, University of Turin, 10-12 September 2008.
Di Fabio F. (2007). "Characterization of yeasts for Montepulciano d'Abruzzo, Colline Teramane DOCG vinificationn. Poster
Communication. 12th Workshop on the Developments in the ltalian PhD Research on Food Science and Technology. Reggio
Calabria, ltaly, 12-14 September2007.
Tofalo R., Di Fabio F., Chaves-Lopez C., Piva A.• Torriani S., Suzzi G. (2007). "Characteristics of osmotolerant yeasts from "Vino
Cotto", a traditional wine produced in the Abruzzo region (ltaly)". Proceedings 8th lntemational symposium of Enology of
Bordeaux. Bordeaux. France, 25-27 giugno 2007.
Di Fabio F. (2006). "VVine from Abruzzo Region Eoology and Microbiology of Production". Poster Communication. 11th Workshop
on the Developments in the ltalian PhD Research on Food Science and Technology. Mosciano Sant'Angelo. ltaly, 27-29
September 2006.
Tofalo R., Di Fabio. F., Chaves-Lopez. C.. Piva A., Suzzi G. (2006). "Yeasts from traditional ltalian Cooked VVine". Proceedings
2Qth lntemational ICFMH Symposium - Food Micro 2006 - Bologna. ltaly, August 29- September 2, p.285
Tofalo R., Chaves-Lopez C., Di Fabio F.• Angrisani R., Suzzi G. (2006). "Detenninazione di Dekken:M3rettanomyoos nel vino oon
metodi roltura-indipendenti. Atti IV Qmv. Naz. AISSA, -Qualità e sostenibilttà delle produzioni agrarie, alimentari e forestali-. 5-6
dioombre 2006, Mosciano S. Angelo (TE). p.227-228.
V. Langella, P. Semprini. F. Di Fabio. B.Pasini, M.T. Falda. F. Panella, S. Calvarese (2003): Indagine preliminare sull'efficacia
dell'estratto di pompelmo nella lotta oontro la peste americana. Vet. lt. Anno XXXIX-47. Gennaio-Marzo 2003. pp 49-54.
Federioo Di Fabio
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ARTA Abruzzo 2-3 febbraio 201 O.
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soluzioni. Presso Università degli Studi di camerino, Centro Universitario di Ricerca per lo sviluppo e la gestione delle risorse
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Scheda di Fonnazione Personale Presso ARTA ABRUZZO: Fonnazione inerente il Sistema di gestione integrato.
Corso di Fonnazione: "La validazione dei metodi analitici: Metodi biologici, chimici e fisici". Organizzato da Agenzia Regionale
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Corso di Formazione: Eusoft®.Lab-LIMS End User. Presso ARTA Abruzzo- Dip.Prov. di L'Aquila 7 ottobre 2008
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garanzia di qualità e SINAL SistemA Nazionale per l'Accreditamento di Laboratori. ARTA Teramo 20-21ottobre2008.
Xlii-Workshop on the Developments in the ltalian PhD Research in Food Science and Technology-Alba (TO) 9-1 1 settembre
2008.
Xli-Workshop on the Developments in the ltalian PhD Research in Food Science and Technology-Reggio Galabria 12-14
settembre 2007.
Convegno: "Legionella un Killer Ambientale" presso ART.A. Abruzzo. Pescara 19/10/2006.
Xl-Workshop on the Developments in the ltalian PhD Research in Food Science and Technology-Mosciano Sant'Angelo 27-29
settembre 2006.
/
Attestato di partecipazione "Legionella un Killer ambientale, strategie di prevenzione e di intervento" 19-10-2006 promosso da
ARTA ABRUZZO.
Corso di Formazione: "Validazione e valutazione dei metodi di calcolo e dell'incertezza di misura in campo biologico". IZSAM
Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G.Gap:>rale" Teramo, 24-26 settembre 2003.
Corso di Formazione "Formazione Fonnatori". IZSAM "G.Caporale" Teramo, 26-29 luglio 2004.
Corso di Formazione "Il Sistema Qualità". IZSAM "G.Caporale" Teramo,13-15 settembre 2004 .
Corso di Lingua inglese Il livello. Centro Territoriale Pennanente per L'Istruzione e la Fonnazione in età adulta, Teramo
gennaio-maggio 2002.
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V
lntcrnational Journal o[ Food Microbiology 130 (2009) 179-187
Contents lists available at ScienceDirect
International journal of Food Microbiology
jou mal homepag e: www. elsevie r. com /loc ate/ijfood micro
f I \I.\ Il R
Molecular identification and osmotolerant profile of wine yeasts that ferment a high
sugar grape must
Rosanna Tofalo a. Clemencia Chaves-L6pez a. Federico Di Fabio a. Maria Schirone a. Giovanna E. Felis b .
Sandra Torriani b. Antonello Paparella a. Giovanna Suzzi a.*
' Dipartimenro di Scienze degli Alimenri, Universitd degli Studi di Teramo, Via C.R. Lerici I. 64023 Mosciano Sanr'Angelo. Teramo. rtaly
Diparrimtnto di Scienze, Tecnologie e Mercati della Vite e del Vino, Università degli Swdi di Verona. Via delle Pieve 70. 37029 San Ro1iano. Verona. lrnly
b
'
l\RTICLE
J NFO
Article history:
Rcceivcd 17 October 2008
Reccivcd in revised form 20 January 2009
Acceptcd 21 Janu.1ry 2009
Keywords:
Osmotoler,111ce
Yeasts
Ethanol tolcrance
Vino cotto
ABSTRACT
The objecrive of rhis srudy was ro cxamine the Saccharomyces and non-Saccharomyces yeast populations involved
in a spontancous fcrmcntation of a tfaditional high sugar must (Vino cotto) produccd in centrai ltaly. Molecular
identification of a tota I of 78 isolates was achieved by a combinati on of PCR-RFLP of the 5.85 ITS rRNA region and
sequencing of the DI /02 domain of the 265 rRNA gene. In addition. the isolates were differentiated by RAPD-PCR.
Only a restricted number of osmotoleranr yeasr species. i.e. Candida apicola. Candida zemplìnina and Zygosaccharomyces bailii, werc found throughout ali the fermentation process, while Snccliaromyces cerevisiae prevailed
after 15 days of fcrmentation. A physiological characterization of isolares was performed in relation to the
resistance to osmotic stress and ethanol concentration. The osmotolerant fcatu rcs of C. apicola. C. zemplinina and
Z. bai/ii were confirmed, while S. cerevisiae strains showed three patterns of growth in response to different
glucosc conccntrations (2%. 20%, 40%and 60%w/v). The ability of some C. apicola and C. zemplinina strains to
grow at 14%v/v ethanol is noteworthy. The find ing that some yeast biotypes with higher multiple stress tolerance
can persist in thc entire winemaking process suggests possible future candidates as starter for Vino cotto
production.
lt> 2009 Elsevier B.V. All rights reserved.
1. lntroduction
"Vino cotto" is a wine produced in the Marche and Abruzzo regions
(centrai Italy) according to traditional procedures that involve a
prolonged ferrnentation of cooked grape rnust. The flow-chart for the
traditional rnanu facture ofVino cotto is showed in Fig. I. Cooked rnust
derives frorn white grapes of the Trebbiano, Passerino or Moscato
cultivars and is obtained by direct heating of the rnust in copper
boilers unti! its volu me is reduced by 10-70%, according to the specific
process. During cooking, the must becornes dark and dense, and
reaching a very high sugar concentration (up to 55% w/v) (Di Mattia
et al., 2007: Piva et al.. 2008). Cooking is carried out below boiling
ternperatures (80-95 °C), and requires long processing times (up to
48 h). To start fermentation, fres h rnust is added to sterile cooked
must, at percentages that depend on the different manufacturing
practices. The indigenous yeasts of the added fresh must conduct the
alcoholic fermentation t hat proceeds slowly fo r about 40 days at room
temperature. Stuck or sluggish fermentations often occur during the
production of Vino cotto. as observed during the fermentation of
• Corrcsponding aulhor. Dipartimento di Scienze degli Alimenti, via C.R. lerici l.
54023 Mosciano S. Angelo (TEJ. ltaly. Tel.: +39 0861 266938; fax: +39 0861 266915.
E-mail address: [email protected] (G. Suzzi).
0168- 1605/S - sce front matter <O 2009 Elscvier B.V. Ali rights rese1vcd.
doi: t0.1016/j.ijfood micro.2009.0 t.024
Traditional Balsamic Vinegar, another ltalian traditional produtt made
with cooked grape must (Salieri and Giud ici. 2005, 2008; Salieri et al.,
2006). These problems derive from the exposure ofye ast cells to high
osmotic conditions of cooked must (hyperosmotic shock) that
determine a rapid loss of intracellular water (Hohmann, 2002),
followed by cytoskeleton collapse (Chowdhury et al., 1992), intracellular damage and subsequent arrest of growth. The majority of yeasts
requires a minimum water activity (aw) of 0.85 for their growth , and
requires only xerophilic yeasts can grow, at slow rate, at aw values
between 0.61 and 0.75 (Martorell et al.. 2005: Salieri and Giudici.
2008). In generai, osmotolerant yeasts are able to retain the ability to
synthesize glycerol as a compatible solute or osmoregulator, and some
yeasts even have active glycerol uptake pumps (Hohmann, 2002).
There are only few studies dealing on the yeast population involved
in the fermentation ofcooked grape musts. Recently, Solieri et al. (2006)
found a complex yeast population in Traditional Balsamic Vinegar, that
includes S. cerevisiae, severa! Zygosaccharomyces species, two species
belonging to the Hanseniasporo genus (Hanseniaspora osmophila and
Har1seniaspora valbyensis), and two Candida species (Candida stellata
and Candida lactis-condensi). Also in high sugar grape musts, such as
special wines from dried, botrytized grapes or late-harvest grapes,
osmotolerant non-Saccharomyces yeasts are commonly found (Mills
et al., 2002). Candida zemplinina has been recently identifìed as a
new osmotolerant and psychrotolerant yeast, fermenting sweet and
R. 'fofalo et al. I International )oumal of fòod Microbiology 130 (2009) 179-187
180
yeast species was possible (Quesada and Cenis, 1995; Torriani et al.,
1999; Bujdoso et al., 2001: Urso et al., 2008). From the results obtained in
the present srudy, it is anticipated that the traditional and regiondependent handling makes Vino cotto a wide genetic source of diverse
yeast species and strains suitable for following starter selection
programmes.
Fresh must from white grapes
Boiling (80-95°C uncil 30-70% reduction)
.o.
Cooling at 35°C
u
2. Materials and methods
Fresh must addition
u
2.1. Wine production
Fermentation (40-45 days at 25°C)
Cooked must was prepared according to Piva et al. (2008). White
grapes ofTrebbiano d'Abruzzo were removed from stalks and pressed.
The fresh Trebbiano must (FfM) (100 L) was concentrated in copper
boilers of 120 Lcapacity to about 70%. The masses were rapidly heated
to boiling point, then the temperature was set at 95 °C for the whole
process (up to 18 h).
Ageing in barre)
u
Bottling
Fig. t. Vino cotto flow chart.
2.2. Analydcal determinations
botrytized musts (Sipiczki, 2003}. Various authors have mentioned that
indigenous yeast species, such as Kloeckera apiculata, C. stellata and
Torolaspora delbroeckii, may have better abilìty than S. cerevisiae to grow
during fennentations conducted at high sugar concentrations (e.g.,
> 200 g/L) (Benda, 1982: Lafon-Lafourcade, 1983). Malacrinò etal. (2005)
reported that strains of osmosensitive species, such as S. cerevisiae, may
possess appreciable capability to overcome osmotic stress and to yield
ethanol by fermentation of grape musts with high sugar concentration
in winemaking conditions. lhese authors suggested to use these strains
as starter for winemaking of partially dried grapes.
Very little infonnation is available about the yeast microbiota ofVino
cotto, as previous researches on this peculiar wine have been focused
more on the technological aspects'°f the winemaking process. Therefore, the aims of this study were to monitor the Saccharomyces and nonSaccharomyces yeast populations during Vino cotto manufacturing and
to characterize the yeast isolates for their tolerance to osmotic stress and
ethanol. Initially, yeast isolates were presumptively identified using
morphological identification on Wallerstein Laboratory Nutrient (WLN)
agar and speciation of the new isolates was carried out by applying two
well-recognized yeast identification procedures based on ribosoma!
RNA (rRNA) gene analysis, i.e. restriction fragment Jength polymorphism
(RFLP} analysis of the JTS1-JTS2 reg_ion (Esteve-Zarzoso et al., 1999) and
sequencing of the genes encoding ttie D1/02 domain of the large (265)
subunit of rRNA (Kurtzman and Robnett, 1998). A combination of the
two procedures was required, since the fast and not expensive ITS-RFLP
approach is not as discriminatory as 265 rRNA gene sequence (Arias
et al., 2002). In addition, differentiation of the isolates at the subspecies
leve! was acquired by applying the random amplification of poJymorphic
DNA (RAPD)-PCR technique. This whole genome PCR sampling method
works by priming at arbitrary sites and results in strain-specific
fingerprints from which ditferentiation of strains belonging to different
Chemical analyses were conducted using the officiai EU Community methods for the analyses of wines (EEC, 1990). The tota)
polyphenols were determined by the colorimetric reaction with the
Folin pocalteau reagent as reported by Di Mattia et al. (2007).
2.3. Fermentation chamcteristics and yeast population dynamics
Vino cotto must (VCM), obtained by mixing 70% cooked must (CM)
added with 30% FfM, was spontaneously fermented by indigenous yeasts
in tanks (120 L) at room temperarure (approximately 18 °C) for 45 days.
Samples were taken from FfM, VCM and during the fennentation
process. For alJ samples, decimai dilutions in sterile physiological solution
(Nad 8.5 g/L) were made and 0.1 ml of each dilution was spread on three
different media: Wallerstein Laboratory Nutrient agar (WLN: Oxoid,
Milan, ltaly), Lysine-medium (LM: Oxoid) and Yeast Peptone Dextrose
agar (YPD; 1% w/v yeast extract, 2% w/v peptone, 2% w/v glucose and 2%
w/v agar). AII media were supplemented with chloramphenicol
(150 ppm}. Plates were incubated at 25 °C for 3-5 days. According to
Pallman et al. (2001 ), the WLN medium plating was used to monitor
yeast population diversity during fermentations. The colony moÌ'phologies are characteristic enough to identify the corresponding yeast's
genus and species. After counting, a tota] of 20 colonies of yeasts with
different colour and morphology was isolateci from WLN plates. The
isolates were purified by repetitive streaking on YPD and then stored
at -20 °C in YPD broth supplemented with glycerol (25% final
concentration).
2.4. Molecular identification of the isolates
Yeast cells were grown aerobically in YPD at 28 °C. DNA was
isolated according to Querol et al. (1992). The 5.8 internal transcribed
Tahle 1
Chemical composition of the musts and Vino cotto
Sample
Fresh Trebbiano must'
Cooked must
Vino cotto must11
Vino cottoe
Reducing sugars
CgM
Tartane acid
(g/L)
Total acidity
(g/L of tartaric acid)
172.5:t21.4
554.5±285
304.5±18.7
64.5±2.21
4.81±0.08
13.32±0.35
7.35±0.21
3.61 :t0.11
7.41±0.28
17.64±0.15
9.42±0.26
8.59±0.37
• Data are the means of 3 repetitions with the relative standard deviations.
b GAE: galic acid equivalent.
e Trebbiano d'Abruzzo must.
d Cooked must added with fresh must.
e Vino cotto must after 40 days of fermentation.
Volatile acidity
(g/L of acetic acid)
0.76±0.05
Ethanol (%)
pH
Total phenolics
(ppm GAEb)
15.35±0.27
3.08±0.03
2.75±0.02
2.94±0.02
3.03±0.02
348±12.34
1754±56.72
1200±31.21
569±16.28
R. Tofato e1 al./ ln1ernacional}o11rnal of Food Microbiology 130 (2009) 179-187
181
25
20
~
s
15
$...
o
u
IO
8
5
o
o
IO
20
30
40
Time (days)
Flg. 2. Fermentation kinetics of Vino cotto must. TI1e results are reported as mean of 3 repetitions; the coefficient of variability was lower than 10%.
'
spacer (ITS) rRNA region was amplified in a Bio-Rad thermocycler
(MyCycler, Bio-Rad Laboratories, Milan, ltaly) using ITSl and ITS4
primers as described previously (Esteve-Zarzoso et al., 1999). The
amplified DNA was digested with the restriction endonucleases Hinjl,
Cfo I, Hael/I and Mbol (Roche Diagnostics, Mannheim, Germany),
according to the supplier's instructions. The PCR products and their
corresponding restriction fragments were separated in 1.5% and 2%
agarose gels, respectively, in 1x TAE (40 mM Tris-acetate, 1 mM EDTA,
pH 8.2) buffer. After electrophoresis, gels were stained with ethidium
bromide, and documented by the Gel Doc 2000 (Bio-Rad, Milan, Italy).
2.5. Sequencing, sequence comparison and pllylogenetic analysis
Amplification of the 01 /D2 domain of the 265 rRNA gene was carried
out using Nlt and NL4 primers as described by Kurtzman and Robnett
(1998). PCR products were gel-purified with GFX™ PCR DNA and Gel
Band Purification Kit (Amersham Biosciences AB, Uppsala, Sweden),
according to the manufacturer's instructions and delivered to Centro
Ricerca Interdipartimentale Biotecnologie Innovative (Padua University,
Padua, Italy) for sequencing. The sequences obtained in FASTA format
were compared with those depo;ited in GenBank DNA database (http://
www.ncbi.nlm.nih.gov/) using the basic Blast search tools (AJtschult
et al., 1997). The D1/D2 sequences analysed in this study were deposited
in the EMBL Nucleotide sequence Database under the accession
numbers C. apicola (FM201316); C. zemplinina (FM201317).
(FM201318); S. cerevisiae (FM201319); Z bailii (FM201320).
Alignment of sequences obtained for the isolates and reference
sequences retrieved from the database was performed with CLUSTALX
(Thompson et al., 1997). Webcutter 2.0 programme, available at http://
rna.lundberg.gu.se/cutter2/, was used to predict restriction sites in ITS
molecular sequences.
2.6. RAPD-PCR analysis
The M13 primer (5'-GAG GGT GGC GGTTCT-3') was used in RAPDPCR amplifications. Reactions were carried out in 25 ~IL reaction mix
containing 2.5 µL of 10x PCR buffer (Invitrogen), 1.5 mM MgCl 2,
200 pM of each dNTP, 20 pmol of primer, 1 U of Taq polymerase and
20 ng of extracted DNA. RAPD-PCR products were visualized on a 1.5%
agarose gel after staining with ethidium bromide and acquired using
the Gel Doc 2000. Conversion, normalization and analysis of RAPDPCR profiles were carried out using Fingerprinting II Informatix™
Software (Bio-Rad). Similarities of bands were analysed using Pearson
coeffìcient, and correlation coefficients were calculated by the
unweightecl pair group method with arithmetic averages (UPGMA).
2. 7. Frequency percentage analysis
Colonies randomly collecred from plates at the highest dilution give a
high probability to pick up strains belonging to the dominant species
(Pulvirenti et al., 2004). The method proposed by Salieri et al. (2006) was
applied to study the species distribution in the analysed samples. lt was
considered how many times each species was detected in the samples,
without considering the strain number belonging to the species. In this
way it was obtained the number of positive samples to each species and
the corresponding frequency, defìned as the number of samples positive
to a species divided by the tota! number of samples expressed in
percentage.
2.8. Sugar and etl1anol tolerance
Overnight cultures of yeasts were centrifuged (3000 xg) at 4 °C for
10 min and cells were washed twice with potassium phosphate buffer
(pH 7.4). Then, cells were suspended in the same buffer to give a
concentration of about 106 CFU/mL An aliquot of 20 ~li.. was inoculated
into Bioscreen e (M-Medical S.r.1 .. ltaly) plates containing 180 µL ofYPD
broth with 150 ppm of chloramphenicol and supplemented with O
(control), 8, 14, or 20% v/v ethanol or 2, 20, 40, 60% w/v glucose and
incubateci at 25 ·e for 100 h. The increase in cell number was determined
by measuring the optical density (O.O.) of cultures at 600 nm.
Table2
Viable counts during Vino cotto production
Counts (log CFU/ml) on"
YPD
WLN
Fresh Trebbiano must
Vino cotto must (VCM)
VCM during fermentalion
Vino colto
6.11 ±0.12
630±0.24
8.20±0.15
5.15±0.11
6.25±0.35
6.55±0.20
8.20±0.25
5.19±0.17
LM
6.30±0.20
6.51 ±fl.45
8.31 ±0.20
5.26±0.25
YPD: yeast peptone dextrose agar. WLN: Wallerstein Laboratory Nutrient agar; LM,
Lysine medium.
• The results are the means of 3 repetitions with the res~ective standard deviations.
182
R. Tofalo et al./ lntentational joumal of Food Microbiology 130 (2009) 179-187
Table 3
Sizes of the 5.85
Pro file
r
Il
lii
IV
rrs rRNA gene amplicons and the restriction fragments of the yeast isolates
No. of
isolates
PCR product
(bp)
Cfo I
Hae lii
Hin/I
31
21
15
11 ..
834
509
473
796
345+365+100
207+187+77
208+110+77
342+232+143
318+231 +171 +130
400+100
448
700+90
367+128
210+103
215f-215
329+220
Restriction fragments (bp) wilh:
Spedes
Mbol
290+90
S. cerevisiae
e apicola
e zempliruna
z baUii
ldentification of these isolates was based on sequence analysis of the D1/D2 domain that revealed 100% identity with the species Z bailii.
a The RFLP pattern of these isolates did not match the data reported in literature (Sol ieri cl ,11., 2006: Esteve-Zarzoso et al .. 1999).
In arder to evaluate yeast growth, the values were analysed over time
according to the Gompertz equation modified by Zwietering et al.
(1990):
subjected to Student's t test to identify significant differences between
yeast species using STATISTICA package (StatSoft Inc., Tulsa, OK, USA).
3. Results
y =A · exp {- exp [ (µmA ·e) ·(À -
t) + 1] }
3.1. Chemicat detenninacions
where y is O.O. value at time t (h), A represents the maximum O.O.
(when t-=), J.lmax is the maximum specific growth rate (as h- 1), and
À is the lag time (h) far O.D. increase. For the modelling with
Gompertz equatìon, means of three replicates and two repetitions
~ere used. In all the cases, the variability coefficient of raw data (cell
load as O.O.) was <5%. The data relative to the growth kinetics were
Vino cotto represents a very peculiar environment, as can be
observed from the chemical parameters of the samples analysed
(Table 1). The fermentation of Vino cotto must started after 6 days
from the fresh Trebbiano must addition to cooked must, and was
carried out slowly far 45 days (Fig. 2), due to the high content of sugars
(304.5 g/L) and polyphenols ( 1200 ppm). At the end of the
% similarily
so
55
60
65
70
75
80
85
90
95
IO
fl, F2. l'J, H. N. F6.
n.~1.1:11111. 1-· /YJ
1191
C. apicola
l'IY.1. 01161-:fll,t.':fl
ru!l.l:'.!.i!I
t'478, 1'480", 1'481,
t'.W
F#.1
l'IS, 1119.110,
('!l. f66.
•"2""·
t'288
Fl9.1'1,F11. I-"
..
t'lll.1'11.1'11.M.'.l'U
F.Ml,HI
FJ1,1:.!JJ
S. cerevisiae
t:Jl:l~
00
Jn
f..Y.S
t:.!.6.!
00
Uli!
~.l,W.IJ.1J.!:ill
J
lii
1'17 '. I ":2. F?.l, l.\t, t:l&/
fl:!1!
f.'<>l.F.1•J
Fl~.
} C. zemplini11a
F491
l'.lt>.~.1'479
E.!-lH~
FI S. I' Il)'. f~(l;!.t'.f76
1'21, F:>T. t:JM
ra1.•·.w.
} Z. hai/ii
1/9'1, l'~83
Flg. 3. Banding patterns were clustered using the UPGMA. with correlation levels expressed as percentage values of Pearson correlation coefficients. Yeasts isolated at different times
are indicated <1s follows: normai character, Fresh Trebbiano must: italics, Vino cotto must; underlined. Vino cotto must after 15 days; bold, Vino cotto must after 45 days of
fermentation. Species validation of representative strains ("') of each cluster included 01 /02 domain sequence analysis.
R. Tofalo et al./ lnternarional]ournal of Food Microbiology 130 (2009) 179-187
100
90
V.
80
B
:'3
70
...o
60
o
.!a
Il)
~
50
="
40
e
30
'é
Il)
~
20
IO
o
Vino couo mu.st
a
S. cerevi.fit1e
Vino cnllo must
aftcr I S days
.,. C. apico/a
Vino collo musi
after 45 day1.
• C. :.emplinina o Z bclilii
fig. 4. Evolution of yeasts during Vino cotto production.
fermentation, the average of alcoholic content was 153.5 g/L and the
reducing sugars 64.50 g/L, confirming previous observations (Di
Mattia et al., 2007).
' 3.2. Enumeration of yeast populations
To study the succession of yeast populations during the Vino cotto
manufacturing, yeasts were detected from FfM, VCM, during and at
the end of the ferrnentation process. WLN agar proved extremely
useful in the isolation and initial presumptive identification of
different yeast species fr9m the samples (Table 2). At the beginning
of the spontaneous fermentation, Vino cotto must exhibited a total
viable count on YPD medium of 6.30 log CFU/ml and a maximum
number of 8.2 log CFU/mL during fermentation, while at the late
stages of the process the viable cells decreased to 5.15 log CFU/ml
Counts on WLN and on LM showed a similar trend to that detected on
YPD medium.
3.3. ldendfication and molecular characterization of yeasts strains [rom
Vino cotto
A tota I of 78 yeasts were isolated from the samples collected during
Vino cotto production, after boiling and addition of FTM, in arder to
focus on the most probably osmotolerant species. Molecular characterization was carried out using a qJmbination of different techniques to
identify and gain information origenomic relatedness among isolates.
Identification was performed by amplifying the 5.8-ITS rRNA, and
subsequent restriction analysis was carried out with Cfo I, Hae III and
Hin/ I endonucleases (Esteve-Zarzoso et al., 1999). The results of PCRRFLP analysis of the 5.8-ITS rRNA are reported in Table 3. Four different
profiles. named from I to IV, were generated, and were compared to the
data set previously described (Esteve-Zarzoso et al., 1999; Villa-Carvajal
et al., 2006; de Llanos Frutos et al.. 2004). Thirty-three isolates showed
the restriction pro file named I, coresponding to the pro file ofS. cerevisiae
CBS 1171T; therefore, these isolates were assigned to this species. The
isolates with profile II showed a pattern similar to the one reported in
literature for Candida apicola (de Llanos Frutos et al .. 2004). Profile lii
corresponded to the pattern described for the species C. zemplinina and
C stellata by Sipiczki (2003): after the additional restriction with MboI.
the isolates generated two fragments of 290 and 90 bp that are typical of
C. zemplinina. Sequence analysis of lTS1-5.8S rDNA-ITS2 region of
selected isolates confirmed the presence and positions of predicted
restriction sites. Isolates with profile IV showed a particular RFLP pattern
that did not match the data reported in literature (Salieri et al., 2006;
Esteve~Zarzoso et al .. 1999).
To achieve a certain identification of the yeasts, the 01/02 domain
of the 265 rRNA gene from two isolates of each 5.85-ITS profile was
sequenced and compared with those available in the EMBL nucleotide
183
sequence database. Ali the sequences obtained displayed similarity
values ranging from 99 to 100% to reference sequences, confirming the
identity of isolates with profiles I, Il, and III as S. cerevisiae, C. apicola
and C. zemplinina, respectively, and allowing the inclusion of the
isolates with pratile IV imo the species Zygosaccharomyces bailii (data
not shown}.
lsolates were further characterized genomically with RAPD-PCR. to
obtain information on the genomic relatedness of isolates and to
determine the probable number of the different yeast strains present
in the samples. Indeed, RAPD-PCR has proved to be an informative
method suitable for the study of a large number of strains in a short
time and has been used successfully for identificati on and intraspecific
differentiation of Saccharomyces and non-Saccharomyces yeast species
(Quesada and Cenis, 1995~ Torriani et al .. 1999; Bujdoso et al.. 2001:
Urso et al., 2008).
The dendrogram based on the numerical analyses of generated
digitized M13 PCR fingerprints is shown in Fig. 3. Four main clusters
were obtained, which corresponded to the species previously identified.
Considering 95% similarity as the arbitrary threshold to define biotypes,
it can be easily observed that 6 biotypes of C. apicola, 13 of S. cerevisiae, 6
of C. zemplinina, and 4 of Z bailii were distinguishable. Moreover, as the
isolates were collected at different stages of fermentation, the RAPD-PCR
analysis allowed the observation of microbiota evolution during
winemaking.
As regards S. cerevisiae, an ampie genetic polymorphism was
observed: three groups can be easily discerned with relatively low
genomic relatedness (less then 75%). In particular, subduster Icontained
almost ali of the strains isolated from fresh Trebbiano must at the
beginning of the fermentation, while subclusters li and Ili contained
almost ali the isolates from Vino cotto must after 15 days of
fermentation. lnterestingly, F25, F66, and F288 isolates, constituting a
single biotype in cluster I, were isolated in FfM (F25) and VCM after
45 days of ferrnentation, thus indicating that this strain was able to
persist during ali the winemaking process. Further, different strains
were present at the same stage of fermentation, indicating a multistrain
process.
Considering the other three species which were found, a remarkable biodiversity was observed for C. zemplinina, C. apicola, and Z.
bailii strains. c. apicola strains were present at ali stages of
ferrnentation, but apparently there was no persistence of a single
Table4
Growth parameters of the yeast isolates from Vino cotto at different glucose concentrations
Glucose
(% w/v)
No.or
isolates
Species
2
6"'/6•*
e apico/a
e zempllnina
6/6
13/13
4/4
20
40
60
0
6/6
6/6
13/13
4/4
6/6
6f6
13/13
4/4
2/6
1/6
0/13
0/4
Growth parameters
/Jmu. (h-1)
Am.\X (0.D)
~
{h)
1.53±0.19a
1.44±0.06b
1.65±0.17c
1.67±0.0Sc
0.05±0.0la
0.06±0.0la
0.04 ±0.02ab
0.05±0.0la
2.66±0.77a
1.21 ±0.4tbc
6.76± 13.54ab
430:t0.98ac
S. cerevisiae
Z bailii
1.35±0.0Sa
1.47±0.0Sb
1.45±0.llb
135±0.0Sa
0.09:!:0.0ta
0.12±0.0lb
0.12:t0.02b
0.10±0.0la
t.16±0.lla
1.63±0.tSb
5.59:t13.2Sabc
2.57±0.SOc
C. apicola
1.11 ±O.Ola
0.02±0.0ta
0.03:!:0.0lb
0.03:!:0.0lb
0.03±0.0lb
0.008±0.002a
O.OlSb
6.24±0.84a
6.72±0.75a
9.34±3.SOb
8.26:!:0.72b
t5.13±233a
9.35b
5. cerevisiae
l.. bailii
e apicola
e zemplinina
e zemplinina
S. cerevistae
z bailfi
C. apicola
C. zempllnina
S. cerevisiae
Z. bailii
1.15±0.0Sa
1.37±0.06b
1.33±0.07 b
0.60±0.12a
0.44b
Results are the means of 3 replicates for 2 repetitions; standard deviations are àiso
indicated; A111..,. maximum abs; /Jm..,.: maximum growth rate: i\: length of lag. -: no
growth.
Means within a column in the same block with different letters are significantly
different (P<0.05).
*: positive isolates. *"': total isolates.
R. Tofalo et al./ lntemational)oumal of Food Microbiology 130 (2009) 179-187
184
genotype. On the contrary, and similarly to what previously observed
for S. cerevisiae, a biotype of C. zemplinina (isolates F36, F365, and
F479) was detected in ali phases of production.
lnterestingly, the predominant species during fermentation were
very different from those observed in FTM, before its addition to CM:
according to the counts obtained in WLN medium, in FfM the
S. cerevisiae yeast population was only 32%, of tota! population while
68% was formed by non-Saccharomyces wine yeasts belonging to the
genera Hanseniaspom, Kloeckera, Metschnikowia and Candida (data not
showni The evolution of the species S. cerevisiae, C apicola and Z bailii
during Vino cotto production was determined also by plate count
(Fig. 4): in Vino cotto must. before the start of fermentation, only the
osmotolerant species C. apicola, and Z bailii were isolated, whereas
yeasts belonging to the species S. cerevisiae and other non-Saccharomyces wine yeasts were not found. During fermentation S. cerevisiae was
the prevalent yeast, whereas at the end only few strains, belonging to
this species, were isolated. C. apicola was the dominant species at the
start and at the end of fermentation (Fig. 4). Solieri et al. (2006)
suggested that yeasts present in cooked must depend on the stochastic
contamination events that occur after cooking. In Vino cotto manufacturing the addition of fresh must contributes to previde the yeast
inoculum. Although S. cerevisiae was not isolated after Vino cotto must
blending, it performed the fermentation. Vino cotto appeared a stressful
environment for the survival of the majority of the primary contaminant
yeasts, so few species can grow and at the end C. apicola was the
prevailing species, followed by Z bailii, C. zemplinina and S. cerevisiae.
3.4. Phenotypes of isolates
Ali the isolates obtained in the present study, belonging to the
species C. zemplinina, C. apicola, Z bailii and S. cerevisiae, were tested
for growth at various concentrations of glucose (2, 20, 40, 60% w/v)
and ethanol (O, 8, 14, 20% v/v). These analyses were performed on ali
the isolates, in arder to verify if the 95% similarity level could be
S. cerevisiae type A
Ethanol
Glucose
'
1.8
...... 1,6
ee
~
e.o
1.8
. f=
.........
1.4
1.2
l
l ~:!
as
11.
[J
.r,
<
40
20
l,2
l
0.8
0.6
0.4
0.2
8e
•
o
o
-e
[J
: di
0,2
1.6
~
lii
•
ee
o
••
••
••a
•
•
~ 0.4
o
111111111
60
Time (hours)
80
1.4
o
100
o
20
40
60
Time (hours)
80
IOO
S. cerevisiae type B
................
.•r~:mac=n
:•
1,8
ee 1,4
8
~
1,2
• ••
•
~ 0,6 - . • •
.o
"'
0.4 • •o
:ac
0.2
t.I
I
u
e 0,8
•
o
ae
1.4
8
1,2
'e
tJ
I
as
0,8
~
0,6
<
0,4
-e
[J
[J
20
1,6
e.o
e
[J
[J
[J
.o
<(
o
1,8
~
1,6
0,2
40
60
lime (hoursl
o
100
80
o
20
40
60
80
100
80
100
Time (hours)
s. cerevisiae type e
ee
o
o
~
t.I
u
e
e:I
-e
~
1.8
1.8
1.6
ee
1,4
1,2
,/
I
0,8
•••·'
••
0.6
~ 0.4
•
••••
20
40
60
lime (hours)
1.2
0.8
100
....
0.6
0.4
0,2
o
80
. .··
'e
d
•
•
8
"ue
-eo
.o
"'
<
1,6
1,4
o
20
40
60
lime (hours)
Fig. s. Pattems of S. cerevisiae growth in responses to dìfferent glucose and ethanol concentrations. Glucose (w/v): ( t) 2%; (•) 20%; (O) 40%; (x) 60%. Ethanol (v/v):
8%: (---) 14%; {-) 20%.
<-=-> 0%; (x)
R. Tofalo et aL / lnternational }ournal of Food Microbiology 130 (2009) 179-187
effectively chosen as threshold for strain delineation. In other words, if
isolates presumptively constituting a single biotype display the same
characteristics, then they can be considered a single strain. otherwise
they have to be considered different strains even ìf genomic similarity
is high, and the 95% chosen as threshold must be critically reeva Iuated and increased.
Data relative to O.O. of three replications for each isolate were
analysed according to the modified Gompertz equation. The predicted
curves fitted well with the experimental points and their regression
coefficients ranged between 0.95 and 0.98. A perfect correlation
between genomic relatedness and phenotypic traits was observed.
therefore we validated the 95% similarity of RAPD-PCR profile as
threshold to delineate biotypes or strains.
Ali the strains belonging to the three osmotolerant species
C. zemplinina. C. apicola, and Z. bailii grew in the media supplemented
with 2. 20, 40% w/v glucose, and most of them grew faster in the
medium with 20% glucose than in that with 2% (Table 4). Glucose at
60% inhibited many isolates and only two strains of C. apicola and one
of C. zemplinina showed some growth.
As shown in Fig. 5, S. cerevisiae strains had three patterns of growth
in response to different glucose concentrations (2%, 20%, 40% w/v),
that we called growth types A, Band C. Type A strains grew better in
' the medium added with 2% glucose, with a decreased growth in media
with higher sugar concentrations. Preferred condition for growth was
medium with 20% glucose for type Band type Cstrains, but they could
be differentiated on the basis of the second preferred condition:
medium with 2% added glucose for type B strains and medium with
40% glucose for type e strains.
C. apicola, C. zemplinina. and Z. bailii strains displayed growth
curves similar to S. cerevisiae type B strains, i.e. best growth in
medium with 20% glucose, and 2% sugar concentration as second
preferred condition. Table 4 shows reports the average of the lag
phase. the growth rate and maximum celi biomass of the strains,
growing at different glucose concentrations. As glucose concentration
increased, a generai decrease in Amax was observed forali the isolates.
C. apicola and C. zemplinina. characterized by a minor Amax overall at
2% and 40% glucose, had a shorter lag phase than Z bailii and S.
cerevisiae, and few isolates showed some growth even in the presence
of 60% glucose. On the contrary, S. cerevisiae and Z. bailii had always
the major lag phase extension and very low O.O. values at 60% glucose
that could not be modelled with the Gompertz equation. C. apicola
Table S
Growth parameters oFyeast isolates from Vino cotto at different ethanol concentrations
Ethanol
(% v/v)
No. of
isolates
Species
o
6•/6**
6/6
13/13
4/4
8
14
20
0
Growth parameters
AmCIJI (O.O)
11miuth- 1)
X(h)
C.apicola
e zemplinina
S. cerevisiae
Z. bailil
1.38±0.09a
1.32±0.tOa
1.33±0.09a
1.56±0.0Sb
0.10±0.03a
0.14±0.0la
0.17±0.0tb
O.lt±0.02a
1.49±0.SOa
3.07±0.25b
3.36±0.18b
7.62:tl.42c
6/6
6/6
13/13
4/4
C. apicota
Czemplinlna
5. cerevisiae
Z bai/ii
0.93±0.0Sa
0.98±0.0Sa
t.01 ±0.07a
0.93:!:0.0Sa
0.05±0.0ta
0.06±0.0la
0.08±0.0tb
0.06±0.0ta
3.44±0.32a
4.15:!:0.51b
3.76±0.2Dab
4.58±0.41b
2/6
2/6
8/13
0/11
C. apicola
C. zemplinina
S. cerevisiae
Z bai/ii
0.50±0.09a
0.54±0.0Sa
0.91 :tO.llb
0.07±0.0ta
0.07±0.0la
0.07±0.02a
3.70±0.17a
8.74±0.21b
10.33±1.34c
0/6
0/6
0/13
0/4
C. apicola
C. zemplinina
S. cerevisiae
Z bai/ii
Results are the means of 3 replicates for 2 repetitions: standard deviations are also
indicated; Am.lX maximum abs; µ.11,,x: maximum growth raie;.\: length oflag. -: no growth.
Means within a column in the same block with different letters are significantly
dilTerent (P<0.05).
*: positive isolates. **: total isolates.
185
and C. zemplinina, growing at high glucose concentrations, showed to
possess more chances that S. cerevisiae and Z. bai/ii to grow in VCM.
Considering ethanol resistance, as expected, ali the S. cerevisiae
strains grew at 8% ethanol, and severa! isolates also at 14% ethanol, as
shown in Fig. 5. C. apicola strains developed at 8% ethanol, with strainspecific growth curves; only three isolates grew slowly at 14%. A
similar behaviour was observed far C. zemplinina, and three strains
grew at 14% ethanol. As a generai trend, ethanol affected yeast growth
by increasing the lag phase, decreasing the growth rate and biomass
yield (Amax) (Table 5). Ali isolates had a similar µmax decrease starting
at 8% ethanol, while a relevant decrease of Amax was observed at 14%
ethanol for ali isolates except S. cerevisiae.
4. Discussion
Ouring Vino cotto production, must cooking determines the
concentration of naturally occurring chemicals in must among which
are sugars, adds, polyphenols, metal ions, and the formation of Maillard
reaction products (Piva et al., 2008). The main objective ofthis study was
to isolate, identify and characterize the predominant indigenous yeast
species and strains during Vino cotto production. Only four species were
identified: S. cerevisiae, C. apicola, e zemplinina and Z. bailii.
As expected, C. apicola, C. zemplinina and Z. bailii showed osmotolerant characteristics, but also some strains of the species S. cerevisiae,
thai!' is not generally considered an osmotolerant yeast, showed this
characteristic. In fact, among the strains of S. cerevisiae isolateci from
Vino cotto, the sugar consumption was different with three different
growth kinetics (Fig. 5) in the presence of 2, 20, 40, and 60% glucose.
Among the studied species, a generai decline in O.O. was observed as the
glucose increased from 200 to 600 g/L; such a trend might be attributed
to a slower proliferation ofyeast cells due to the osmotic etfectgenerated
by high glucose concentrations (Thomas and lngledew, 1990; Zhao and
Un, 2003 ). Strains of the species Z. bailii are known to possess unusual
physiological characteristics, such as resistance to weak-acid preservatives and extreme osmotolerance up to 4 Mglucose (72% w/v) (Martorell
et al., 2007). In this study, the Z bailii strains from Vino cotto exhibited
lower growth rates at high sugar concentrations than C. apicola and C.
zemplinina, and had growth kinetics like those of S. cerevisiae type B
strains, that grew faster at 20% glucose than at 2%. As regards C
zemplinina, Sipiczki (2003) reported that isolates from sweet botrytized
wines were able to grow at 50% glucose and to have some growth even in
the presence of 60% glucose. Similar physiological characteristics were
detected in some of our strains of C. zemplinina, showing also the same
osmotolerant growth kinetics of C. apicola and S. cerevisiae type B.
Among the non-Saccharomyces wine yeasts, K. apiculata and C. stellata
have been reported to possess a better ability than S. cerevisiae to grow
during fermentations conducted in a chemically defined grape juice
medium with high sugar concentrations of more than 200 g/L
(Charoenchai et al.. 1998}. Our data, obtained in YPD medium, showed
that C. apicola and C. zemplinina can grow Jike or better than S. cerevisiae
in media with high sugar contents (Table 4). but they were not able to
dominate fermentation (Fig. 4). In fact, S. cerevisiae had the higher A
values up to 40 days. This finding might be attributed to elevated and
combined osmotic effects, due to the presence of high glucose
concentration and the accumulation of ethanol during ferrnentation of
Vino cotto up to 15-16% (v/v).
As expected, S. cerevisiae was the most ethanol-tolerant species, with
some differences depending on the strain: in fact, only eight strains were
able to grow at 14% of ethanol with different but high cell yield. On the
other hand, Z bailii was the most sensitive species. lt is well known (Fleet
and Heard, 1993: Boulton et al., 1996: Cocolin et al., 2000) that nonSaccharomyces wine yeasts are less tolerant to ethanol than Sacchafomyces. The major target of ethanol is the plasma membrane, altering the
membrane organization and permeability (Alexandre et al., 1994;
D'Amore et al., 1990), and consequently inhibiting glucose transport
and fermentation rate under enologica! conditions (Ansanay-Galeote
~
186
R. 1bfalo et al. I lntemational )oumal of Food Microbiology 130 (2009) 179-187
et al., 2001 ). Ethanol stress represents toxicity through intracellular R05
generation in addition ~o deleterious damage to celi membrane and
functional proteins (Costa et al.. 1997). Many ethanol protectants such as
trehalose and various plasma membrane components, including ergosterol, phosphatidylinositol, stearic acid, palmitic acid, as well as
phospholipid palmitoleic acid, in addition to inositol, praline and
tryptophan, might result in an even higher resistance to ethanol stress
(Mansure et al., 1997; Inoue et al., 2000; Furukawa et al., 2004: Takagì
et al., 2005, 2007; Lei et al., 2007: Hirasawa et al., 2007).
As regards the composition and dynamics of yeast populations
during the production of Vino cotto the 5.85-ITS restriction profile of
ali Z bailii isolates was not in agreement with the data reported in
literature. Nevertheless, they were clearly identified by 01 /02 domain
sequencing. An unusual polymorphism for 5.8S-IT5 DNA has already
been reported for other species. Fernandez-Espinar et al. (2000) and
Suzzi et al. (2006) described an atypical polymorphism for 5.85-IT5
DNA for 5. cerevisiae strains. 5equence divergence which can be
observed in IT5 regions· is usually higher than in ribosomal RNA
encoding genes; in fact, Salieri et al. (2006, 2008) reported similar
results far Z. rouxii and found a new putative species highly relateci to
Z. rouxii on the basis of IT5-region. Therefore, the sequence heterogeneity displayed by the Z bailii isolates from Vino cotto may indicate
ttaxonomic peculiarity, whose investigation was out of the aim of this
paper and will be deepened in a future study.
RAPD-PCR fingerprints of the isolates showed a different genetic
homogeneity within the four species (29 profiles for 78 isolates) but,
most importantly, they allowed to follow the dynamics of the
population, showing that only few biotypes were present during ali
the process: in particular, Of'llY one S. cerevisiae strain was shown to be
present either in fresh Trebbiano must and at the end of fermentation.
with an intermediate osmotolerant behaviour. The relatively reduced
biodiversity in terms of yeast species and strains could be explained if we
consider that the selective pressure exerted by the environmental
conditions encountered by microbiétl cells during Vino cotto production,
such as high osmotic pressure and high concentration of some must
compounds, account far the consolidated dominance of selected species
of yeasts and strains.
In conclusion, the application of a multiphasic approach has lead
towards a better understanding of the microbial ecology of this
particular fermentation process. Indeed, the combination of microbiological, physiological and genetic analyses allowed to profile
population dynamics and to characterize single strains associateci with
the fermentation of cooked musts.. Nevertheless, further analysis are
required in future studies to discove·r the species and strains that give the
major contribution to the production and final quality ofVino cotto.
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l3 1h Workshop on thc Dei•elopments in the ltalian PhD Research on Food Science Technology and
Bioteclmology, University of Turin, 10-12 Scptember 2008
Saccharomyces cerevisiae biodiversity and dynamics during Montepulciano
·
"
d'Abruzzo
Federico Di Fabio ([email protected])
Dept. Food Science, University ofTeramo, Mosciano Sant' Angelo, ltaly
Tutors: Prof. M. Pisante, Prof.ssa G. Suzzi
In the present PhD work the resulls obtained from experimental winemaking trials with three aulochthonous
Saccharomyces cerevisiae strains (S437, S441, S615) previously characterized as potential starters for
Montepulciano d'Abruzzo "Colline Teramane DOCG" wine are reported. The musts were crushed and directly
inoculated with a final concentrati on of 106 cells/ml of 48h pure cultures of selected yeasts and of a commerciai
strain, as a control. The musts were obtained by grapes harvested in three different homogeneous areas of
Teramo (GIS technology). All the musts were added with 80 ppm S02 and fermentations were followed by
sugar consumption. The microbial dynamics were determined by PCR-DGGE. Microsatellite PCR was used for
discriminate between wine starter strains. The starter S615 resulted to perform a faster fermentation than other
yeasts. The miscrosalellite analyses indicated that strain S615 possesses a better capacity to dominate native
yeast population than other tested starters. The selected yeasts were able to give wines with a locai character,
being well adapted to specific fermentation and winemaking conditions.
'
Biodiversità e dinamiche dei ceppi Saccharomyces cerevisiae durante la produzione del
vino Montepulciano d'Abruzzo
Nel presente lavoro sono riportati i risultati delle vinificazioni sperimentali ottenuti attraverso l'impiego di
Saccharomyces cerevisiae (S437, S441, S615) preventivamente caratterizzati in base alle loro caratteristiche
enologiche.. I mosti sono stati pigiati ed inoculati con i ceppi selezionati, fino ad avere una concentrazione di 106
cellule/ml. Uno starter commerciale è stato impiegato come controllo. E' stata seguita la cinetica di
fermentazione dei singoli vini e le dinamiche dei ceppi di S. cerevisiae mediante PCR-DGGE. Il ceppo S615 ha
dominato le tre fermentazioni ottenute dalle tre aree. I lieviti selezioni hanno prodotto vini con caratteri di
tipicità, in quanto. meglio adattati alle condizioni di vinificazione.
Keywords: Saccharomyces cerevisiae, winemaking, yeast.,
1. Introduction
Traditionally, wine fermentation has been carried out in a spontaneous way by indigenous yeasts present on the
grapes when harvested, or introduced from the equipment and celiar during lhe vinification process (Mortimer
and Polsinelli, 1999; Ciani et al. 2004). In the past years, strains of Saccharomyces cerevisiae have been selected
for their oenological properties and are used as starters in winemaking to carry out alcoholic fermentation with
success. During conducted must fermentation indigenous yeasts are also present, belonging to the genera
Kloeckera, Hanseniaspora, Candida, Pichia and other non-Saccharomyces that grow in the early stages of
fermentation but also belonging lo native S.cerevisiae which are able to complete the process. The natural and
starter strains of S. cerevisiae, involved in fermentation, play an important part in the characteristics of the final
product. In fact the composition and sensory quality of the resulting wine are due to the diversity of S. cerevisiae
strains and their dominance in fermentation (La Jeune et al. 2006). lt should be noted that the use of the selected
S. cerevisiae starter cultures might not necessarily prevent the growth and metabolic activily of some species
of,e.g. Hanseniaspora and Candida (Heard, Fleet, 1985), and they can be found in later stages as well, or even al
the end of wine fermentations. Jemec and Raspor (2005) studied the influence of the initial S. cerevisiae cell
concentration on yeast population dynamics and fermentation kinetics in pure and composite cullures with nonSaccharomyces yeast (H. uvarum, Candida stellata and M. pulcherrima) and found that high S.cerevisiae
concentration produced wine with the best sensorial properties. Even if commerciai S. cerevisiae strains could be
inoculated in grape must in order to establish a high population and accomplish a well-controlled must
fermentation, many wine-researchers and winemakers prefer the use of locai, autochthonous, selecled strains of
S. cerevisiae as starters (Martini, Vaughan-Martini, 1990). In fact active dried yeasts seem lo reduce the
biodiversity of strains that perform natural fermentation, and , as a consequence of this, to reduce the resultirig
wine complexity (Frezier, Dubourdieu, 1991). The native S. cerevisiae are better acclimated to micro-area
conditions of the wine production region (Martini, Vaughan-Martini, 1990) and can more easily dominate on the
natural flora (La Jeune et al. 2006). However the problems of assuring the dominance of a specific strain during
fermentation and of controlling its presence during ali the process remain. Molecular methodologies can assist
winemakers to collect rapid information regarding the composition and dynamics of yeast species and strain
154
13lh Workshop on the Developments in the ltalian PhD Research on Food Science Tech110/ogy and
Biotechnology, University of Turin, 10-12 September 2008
prevalence (Urso et al. 2008). Final aim of PhD lhesis was monitored the biodiversity of yeast populalions of
Montepulciano d'Abruzzo musts inoculated with autochthonous S. cerevisiae selected in the defined vineyards
of Teramo area. The ability of. the inoculated strains to dominate the natural yeast populations and conduct
fermentation was evaluated by microsatellite PCR.
2. Materials and Methods
2.1 Bioclimatic and geomorphologic analysis.
The climatic database, provided by the "Centro Agrometeorologico Regionale" (ARSSACAR), has been
elaborated by geostatistic techniques (Castrignanò, 1996) for the spatialization of the bioclimatic indexes of
Winkler. The recognition of the homogeneous zones has been carried out using. the following thematic maps as
layers: elevation ; exposure; inclination; illumination; wetness index; NDVI; thermic index of Winkler.
'
2.2 Yeasts and culture conditions.
The following strains were used in this study: S437, S441, S615 (Collection of Department Food Science,
Teramo, Italy), S. cerevisiae DBVPG 6171T (Industriai Yeasts Collection of Perugia, Italy) Saccharomyces
bayanus DBVPG 6173T, Saccharomyces paradoxus DBVPG 6411T,Saccharomyces pastorianus DBVPG
6047T, lssatchenkia terricola DBVPG 7161T, Brettanomyces bruxellensis MUCL 27700T (BCCM/MUCL
Agro-Industrial Fungi and Yeast Collection, Université Catholique de Louvain, Louvain-La-Neuve, Belgium)
and Debaryomyces hansenii CBS 767T, (Centraalbureau voor Schimmelcultures, Baarn, The Netherlands). Ali
yeast strains were preserved on YEPD-agar (1 % yeast ex.tract, 2% glucose, 2% peptone and 2% agar) slants,
stored at 4°C and subcultured every 3 months.
2.3 Vinifications and sampling.
Three strains of S. cerevisiae (S437, S441, S615), isolated from Montepulciano d'Abruzzo musts as reported by
Suzzi et al. (2008) and selected on basis of their oenological features, were inoculateci (final concentration of 106
cells/ml) in three Montepulciano musts, obtained by grapes harvested in three different areas of Teramo. A
commerciai wine y~ast preparation of S. cerevisiae (L404) was used as a contro!. Then, twelve fermentations
were carried out in a winery of Teramo area (ltaly) according to the usual "Montepulciano d'Abruzzo"
winemaking procedure. Sulfur dioxide (80 ppm) was added at the beginning of fermentations. Samples were
aseptically taken in duplicate and collected at the beg.inning, after 24h and at the end of alcoholic fermentation.
The fermentation were fojlowed by sugar consumption.
2.4 Saccharomyces spp. isolation.
Each sample was plated out on WLN medium (Ox.oid). Plates were incubated at 28°C for 5 days for colony
development. After viable counts, only the isolates that presumptively belonged to the species S. cerevisiae,
according to the colonies colour on WLN agar, were purified by repetitive streaking on YPD agar. The purified
isolates were maintained at-80°C in YEPD broth containing 20% (v/v) glycerol.
2.5 DNA extraction.
Two methods were use<f'to obtained genomic DNA. Total genomic DNA of yeast strains, was extracted and
purified from 7-ml cultures according to the method of Querol et al. (1992). For the PCR-DGGE analyses, DNA
was extracted directly from grapes and musts using the PowerSoil DNA Isolation Kit (MoBio Laboratories). For
this purpose, a quantity of 10 ml of each sample, in duplicate, was centrifuged to collected more cells. The DNA
was then extract according PowerSoil DNA Isolation Kit manufacture' s protocol (MoBio, Laboratories).
Quantification of total DNA was achieved using a VersaFluor fluorimeter and a Fluorescent DNA Quantitation
Kit (Biorad, USA).
2.6 PCR amplification.
PCR-DGGE was canied out on the DNA isolated from grape must samples and bulk cell suspensions. The DlD2 region of 26S rRNA gene was amplified by PCR using with primer NLl-GC (5'-(GC)
CATATCAATAAGCGGAGGAAAAG-3') and the reverse primer LS2 (5'-ATICCCAAACAACTCGACTC3'). A GC-clamp which is needed to avoid duplex DNA artifacts was added to primer NLl (Cocolin et al.,
2000). PCRs were performed in GeneAmp PCR System 9700 (Applied Biosystem) programmed as follows:
initial denaturation at 95°C for 5 min, followed by 30 cycles of 1 min at 94 °C for denaturing, 45s at 52°C for
annealing, 1 min at 72 for synthesis, and a final extension step of 7 min at 72 °C.
v'
,,
2.7 DGGE analysis.
PCR products obtained from bulk celi suspension and directly from grapes musts, were analyzed by DOGE by
using a Bio-Rad Dcode apparatus and the procedure as described by Cocolin et al. (2000). PCR samples were
applied directly onto 8% (wt/vol) polyacrylamide gels in lx TAE running buffer and a denaturing ~adient from
20 to 60% of urea and formamide. The electrophoresis was performed at a constant voltage of 120 V for 6h with
155
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L3'11 Workshop on tlH.: Developments in the ltalia11 PhD Research on Food Science Teclznology and
Bioteclznology, University of Turin, I0-12 September 2008
I
I
a conslanl temperature of 60°C. After electrophoresis the gels wcrc stained with ethidiu m bromide for 15 min,
rinsed for 20 min in distillcd water and photographed under UV transillumination. Normalization of Lhe gel was
performed by using band ladders of reference strains DNA in two lanes in ali gcls. Therefore, DNA originating
from: pure cultures of type strains w~s mixed in equa! volumes of same concentration and used as a reference
ladder, after the corrcsponding PCR amplicons were positioned in a DGGE gel with the appropriate gradient.
I
2.8 DNA sequencing.
For the sequencing of DGGE bands, bands of interest were excised from the gels with a sterile biade, nùxed with
50 µI of sterile water, and incubated overnight at 4 °C to allow the DNA of the bands to diffuse out of the
polyacrylamide gels blocks. Two microliters of this solution were used to reamplify the PCR product with NLlLS2 (withoul GC-clamp) primers pair. Then, PCR products were purified by use of the GFXTM PCR DNA and
Gel Band Purification Kit (Amersham Biosciences AB, Uppsala, Sweden) following the manufactures
instructions, and delivered to C.R.LB.L (Padua University, Italy) for sequencing. Searches in the GenBank
database were performed with the BLAST program (Altschul et al. 1997) to deternùne the closest known
relatives of DI region of the 26S rRNA sequences obtained.
2.9 Molecula r identification S. cerevisiae strains.
ldentification of the isolates was ascertained by PCR-RFLP of ITS 1-5.8S rDNA-ITS2 region as described by
Esteve-Zarzoso et al. ( 1999) and Tofalo et al. (2007).
2.10 Microsatellite PCR fingerprinting.
'
Strains of the S. cerevisiae were analysed by microsatellite PCR fingerprinting, as described by Vaudano,
Garcia-Moruno (2008).
3. Results and discussion
;I
·I
1.
The geostatistic elaboration of the bioclimatic data indexes of Winkler and Huglin have allowed to map the
climatia conditions of the zone under investigation for the period corresponding to the vine growth. Three main
zones have been identified which delimitate the zone of the Montepulciano d'Abruzzo "Colline Teramane
DOCG" production. Zone I and 2 cover from the coast to the hills, while zone 3 cover the highest quotes to
1000 m a.s.I.. Zone 3 shows the lowest vegetative index and energy potential, while zone 2 shows the best
energy conditions (Fig. 1). Starter S. cerevisiae strains (S437, S441, S615), were inoculated in musts, obtained
by grapes harvested in three homogeneous areas of Teramo, as
reported in Methods. The commercial strain S. cerevisiae L404 was
used as a contro!. All fermentations were carried out at enviromental
temperature.
Fig.l. Tue three zones identified within Montepulciano d'Abruzzo "Colline
Teramane" DOCG production territory.
•
Zolll! l §i Z"AlOè 2 •
lor.~ 3
The average must temperature during the fermentations, determined at
middle of the day, was 18±3 (°C). At the end of fermentation, the
wine contained residuai sugars ranging from 4.2 to 6.4 g r' with pH
values between 3.35-3.50 and tota! acidity 4.8-5.2 as tartaric acid gr' ·
The first sensory evaluation of the wines did not evidence off flavours. As showed in Figure 2 the tested strains
fennented must in similar way during the first 8 days but then the strains S615 and the contro! strain 404 were
able to fermenl faster and at the end they grcw better than other ones.
S615
S441
S437
lA04
y= - l,L35x+
21,67
y=-l,020x+
21,98
y=-0,964x +
21,61
y=-l,l38x+
21,87
Su gar Consum ption
+S615
• L404
S437
I::
· S441
IO
16
12
18
20
'
daysol lermentat lon
Fig.2 Linear regression of sugar consumption by yeasts.
156
13'" Workshop on the Developments in the ltalian PhD Research on Food Science Technology and
Biotechnology, University of Turin, I 0-12 Seplember 2008
Informalions on yeasl population dynamic during vini (!calions were obtained extracting DNA directly from
samples, followed by the PCR-DGGE of amplicons obtained wilh the primers LS2/NL! -GC (Cocolin et al.
2000). Figure 3 shows the DOGE fingerprints obtained by differences were observed in the DOGE profìles when
the triplicate · samples were analysed (data·· not shown). A few selected bands were excised from the
polyacrylamide gels and further subjected to DNA sequence analysis. The closest relatives, the per cent identities
are reported in Tab. l The fingerprints obtained by direcl sample analysis consisted of 8 different bands (Fig.3).
In generai, S. cerevisiae (band 6) was the dominant species lhroughoul all fermentation in ali the 12 wines. Also
bands 2, 3 and 5 were visible throughout ali fermentation and their closest relalives were Hanseniaspora spp. ,
Cryptococcus magnus and Candida stellata, respectively. The closesl relative corresponding to band 8 was
Aspergillus sydowii and appeared only at the start of fermentation in the must inoculated by the strain S441. No
significant differences among the different fermentations were observed in term of species composition (data not
shown).
The use of PCR-DGGE of nucleic acids from musts and wines had a great potential for the profiling of both the
yeast diversity and changes occurring during the wi ne fermentalion. In fact the capacity of the starters to
dominate the must fermentation is of interest in the wine-making process.
'
Fig.3 PCR-DGGE profiles (20 to 60% denaturant gradient) of
grape must samples during winemaking (Zone 3). The ladder (M)
consisted of (A) S. cerevisiae, (B) S. bayanus, (C) H.uvarum, (D)
D.hansenii (E) S. paradoxus.
Purification and sequencing of faints bands (w) did not prove to
be successful.
Tab.1 Sequencing results from the bands cut from the yeast denaturing gradient gel electrophoresis (DGGE) gels.
%
Accessi on
Band* Closest relative
ldentitv
number
* Band numbers are indicated in Fig.3
AB294409.1
1
Eremothecium ashbyi 92 %
92%
2
Hanseniaspora spp.
AY953954
Cryptococcus
97%
EF 126360. l
3
;:.
maRnus
4
Funga! endophyte
EF420077. l
97%
A tota! of 240 isolates were obtained from
5
Candida stellata
AY60761
97%
the twelve vinifications. After identification
Saccharomyces
EU386754. l
6
99%
only Saccharomyces
by PCR-RFLP,
cerevisiae
cereviszae strains were subjecled to
Penicillium
PCR
fingerprinting
as
microsatellite
7
DQ658168.l
97%
canescens
described
by
Vaudano,Garcia-Moruno,
Aspergillus sydowii ·
98%
8
AM883163. l
(2008). Microsatellite sequences are DNA
sequences made of short tandem repeated motifs (one lo six bases) which show variation in the number of
repeats. These sequences are present in eukaryote and prokaryote and virus genomes. They are often used as
genetic markers in studies of genetic mapping or population genetics (Legras et al. 2005). The three starter
strains tested were clearly distinguishable by their microsatellite profiles (Fig.4). In this way, it was simulated
the competition occurring in vinification processes between the autochthonous starter strain and the indigenous
ones. The miscrosatellite analyses indicated that strain S615 possesses a better capacity to dominate native yeast
population than the other tested starters in ali vinifications. In fac t its presence was determined through ali three
must fermentations. Of interest was the presence of wild Saccharomyces during the must fermentations
performed by the commercia! strain L404 and the autochthonous starter strains S437, S441, whereas starter
strain S615 resul ted lo be alone. Probably strain S615 was better adapted to the chemical and physical conditions
of must during the final phases of fermentation including low nutrient supply and high alcohol concentrations. In
addition sensoria( analyses indicated that the starter strain S6 15 developed flavours and aromas improving the
'
bouquet of Montepulciano d' Abruzzo wine (data not shown).
157
13th
Workshop on lhe Developments in the ltalian PfzD Researclz 011 Food Science Technology and
Bioteclznology, Uni versity of Turin, 10-1 2 Scptember 2008
<l.~6,.J
8.9,)0 .ll 12
~~-
.
13 14 15 1617 18
.
B M
Fig. 4. Electrophoretic pattems of strains isolated
from 5404 vinification (l-2-3-9-10-12-1 3-1 7);
S437 vinilìcatio n (4-5-6), S 615 vinificatio n (8-11 14- 15); S441(7- I 6).
4.Conclusion
'
The data obtained in the last year of my PhD can contribute to the knowledge of yeast population and species
dynamics present in musts during fermentation. by using a multiphasic approach to highlight the ecology of
yeasts during vinification of Montepulciano d 'Abruzzo grapes. Culture-indipendent methods were used to profile
the yeasts communities during vinification. Moreover, the ability to take over the fermentation of the
autochthonous S. cerevisiae and starter culture was determinated also by PCR microsatellite No differences
among the yeast dynamics during the twelve fermentations were found. The results proved a nd confirmed
clearly the importance of used of selected autochthonous yeast strains to conduct the alcoholic fermentation and
to provide the typical aroma and quality of this wine.
5. References
Altschult S.F., Maddelen T.L., Schaffer A., Zhang J., Miller W., Lipman, D.J., 1997. Gapped BLAST a nd PSIBLAST: a new generation of protein database search programs. Nucleic Acids Resarch, 25: 3389-3402.
Castrignanò A., 1996. Utilità della geostatistica in agraria. Bollettino della SISS, 7: 87-92
Ciani M., Mannazzu I., Marinangeli P., Clementi F., Martini A., 2004. Contribution of winery-resident
Saccharomyces cerevisiae strains to spontaneous grape must fermentation Antonie van Leeuw 85: 159-164.
Cocolin L., Bisson 'L.F., Millis D.A., 2000. Direct profiling of the yeast dynamic in wine fermentation. FEMS
Microbio!. Lett. l 89: 81 -87
Esteve-Zarzoso B., Belloch C., Uruburu F., Querol A., 1999. Ide ntification of yeasts by RFLP analysis of the
5.8S rRNA gene and the two ribosoma! internal transcribed spacers. lnt.J. Syst. Bacterio!. 49: 329-237.
Frezier V., Dubordieu D., 1992. Ecology of yeast strain Saccharomyces cerevisiae during spontaneous
fermentation in a Bordeaux winery. Am. J. Enol. Viticult. 43: 375-380.
Heard G.M., Fleet G.H., 1985. Growth of natural yeast flora during the ferme ntations of inocula ted wines. Appl.
Environ. Microbio!. 50: 727-728.
Jemec K.P., RaspcSt P., 2005. Initial Saccharomyces cerevisiae conce ntration in single or composite cultures
dictates bioprocess kine tics. Food Microbio!. 22: 293- 300
La Jeune C., Enry C., Demuyter C., Lollier M ., (2006) Evolution of the population of Saccharomyces cerevisiae
from grape to wine in a spontaneous fermentation. Food Microbio!. 23: 709-716
Legras J. L., Ruh O., Didier M., Karst F., 2005.Selection of hypervariable microsatellite loci for the
characterization of Saccharomyces cerevisiae strains. lntern. J. of Food Microbio!. 102: 3- 83.
Martini A., Vaughan-Martini A., 1990. Grape must fermentation: past and present. In Yeasts technology (Eds
Spencer J. F. T. D ., Spencer, M.) pp. 105-123. Berlin, SpringerVerlag.
Mortimer R., Polsine lli M., 1999. On the origins of wine yeast.. Ann. Res. Microbio!. 150: 199-204.
Querol A., Barrio E., Huerta T ., Ramon D. 1992. Molecular monitoring of wine fermentations conducted by
active dry yeast strains. Appl. Environ. Microbio!., 58: 2948-2953.
Suzzi G., Tofalo R., Chaves-Lopez C., Ramazzotti S., Stagnari F., Di Fabio F., E. Barca, Castrig nanò A., Pisante
M ., 2008. Isola tion and selectio n of Saccharomyces cerevisiae from Montepulciano d 'Abruzzo "Colline
Teramane" areas with different microclimatic characte ristics. Proc. OIV 2008, 15-20 June, Verona, Italy.
Tofalo R., Torriani S., Chaves-L6pez C., Martuscelli M., Paparella A., Suzzi G. 2007. A survey of
Saccharomyces populations associated with wine fer mentations from the Apulia region (South Italy). Ann.,
Microbio!. 57 (4): 545-552.
Urso R., Rantsiou K., Dolci P., Rolle L., Comi G., Cocolin L., 2008. Yeast biodive rsity and dynamic during
sweet wine production as de termined by molecular methods. FEMS Yeast Res: I-IO.
Vaudano E., Garcia-Moruno E., 2008. Discrimination of Saccharomyces cerevisiae wine strains using
microsatellite multiplex PCR and band p attern analysis. Food Microbio!. 25: 56-64.
158
Jz111 Workshop on the Developments in the Ita/ian PhD Research on Food Science Techno/ogy and
Biotechnology, University ofReggio Calabria, 12-14 September, 2007
Characterization of yeasts for Montepukiano d'Abruzzo
"Colline Teramane DOCG" vinification
Dept. Food Science and Technology, University ofTeramo, Mosciano Sant'Angelo, Italy
Tutors: Prof. M. Pisante, Prof.ssa G. Suzzi
In tbe present paper the characterization of yeasts isolated during spontaneous fermentations of musts from the
wine-producing areas of Montepulciano d'Abruzzo "Colline Teramane DOCG"is reported. Among 400 different
isolates, 35 strains, belonging to the species Saccharomyces cerevisiae were selected as starters for
Montepulciano fermentation, for their useful oenological properties, such as fermentative power, aromatic
compounds production, S02 and ethanol resistance . Tue diversity of natural S. cerevisiae population was related
to two vineyards ofMontepulciano under different agronomie practices.
Caratterizzazione e selezione dei lieviti per il Montepulciano d' Abruzzò
"Colline Tramane DOCG"
Nel. presente lavoro sono stati caratterizzati ceppi di lievito isolati da fermentazioni naturali nell'area di
produzione del vino Montepulciano d'Abruzzo "Colline Tramane DOCG" . . Tra i 400 isolati, sono stati
' selezionati 33 ceppi, che sulla base delle principali caratteristiche di interesse enologico, saranno utilizzati come
starter nella produzione del vino. La selezione è avvenuta uti.Iizzando mosti di uva provenienti da vigneti con
t:. cÌifferèiiti pratiche agronomiche.
;
,
Kèy.words: authochtonous yeasts, biodiversity, spontaneous fermenta ti on, Montepulciano d'Abruzzo Colline
teramane "DOCG".
1. lntroduction
'~d!''./r
~r pi.aio activities in this PhD thesis were:
~<~·
···
.
Identification o f yeasts isolated from grape musts obtained in vineyards with different agronomie
practices in wine-producing area ofMontepulciano d'Abruzzo "Colline Teramane DOCG".
Differentiation and characterization of the yeasts isolated for their oenological traits.
· . i~.Materials and methods
16:.::1.
•
Thç1grapes were harvested in 14 homogeneous environmental conditions but under different soil tillage systems,
·cutting up of spontaneous vegetation and conventional tillage. The samples were stored at 10°C unti!
}eiWèntations (max 3 hours). Microvinifications of 14 different musts (juice with skins) were carried out with
anèl ~~ithout sulphur di~xide ( lOOppro) and fermentation was monitored by measurements of C02 loss and
:,oxpressed as percentage. Yeasts were isolated at different times of fermentation in WL agar and colonies were
pre~~.'~P according the different colours as suggested by Cavazza et al. 1992.. The isolates were characterized
fo1lo~g tbe usual criteria for spore formation and morphological and physiological test (Bamett et al., 2000).
lden~cation o~ the ~solates was ascertained by PCR-RF~P of 5.8-ITS region of the rRNA gene (Esteve-Zarvoso
et all'\1999). D1gesttons of PCR method, perfonned w1th Haelll, Hinf I and Cfof were used to confirm the
presen~~ of S.cerevisiae isolates.
~e ~~t~, belonging to Saccharomyces sensu stricto, isolated during initial, middle and at the end of alcoholic
ennentations, were studied for some biotechnological characteristics (fermentation activity, ethanol production,
suiph).!:.dioxide and hydrogen sul fide production, ~-glucosidase activity).
3· Résults and discussion
~:1 Spo~tancous fermentations
~gure ;l:..~hows the ferme ntation kinetics of two different musts with and without S02, as reported in the legend.
~en~t. the fermentation of the musts M and M+, obtained from vineyards cutting up of spontaneous
faster than that of must P, obtained from vineyards cutting up of conventional tillage. These
difli ~\l~ge~ted that yeasts with different growth capacity were present in the two musts and probably a
et:eQ ,d1str1bution of the indigenous yeast species occurred.
~~ql,l was
389
11
12' Workshop on the Developments in the /talian PhD Research on Food Science Technology and
Biotechnology, University of Reggio Calabria, 12-14 September, 2007
Figure 1. Thc ferrnentation kinetics of two different musts with and without S02
M)Grape musts obtained from vincyards
cutting up of spontaneous vegetation;
M+) Grape musts added with S02 obtained
from vineyards cutt ing up ofspontaneous
vegetation;
P)Grape musts obtained from vineyards
cutting up of conventional tillage;
P+) Grape musts added with S02 obtaincd
from vineyards cutting up of conventional
tillage.
20
15
-M
-M+
•
p
5
- - P+
o
6
11
16
21
26
31
36
days
3.2 Yeasts iden tifi cation
Among 400 yeasts isolated on WL agar, only the isolates that presumpti vely belonged to the species s.
cerevisiae acco rdi ng to the colonies colour on WL ancl lo ascospore formation were further stuclied: 56 isolates
from musts M and M+ and 40 from P and P+. Yeasts identification performed by PCR-RFLP, ev iclenced that the
isolates belonged lo Saccharomyces cerevisiae, Pichia fermentans and Hanseniaspora uvarum, as reported in
table I.
Table 1. Lengths ofthe PCR products and the restriction fragments obtai ned for yeasts isolated during wine ferme ntation.
Il
lii
Number of
strains
Species
88
6
2
S.cerevisiae
H.11vmw11
Pichia èrmenta11s
Amplificd
880
750
450
Rcstriction fragments
Co l
365+365
3 15+ 305+ 105
170+ 100
Hae lii
320+ 180+ 150
750
350+80
Hin I
365+ 155
350+200+180
260+200
3.2 Oenological characteristics
Ln orcler to select Saccharomyces cerevisiae useful for Montepulciano vinification, the strains possessing the
highest fenve ntation power at two clays (62 > 2 g/ 100 ml) and at the end of fermentation (6 15 >1 1 g/100 ml)
were further studiecl. As reportecl in table 2, only 35 strains possessecl th is useful oenological characteristic. Ln
addition the selection was performed also for I-I2S and 50 2 formation, aromatic compound production and ~
glucosidase activity
n°strains
~2 >
2e:C0 2/1 OOml
~t s > 11 gCO~/ I OOml
Sum 6.2+6.1;
Table 2. Distribution of high fermentation powcr in S. cerevisiae strains.
·
43
39
35
The 35 selected strains were inoculated in must with di fferent amounts of S02 (50 and 80 ppm) and with 50 g/I
of glucose in order to detect strains resistant to 502 and ab le to growth in an high gravity must. Ali the tested
strains had a 6 2 higher than 2 g/ I00 ml of must aclcled with 50 ppm 502 and 22 wi th ~O ppm S0 2. The presence
of glucose selectecl ònly three strains. At the end of fermentation twelve strains had a 6 15 higher than.11 g/100
ml of must aclded with 50 ppm S02 and 4 with 80 ppm. Many stra ins showed a good fenn entation performance
with glucose. Only one strain produced traces of H2S, whereas the others produced medium or high quantities.
O n thc bas is of thcsc rcsu lts fiv e strains werc choscn and they will be used as starters for scaling up Montepulciano wincmaking in the
Vintage 2007 .
4. References
Barnet JA, Payne RW, and Yarrow D (2000) Yeast: Characteristics and Identification. Third ed Cambrige
University Press Cambrige, pp. I 150.
Cavazza A, Grando MS, Zini C (1992) Rilevazione della flora microbica di mosti e vini. Vignevini. 9: 17-20. h
Esteve-Zarzoso B, Belloch C, Uruburn F, ami Querol A (1999) /dentification ofycast by RPFL analysis-oft .~
5.8S rRNA gene ancl the two ribosoma! internal trascribed spacer. /nternational Journal of Systematic
Bacteriology 49: 329-337.
390
11.75
"CENO 2007"
sth Intemational Enology Symposium
Bòrdeaux June, 25, 26 and 27, 2007
CHARACTERISTICS OF OSMOTOLERANT YEASTS FROM "VINO COTTO",
A TRADITIONAL WINE PRODUCED IN THE ABRUZW REGION (ITALY)
R. TOFAL0 1, F. DI FABI0 1, C. CHAVES-LOPEZ1, A. PIVA1, S. TORRIANI2 , G.
suzz1 1
1
Department of Scienze degli Alimenti, University of Teramo
C.R. Lerici, 1, 64023 Mosciano S. Angelo (TE), Italy.
2
Department Scientifico e Tecnologico, University ofVerona
Strada Le Grazie 15, 37134 Verona, Italy.
· e.mail: [email protected]
'
(11.!!i''r''.
.
'
"Vino cotto" is a ·typical sweet wfue produced from cooked musts by a prolonged
ferm.entatfon. This wine is obtained by clirect heating of must in copper boilers until its
volume is reduced of 10-60 %, and it beéomes dark and dense. ·wnd autochthonous
yeasts perform the alcoholic fermentation tbat proceed slowly for about 40 days at room
te~perature. Tue must considered in this study was obtained from Trebbiano cultivar;
after cooking the must characteristics were as follow: sugar content of about 55 %, pH
2. 72, total acidity 17.6 g/l. A total of 78 yeasts were isolated during fennentation from
cooked must samples added or not with 802 and identified by PCR"".RFLP analysis of the
region spanning the internal transcribed spacers (5.8S-ITS rRNA). Tue restriction
analysis showe.d four fragment proftle groups that were identified as Saccharomyces
cerevisiae (31 isolates), Candida apicola (22), Candida zemplinina (16) and
Zygosaccharomyces bailii (9). The prevalence of these yeasts was confirmed by DGGE
of PCR products of a region of 26$ -rDNA obtained from the total DNA extracted
directly from the cooked must samples. An intraspeci:fic differentiation of the isolates
was also conducted by RAPD-PCR withprimer Ml3. Because ofthe detection ofyeast
species usually considered as osmotolerant, in particular C. zemplinina, C. apicola and
Z. bailii, the isolates were tested for the ability to grow in a synthetic medium added
with increasing .concentrations of glucose and ethanol. Ali the isolates developed in
presence of 2, 20 and 40 % glucose; most of them grew faster with 20 % glu~ose than
with 2 %; 60 % glucose was inhibitory for many isolates and only four isolates of C.
apicola and one C. zemplinina grew poorly. As expected, ali the S. cerevisiae isolates
grew with 8% ethanol, and several isolates with 14 % ethanol, whereas only few
Saccharomyces exhibited scarce growth with 20 % ethanol. Candida apicola isolates
developed only witb 8 % ethanol, even if with different growth kinetics; only two C.
apicola isolates grew slowly with 14 % ethanol and showed poor growth with 20%
ethanol. Our results underline that chemico-physical conditions of cooked must (high
sugar level, high acidity and polyphenol content) can select a particular biota of
osmotolerant yeasts. In particular, the yeasts more tolerant to high sugar concentrations
differed from those that grew better at high alcohol concentrations, confirming that the
response of yeast cells to hyperosmotic shock, stimulated by the two stress factors,
follows different mechanisms.
177
11
l l 1 Workshop on the Developments in the Italian PhD Research in Food Science and Technology
University ofTeramo, Mosciano Sant' Angelo, 27-29 September, 2006
Wine from Abruzzo Region
Eèology and Microbiology of Producti9n
Dept. Food Science and Technology, University ofTeramo, Mosciano Sant' Angelo, Italy
Tutor: Prof. M. Pisante, Prof.ssa G.Suzzi
This PhD research project is aimed to characterize the yeast isolated during spontaneous fermentations of musts
from Montepulciano d'Abruzzo grapes grown in experimental vineyard and to characterize indigenous yeasts
isolated from musts used for production of cooked wine, a typical alcoholic beverage of Abruzzo region.
Vini Abruzzesi:
Ecologia e Microbiologia delle produzioni
L'obiettivo del progetto di dottorato è rivolto alla valutazione delle biodiversità dei lieviti isolati durante la
fermentazione spontanea di mosti di uve Montepulciano d'Abruzzo provenienti da vigneti sperimentali, e dei
lieviti isolati da mosti d'uva utilizzati per la preparazione del vino cotto, prodotto tipico Abruzzese.
1. Introduction
Every vineyard's soil will have different characteristics. The ability to optumze winegrape production
necessitates an understanding the factors that influence grape quality and viticultura! production systems. The
first objective ofthis PhD thesis is to identify the amount ofvl).fiability in soil microbial biodiversity related with
different environmental conditions and site-specific vineyards management on yeast biodiversity in
Montepulciano d'Abruzzo. The aim is to examine soil health in the vineyard by monitoring the population
dynamics and diversity of the soil microbial community under different microclirnate conditions and vineyard
cultura! practice§, with an emphasis on microbial populations capable of suppressing soil borne disease. Soil
health can be seen as implying "ecosystem sustainability, diversity, functional connectedness, aod resilience in
response lo a disturbance or stress"(Benitez et al., 2005; Franchini et al., 2006). The composition of the
vineyard yeast biota can vary according to the ~ limatic conditions, the grape variety and the vinification
techoology. Since the. unique flavour of each wine is the result of multivariate interactions between the
geography, climate, cultivar, vineyard management and oenology, in recent years, much work has been carried
out on the ecology of wine yeasts, in order to ascertain the existence of strains typical of one ecosystem and also
to carry out selection programmes of the yeasts representative of each wine-producing zone (Frezier and
Dubordieu, 1992; Romano et al., 2003). The quantitative production of secondary aromatic compounds of
fermentation is of great interest to enhance the varietal character of the wine and optimize winemaking, as well
as the enzymatic activity of yeasts, such as esterases (Rosi and Bertuccioli, 1989), P- glucosidase (Rosi, et a(
1994), proteinase (Ubeda et al., 1998) and pectic enzymes (Gainvors et al., 1994). Some ofthese strains may be
responsible for givingjhe wine the unique characteristics from the region of its production (Pretorius, 2000). The
natural yeast biota see·ms to be made up of relatively small, genetically isolated yeast subpopulations that can be
distinguished by both their genetic markers and their pbenotypic characteristics (Nadal et al., 1996). This study
was undertaken to evaluate the genetic and biotechnolog ical characteristics of the indigenous Saccharomyces
sensu stricto populations from musts of different areas of the Abruzzo region. The representative yeast biotypes,
that can be individuated, would be useful as inoculants in vinifications carried out in that specific oenological
area.
2. PhD Thesis Objectives and Milestones
Within the overall objective this PhD project can be subdivided into the following activities according to the
Gantt diagram given in Table 1:
1. Bibliographical research on climatic and agronomie conduction in the vineyard and on yeast taxonomy and
biotechnology.
2. Soil and clirnate characterization, vineyards management and wine-growing production.
3. Montepulciano d'Abruzzo vinification. The grapes will be harvested in different environmental conditions
under two soil tillage systems, cutting up of spontaneous vegetation and conventional tillage. The samples
will be stored at l 0°C unti I fermentations and rnicrovinifications of the juice plus skins or not, will be
carried out with and without sulphur dioxide. Yeasts will be isolated at different times of fermentation, (i)
0% (ii) 50% (iii) I 00% sugar depletion, and the wines obtained will be characterised for aromatic secondary
compouhd production.
501
I I1" Workshop on the Developments in the l talian PhD Research in Food Science and Technology
University ofTeramo, Mosciano Sant' Angelo, 27-29 September, 2006
4.
Yeasts belonging to the species Saccharomyces sensu stricto will be characterised for the major enologica!
trai ts, such as ethanol production, secondary compound formation, S02, H2S, metabolism of organic acids
'
growth capacity, resistance of antimicrobial compounds (Vincenzini et al., 2005).
5. Cooked wine vinification. The samples will be obtained from wineries located in different areas of Abruzzo·
the mu"sts will be fermented in laboratory, at room temperature wi th and without S02. At different times'
serial diluited samples will be seeded onto plates for strain isolation, and isolates will identified and studied
for technological traits.
6. Molecular identification and typing yeast: identification of the isolates will be ascertained by PCR-RFLP of
5.8S-ITS region of the rRNA gene and species-specific multiplex PCR (Esteve-Zarzoso e/ al., 1999; De
Llanos Frutos et al. , 2004). Individuai isolates were differentiated by RAPD-PCR and AFLPs {Barros
Lopes et al., 1999).
7. Freeze-drying: Yeast strains with desirable features will be studied for suitability mass culture, freeze-drying
distribution and rehydration, and the strains possessing the best technological traits will be dehydrated to
realized a yeast collection for optimizing the winemaking of Montepulciano d'Abruzzo.
8. Chemical analysis: The main chemical characteristic, such as, pH, Brix degree, tiratable acidity, phenol and
aroma will be done for characterize musts and wine sample.
9. Statistica! analysis of date, will be performed using statistica! software.
10. Writing and Edition ofthe PhD thesis, scientific papers and oral and/or poster communications.
Table 1:
Canti dia ram {or lhis PhD lhesis roject.
Activity
Months
1- Bibliographical
rcscarch
2- Parnmeter for
3.
Selected References
l .Barros Lopes M, Rainieri S,
Henschke PA, Langridge P, ( 1999).
AFLP fingerprinting /or analysis of
y east genetic variation. International
of
Systematic
and
Journal
4-Yeast isolation
Bacteriology 49 : 915- 924.
6-Molecular
2.Benites J, Pisante M, Stagnari F,
identification
(2005). lntegrated soil and
(Eds)
9-Study of
water management /or orchard
oenolo ical traits
IO- Chcmical analysis
development. Role and .lmportance.
of musts and wine
FAO Land and Water Bulletin No 10.
11-Statistical analysis
3.De
Llanos Frutos R, Fernandez12- Writing and 4
Edition of the PhD
Espinar MT, and Querol A, (2004).
thcsis
Jdentification of species of lhe genus
Candida by analysis of the 5.8S rRNA gene and two ribosoma! internal trascribed spacer. Antonie van
Leeuwenhoek 85: 175- 185.
4.Esteve-Zarzoso B, Belloch C, Uruburu F, and Querol A, (1999). Jdenlijìcation ofyeasl by RPFL analysis of
the 5.8S rRNA gene and the two ribosoma/ internal trascribed spacer. International journal of Systerpatic
Bacteryology 49: 329-337.
5.Franchini J C, _Crispino C C, Souza R A, To1Tes E, Hungria M, (2005). Microbiologica/ pararnelers as
indicators of soirq_L1ality under varioys management and crop rolalion system in southern Brazil .So il e Tillage
Research. xxx (2006) xxx, Article in Press.
6.Frezier V, Dubordieu O, ( 1992). Ecology of y east strain Saccharomyces cerevisiae during spontaneous
f ermentation in a Bordeaux wine1y. American Journal ofEnology and Viticulture 43: 375 - 380.
6.Gainvors A, Frezier V, Lemaresquier H, Lequeart C, Aigle M, Be lardi A, ( 1994). Detection of
polygalacturonase, pectin-lyase and pectin-esterase activities in a Saccharomyces cerevisiae strain. Yeast 10:
13 11-1 3 19.
7 .Nadal D, Colomer 8, Pina B, ( 1996). Molecular polymorphism distribution in phenotypically distinct
populations of wine yeast strains. Applied and Environmental Microbiology 62: 1944- 1950.
8.Pretorius I S, (2000). Tailoring wine yeast /or the new 111il/enniwn: nove/ approaches to the ancient art of
winemaking. Yeast 16: 675-729.
9.Romano P, Caruso M, Capece A, Lipani G, Paraggio M, Fiore C, (2003). Metabolic diversity of
Saccharomyces cerevisiae strains from spontaneously f ermented grape musts. World Joumal of Microbiology
and Biotechnology 19: 3 11 -315.
IO.Rosi I, Bertuccioli M, ( 1989). Esterase activity in wine yeasts. In : Dunod (Ed.), Actualités oenologiques 89.
Techniques and Documentation, Paris, pp. 206-2 11 .
I I. Rosi I, Vinella M, Domizio P, ( 1994). Characlerisation of f3-glucosidase activity in yeasts of oenological
origin. Journal of Applied Bacteriology 77: 519-527.
12. Ubeda Iranzo J F, Briones Perez A I, Izaquierdo Cai\as P M, ( 1998). Study of the oenolog ical characteristics
and enzymatic activities of wine yeasts. Food Microbiology 15: 399-406.
13.Vincenzini M, Romano P, Farris G A, (2005). Microbiolog ia del vino. Casa Editrice Ambropiana 2005.
~ptember 2nct 2006
EOO•,>
MJCJ1)
FoodMicro 2006, Bologna, August 29th - September 2nct 2006
2006
=:;;;;-~--
3.1 Fermented foods: traceability, labels and role ofnative microbes (P).
g Rhizopus oligosporus
Yeasts from traditional Italian Cooked Wine.
Rosanna Tofalo 1, Federico Di Fabio 1, Clementia Chaves-Lopez 1, Andrea Piva 1, Giovanna Suzzi 1•
>me Science, Sardar Patel
'Dipartimento di Scienze degli Alimenti - Università degli Studi di Teramo- Mosciano S. Angelo,
TE, Italy.
Keywords: Vino cotto, wine yeast, PCR-RFLP, DGGE.
:ractive flavour, texture and
y enzymatic degradation of
:ularly soluble nitrogenous
·ield, nutrient analysis, fatty
ittem were studied for soy
preparation of soy tempeb.
t0.83, 11.15, 4.65 and 38.88
npeh sundried for two days.
l and 8.34 for raw soybean
leic aci,? and linolenic acid
increase. No changes were
. Tota! free amino content
!mpeh on dry weight basis.
ientation from 7.27 to 4.09,
mtent increased (marginally
5y, New Delhi is gratefully
''Vino cotto" is a sweet wine produced after a long fermentation of cooked musts in some area of
centrai Italy, such as Abruzzo and Marche regions. It is traditionally produced fro m white grapes
mainly of Trebbiano, Passerina, Montonico and Moscato cultivars. Tbe cooked must is obtained by
direct heating in copper boilers unti I the volume is reduced at percentages ranging between 10-60%,
wben it becomes dark and dense. The alcoholic fermentation, carried out by yeasts originating from
processing equipment, starts after 10 days at room temperature and Iasts about 40 days. In this study
yeast populations were examined by culture-dependent and -independent methods during the
fermentation of cooked musts with and without S02 addition. The yeasts isolated were
presumptively identified using traditional tests and the species were subsequently confirmed using
PCR-RFLP analysis of the region spanning the internal transcribed spacer (5.8S-ITS rDNA) with
different endonucleases. During tbe first phase of fermentation Saccharomyces cer_evisiae was not
detected, whereas Candida spp. and P-ichia spp. represented respectively 43% and 56% of tota!
isolates; after 10 days S. cerevisiae was the dominant species (80%), while at the end of
fermentation its presence decreased to less than l 0%. On the otber hand, Zygosaccharomyces bai/ii
was tbe dominant specie during ali process in cooked wine added with S02. These results were
confirmed by PCR-DGGE of a portion of the 26S rDNA of the tota! DNA isolated from cooked
musts and the strains were differentiated by RAPD-PCR. Ali yeasts were also characterized for
biotechnological traits.
... :.
285
'
'
'
DETERMINAZIONE DI DEKKERAIBRETTANOMYCES NEL VINO CON METODI
COLTURA-INDIPENDENTI
Tofalo Rosanna 1, Chaves-Lopez Clemencia1, Di Fabio Federico 1, Angrisani Roberto', Suzzi Giovanna'
1
Dipartimento di Scienze degli Alimenti - Università degli Studi di Teramo - Mosciano S. Angelo (TE), Italia
e-mail: [email protected]
Introduzione
Dekkera bnaellensis (forma anamorfa di Brettanomyces bruxellensis) è un lievito deteriorante
soprattutto di bevande alcoliche e bibite (Deak e Beuchat, 1996). La sua presenza è importante
nell'industria enologica, soprattutto nella produzione dei vini rossi affinati in "barrique", dove
può produrre composti fenolici maleodoranti, quali il 4-etilfenolo e il 4-etilguaiacolo. Spesso,
tuttavia, nel vino vengono determinati alti livelli di questi fenoli, mentre non si trovano affatto o
solo basse concentrazioni di cellule di Dekkera/Brettanomyces. Con i metodi fenotipici
occorrono 1 o 2 settimane per identificare questa specie (Rodrigues et al., 2001). Lo sviluppo di
tecniche molecolari ha permesso di differenziare velocemente le colonie di Dekkera isolate da
vino, utilizzando RAPD-PCR o PCR specie-specifiche (Egli e Henick-kling, 2001).
' E' ormai accertato che lieviti Dekkera/Brettanomyces possono essere presenti in numerosi mosti
e vini, provenienti dall'ambiente esterno e/o dalla cantina. La loro presenza non significa
necessariamente che il vino prodotto sarà deteriorato o di scarsa qualità. Tuttavia, i vini
contaminati, possono essere considerati vini a "rischio", soprattutto quelli che andranno
all'invecchiamento. In questo studio si è voluto mettere a confronto due tecniche molecolari
(PCR-DGGE e Real-Time-PCR) per quantificare la presenza di Dekkera/Brettanomyces in mosti
e vini.
Metodologia
I ceppi tipo delle specie Saccharomyces bayanus DBVPG 6171 T (Dipartimento di Biologia
Vegetale, Università di Perugia), S. cerevisiae DBVPG 6173 T' S. paradoxus DBVPG 6411 T, S.
pastorianus DBVPG 6047T e Hanseniaspora uvarum DBVPG 6718\ Brettanomyces anomalus
MUCL 27704 (Mycotheque de l'Universite Catholique de Louvain), B. bruxellensis MUCL
27700, Pichia anomala MUCL 20294 sono stati usati come riferimento. Per valutare la
sensibilità e la riproducibilità delle tecniche la determinazione del ceppo B. bnaellensis è stata
eseguita inizialmentè· in terreno sintetico ( 1% di estratto di lievito, 2% di peptone e 2% di
glucosio) e poi in vino inoculato con il lievito. Il DNA genomico totale delle colture pure e dei
vini è stato estratto con il metodo descritto da Querol et al. (1992). La PCR-DGGE è stata
effettuata secondo quanto riportato da Prakitchaiwattana et al. (2004). La Real-time-PCR è stata
eseguita in accordo con Phister e Mills (2003 ).
Risultati e discussione
Per la ricerca del B. bruxellensis, due tecniche molecolari coltura-indipendenti PCR-DGGE e
Real-Time-PCR sono state utilizzate. Brettanomyces bruxel/ensis è stato diluito ed aggiunto al
vino per ottenere campioni di vino con livelli noti di lievito. Per ogni campione è stato eseguita
la conta in piastra. I prodotti di PCR sono stati separati e rilevati mediante gel DGGE, che ha
permesso la separazione in base alle differenze di sequenza. La coppia di primer utilizzati
amplificava un frammento molto variabile della regione 26S, che ha permesso facilmente di
differenziare le specie esaminate. L'intensità delle bande è stata quindi correlata con la conta in
piastra: è stato possibile determinare fino a 102 ufc/ml di lievito.
L'analisi successiva ha rilevato tramite Real-Time PCR la presenza di B. bruxellensis,
considerando le stesse condizioni adottate per la PCR-DGGE. La Real-Time-PCR permette
tramite la registrazione della fluorescenza di monitorare il processo della reazione di PCR
227
durante il suo svolgimento. Le curve relative a vini con inoculo maggiore sono le prime ad
innalzarsi, e quindi ad essere rilevate con un CT (threshold cycle) pari a 19.4. I vini con minore
inoculo hanno un valore di CT maggiore. Gli altri ceppi tipo impiegati nel lavoro non hanno
presentato prodotti, confermando che il metodo usato è fortemente discriminante a livello di
specie. Riportando graficamente i CT ottenuti dai vini contaminati con il numero di cellule
2
ottenute dalla conta in piastra si è ottenuto un'ottima correlazione con un R pari a O. 9923
(figura 1).
22,5
22
21,5
..
u
21
20,5
Figura l. Detenninazione dell'efficienza di
QPCR e limite di rilevabilità di B.
buxellensis in vino.
"1
I
y = -0,4455x + 22,099
i
ji
2
R =0,9923
I
i
1
201
19,5 i
19
i
.l-----~,----~---~ ~-~
o
2
3
4
5
6
7
Log (ufc)/ml
Il limite rilevabile è risultato essere 1 ufc/ml di vino. L'utilità di questa tecnica, più della PCRDGGE, è fac_ilmente intuibile, in quanto permette di rilevare e di numerare in breve tempo e
facilmente la presenza di B. bruxellensis. L'applicazione della Real-Time-PCR rende possibile la
tracciabilità delle specie coinvolte nei processi di trasformazione che si realizzano durante la
vinificazione e decidere gli opportuni interventi. Saper diagnosticare in tempo una diffusione non
controllata di certrìnicrorganismi significa minimizzare e controllare il rischio della comparsa di
alterazioni.
Bibliografia
Deak, T. e & Beuchat, L. (1996). Handbook of food spoilage yeasts. CRC Press, New York,
NY.
Egli, C.M. e & Hep}ch-Kling, T. (2003). Identification of Brettanomyces/Dekkera species based
on polymorphism iii the rRNA Internal Transcribed Spacer region. Am. J. Enol. Vitic. 52: 241247.
Phister, T. G. and & Mills D.A. (2003). Real-Time PCR Assay for Detection and Enumeration of
Dekkera bruxellensis in Wine. Appl. Envir.Microbiol. 69:7430-7434.
Prakitchaiwattana, C.J., Fleet, G.H., Heard, G.M. (2004). Application and evaluation of
denaturing gradient gel electrophoresis to analyse the yeast ecology of wine grapes. FEMS Yeast
Res. 4(8):865-877.
Querol, A., Barrio, E. Ramòn, D. (1992). A comparative study of different methods of strains
characterization. Syst. Appl. Microbio/. 15: 439-446.
Rodrigues, N., Goncalves G., Pereira-da-Silva, S., Malfeito-Ferreira, M., Loureiro V. (2001).
Development and use of a new medium to detect yeasts of the genera Dekkera/Brettanomyces J.
Appl. Microbio/. 90(4):588-599.
228
INDAGINE PRELIMINARE SULL'EFFICACIA
DELL' ESTRATTO ~DI SEMI DI POMPELMO
NELLA LOTTA CONTRO LA PESTE AMERICANA
1
V. Langella 1 , P. Semprini 1 , F. Di Fabio 1, B. Pasini2 , M.T. Falda2 , F. Panella 3, S. Calvarese'
Istituto Zooprofilattico Sperimentale dell 'Abruzzo e del Molise "G. Caporale", Teramo - Italia
2
Apicoltura Pasini, Campagnatico (Gr) - Italia
3 Unione Nazionale Associazioni Apicoltori Italiani, Lanciano (Ch) - Italia
RIASSUNTO
Il presente progetto pilota ha avuto la finalità di verificare in vitro la capacità inibente del!' estratto dei semi
di pompelmo nei confronti del!' agente responsabile della peste americana delle api Paenibacillus larvae
subsp. larvae e misurarne l'efficacia "in campo" su alveari infettati sperimentalmente.
Sono state utilizzate 21 famiglie infettate con sospensione batterica di un ceppo di Paenibacillus larvae
subsp. larvae alla concentrazione di 10 miliardi di spore/ml e 10 famiglie con infezione naturale
diagnosticata mediante visita clinica. L'apiario è stato suddiviso in 3 gruppi: al primo gruppo è stato
somministrato l'estratto di pompelmo sotto forma di olio, al secondo sotto forma di polvere sciolta
~in soluzione zuccherina e al terzo, di controllo, con sulfatiazolo. I risultati hanno evidenziato un buon livello
di infezione delle famiglie con una percentuale del 71 % dopo 20 giorni e del!' 86% dopo 40 giorni
dal!' infezione. I risultati delle prove di laboratorio hanno evidenziato una buona capacità inibente
cieli' estratto di semi di pompelmo nei confronti del Paenibacillus larvae subsp. larvae con un alone
di inibizione di 8 mm e di 2 mm del/' olio e della polvere rispettivamente alla diluizione di 1 :80.
La sperimentazione effettuata può essere considerata una traccia importante per lo sviluppo di un ulteriore
progetto di ricerca che comprenda un numero cospicuo di alveari, l'integrazione con altri eventuali fattori
come la capacità igienica della famiglia e l'esame di ulteriori prodotti disponibili in commercio.
PAROLE CHIAVE
Estratto di semi di pompelmo, Paenibacillus larvae subsp. larvae, Peste americana.
t
(
Introduzione
La peste ameri cana·,è una
malattia sostenuta dal batterio
Paenibacillus larvae subsp. larvae che colpisce la covata causando un'alta mortalità delle larve.
Questa patologia porta alla distruzione delle colonie anche se sono
documentati casi in c ui queste,
dopo aver mostrato lievi segni
della malattia, si sono riprese per
un periodo indefinito. Le spore
sono trasmesse dalle api adulte
alle larve durante le operazioni di
nutrizione e pulizia dei favi, quindi il comportamento igienico della famiglia può giocare un ruolo
importante nel decorso de ll a
malattia.
I mezzi di lotta possono essere
molteplici; da azioni radicali
come la distruzione delle colonie,
a metodi chemioterapici come il
PRELIMINARY INDAGATION
trattamento con Sulfati azolo e
ON THE EFFICIENCY
OF GRAPEFRUIT SEEDS ·
ossitetracicline che determinano,
però, problematiche legate alla
EXTRACT AGAINST
AMERICAN FOULBROOD
residualità nel miele. Appare evidente, pertanto, l'importanza del1' utilizzo di sostanze o prodotti
alternativi e/o naturali che riduca- , Summary
This pilo! project aims al verifyi ng in
no al minimo l 'impatto con la ·j
vitro the inhibiting a bility of the grapesalubrità degli alimenti (2, 3) ..
fru it seeds extract against PaenibacilE' noto che l'estratto di semi di lus larvae subsp. larvae, responsible
pompelmo ha potere battericida e for American foulbroo d and then
so luzioni commerciali hanno verify its effectiveness on experi men~
dimostrato che è attivo nei confron- tally infected bee-hives. Twenty-one
fam il ies infected with a bacter ia l
ti di numerosi germi quali Salmosuspension of a Paenibacillus larvae
nella spp, E. coli, Listeria monocysubsp. larvae strain al a concentration
togenes e Candida alhicans.
of 1.000 million spores/ ml, and l O
L'effetto inibente è dovuto families natura lly infected and diagnoprincipalmente ad alterazioni della sed th rough clinica! insp.ectiòn, were
membrana cellulare con inibizione used . The apiary was divided into 3
della respirazione cellulare e con- groups: the firs t one was treated with
seguente modificazione della per- grapefru it see ds extra Tct o il , the
49
;.
ANNO XXXIX
meabilità della cellula (1, 5, 8, 10)~
L'obiettivo del presente lavoro
è stato quello di verificare in laboratorio la capacità inibente dell'estratto nei confronti del Paenibacillus /arvae subsp. larvae e misurarne l'efficacia "in campo" su
alveari infettati sperimentalmente.
Materiali e metodi
Prove di inibizione in vitro
La capacità inibente dell 'estratto di semi di pompelmo è stata testata "in vitro" nei confronti
del Paenibacillus larvae subsp
larvae, Bacillus subtilis BGA, B.
cereus 11778, B. cereus K250 e
Micrococcus luteus 9341.a,
secondo la procedura IZS TE
B3.1.2 SOP 009. L'estratto di
pompelmo è stato saggiato a conc en trazioni scalari con acqua
distillata sterile e in base alla misura dell'alone di inibizione ne è stata valutata la capacità inibente.
Preparazione
della broclocoltura per l'iniezione
Il ceppo batterico utilizzato per
l'infezione, Paenibacillus larvae
subsp. larvae ATCC 9545, è stato
coltivato secondo la procedura IZS
TE B 5.1.1 SOP 003. La pas~ batterica ottenuta è stata titolata
secondo la procedura IZS TE
B3.1.2 SOP003 e successivamente
diluita fino a ottenere una concentrazione finale di 10 miliardi di
spore/ml (9).
Per l'infezione ad ogni alveare
sono stati somministrati, sotto forma di spray, 60 ml di sospensione
formata da 30 ml di brodocoltura
batterica e 30 ml di soluzione zuccherina al 25% (4, 7).
Modello sperimentale
La prova sperimentale è stata
svolta a Campagnatico, località
della maremma toscana a 275
metri sul livello del mare in provincia di Grosseto, utilizzando un
apiario di 31 alveari in arnie
:~:ACJ·:cSUU.'EFFICAC!A
:)ELL"ESTRJ\TfO Di SEM!
:~ ...•
:: e:::.MC
Dadant-Blatt.
~ second one with grapefruit seeds
La sper(mentazione è stata ~ extract powder in sugar solution, the
svolta dalla metà di settembre alla 'Q third one - the control group - was
fine di novembre 2002.
1··
treated with Sulfathiazole. Results
·
·
d
li
·
ta
·
showed a good infection level of fami,.
Ali lillZIO e a spenmen z10 ne
lies, 71 % after 20 days and 86%
le famiglie sono state controllate e .; after 40 days from infection. Results of
di ciascuna ne è stata valutata la
lob tests showed a good inhibiting
forza, la potenzialità produttiva, la
ability of the grapefruit seeds extract
raccolta del polline, la raccolta del
against Paenibacillus larvae subsp.
propoli e l'aggressività.
larvae, with an inhibiting halo of 8
E' stata valutata, inoltre, la ~ mm and 2 mm of oil and powder
capacità igienica delle famiglie \ respectively, at 1:80 dilution. This
i
reseorch con be considered on impor-
poichè questo parametro può con- ~ tant step for the development of a
siderarsi importante nell'aiutare il f; new research project involving a larrisanamento di un alveare durante
ge number of hives, the integration
il trattamento terapeutico (i risulwith other factors such as the family
tati saranno oggetto di prossima
hygienic ability, and the study on
pubblicazione).
other commerciai products.
L'apiario è stato diviso ..in due
KeyWords
gruppi:
American foulbrood, Grapefruit seeds
- Ventuno famiglie sane sono
extract, Paenibacillus larvae subsp. larvae.
state infettate sperimentalmente
lntroduction
con 300 miliardi di spore per
American foulbrood is caused by
alveare con spray distribuito sul
the Paenibacillus larvae subsp.
lato di ogni favo (contrassegnate
larvae, affecting the brood and thedal n. 1 al n. 21) (4, 6).
refore causing a high mortality among
- Dieci famiglie con infezione
larvae. This pathology con lead to the
naturale diagnosticata mediante
destruction of colonies, even if there
visita clinica (contrassegnate dal
are cases in which lightly-affected
n. 22 al n. 31).
colonies recovered far on indefinite
period of time. The spores are tran- ·
Per verificare lo stadio di infesmitted
to the larvae by the adult bees
zione sono stati effettuati campiowhile cleaning the combs, thus the
namenti di favi di covata a distanhygienic behaviour of the Family con
za di 20 e 40 giorni dall'inizio
play on essential role in the developdell '.esperimento.
ment of this disease.
~rove
sperimentali
in apiario: trattamenti
Prima del trattamento sperimentale sono state eseguite alcune prove preliminari al fine di
determinare la concentrazione
ottimale del principio terapeutico,
eventuali fenomeni di tossicità
per le api e la sua appetibilità in
funzione della forma di somministrazione (candito, soluzione zuccherina, acqua). Sono stati formati tre gruppi mediante estrazione a
sorte.
Primo gruppo: dieci alveari, di
cui 7 infettati sperimentalmente e
3 con infezione naturale, sono stati
50
There are many ways to fight this:
from radical actions, like destroying
the colonies, to chemotherapeutic
actions, like treating the colonies with
sulfathiazole and oxytetracyclines
although these treatments can determine problems of residuality in honey.
Therefore, it is obviously important
to use alternative and/or natural products or substances to hove the smallest impact on food healthiness (2, 3).
Grapefruit seeds extroct is well
known as a bactericide and commerciai solutions have demonstrated that
it works against many germs like Salmonella spp., E. coli, Listeria monocytogenes and Candida albicans. The
inhibiting effect is mostly due to the
alteration of the cell membrane inhibi-
Tabella 1: Test di inibizione in vitro.
Table 1: "ln vitro" inhibifing test.
Olio semi pompelmo
Gropefruit seeds oil
tal quale/plain
J
l
B. sub61is
B. cereus
BGA
11778
B. cereus
K250
M. lute~s
9341.a
025mm
012mm
0 lOmm
010mm
010mm
1:10
016mm
010mm
0 10 mm
010mm
010mm
1:20
0 12mm
010mm
010mm
010mm
010mm
1:.40
0 lOmm
06mm
06mm
06mm
06mm
1:80
0Bmm
03mm
03mm
03mm
03mm
B. sub6/is
8. cereus
BGA
11778
8. cereus
K250
M. luteus
9341.a
Materials and methods
Gq>.seeds~
Paenibacillus larvae
subsp. larvae
tal quale/plain
016mm
08mm
08mm
08mm
08mm
1:10
012mm
06mm
06mm
06mm
06mm
1:20
010mm
06mm
06mm
06mm
06mm
1:.40
018mm
06mm
06mm
06mm
06m
1: 80
02mm
02mm
02mm
02mm
02mm
The inhibiting ability of the grapefruit seeds' extract hos been tested in
vitro agoinst Paenibacillus larvae subsp. larvae, Bacillus subtilis BGA, B.
cereus 11778, B. cereus K250 and
Micrococcus luteus 9341 .a, according to the IZS TE B3. l.2 SOP009
procedure. Grapefruit extract has
been tested in scalar concentrations
with sterile distilled water and the inhibiting ability has been calculated on
the basis of the size of the inhibition
hai o.
Pclv.semi~
'
..
Paenibacillus farvae
subsp. larvae
ting the cell respiration and consequent modification of the cell permeability (1, 5, 8, 1O).
This study aims at verifying in
laboratory the inhibiting ability of the
extract against the Paenibacillus larvae subsp /arvae and then verify its
efficiency directly on experimentally
infected bee-hives.
trattati con olio di estratto di semi
di pompelmo (2 g in 50 ml di soluzione zuccherina al 50% per alveare per 3 volte a distanza di 7 giorni). La sospensione è stata spruzzata sui favi coperti dalle api.
Secondo gruppo: dieci alveari, di
cui 7 infettati sperimentalmente e 3
con infezione naturale, .. sono stati
trattati con polvere di estratto di
semi di pompelmo (2 g in 50 nù di
soluzione zuccherina al 50% per
alveare per 3 volte a distanza di 7
giorni). La sospensione è stata
spruzzata sui favi coperti dalle api.
Terzo gruppo: sette al~.ari, di cui
3 infettati sperimentalmente e 4
con infezione naturale, sono stati
trattati con Sulfatiazolo (1 g in
300 ml di soluzione zuccherina al
50% per alveare per 3 volte ·a
distanza di 7 giorni).
Quattro alveari sono stati
esclusi dai trattamenti in quanto
una famiglia era diventata orfana
mentre le restanti tre erano risultate sempre negative all'esame
batteriologico dei primi due campionamenti.
Alla fine dei tre trattamenti
sono stati prelevati porzioni di
favo di covata, su telaini diversi,
per la ricerca di Paenibacillus larvae subsp. larvae. Negli alveari
dove la covata era assente la ricerca è stata eseguita sul miele.
Risultati
*
lnhibition tests in vitro
La Tabella 1 mostra i risultati
dei test di inibizione "in vitro" dell'estratto di pompelmo. Come si
nota la capacità inibente del prodotto, sia sotto forma oleosa sia in
Preparation of the broth
medium lor inlection
polvere, nei confronti del PaenibaThe bacterial stroin used for the
cillus larvae subsp. larvae risulta
Paenibacillus larvae subsp.
infection,
maggiore rispetto a quella degli
larvae ATCC 9545, has been cultivaaltri ceppi. Infatti, ilprodotto tal
ted according to the IZS TE B 5.1. l
quale e quello diluito mostrano
SOP 003 procedure. The resulting
una capacità inibente quasi dopbacterial poste has been titered accorpia nei confronti degli altri germi;
ding to the IZS TE B3. l .2 SOP003
in particolare, la forma oleosa alla
procedure and then diluted to get a
final concentration of 1.000 million
diluizione di 1:80 presenta un alospores/ml (9).
ne di inibizione pari a 8 mm di
For the infection, every hive has
diametro rispetto ai 3 mm degli
been sprayed with a 60 ml suspenaltri ceppi.
sion made of 30 ml of bacterial broth
I risultati del primo campionamedium end 30 ml of sugar solution
mento, effettuato dopo 20 giorni
at 25% (4, 7).
dall'infezione per verificare la
capacità infettante delle spore,
Experimental model
sono riportati nella Tabella 2.
The experiment has taken piace in
Ovviamente, gli alveari con · Campagnatico, a village in the south
infezione naturale sono risultati
of Tuscany at 275 metres above sea
tutti positivi all'esame batteriologico mentre su 21 alveari infettati
sperimentalmente, 15 risultano
positivi e 6 negativi con una percentuale di positività del 71 %.
Dai risultati del secondo campionamento, effettuato dopo 40
giorni dall'infezione, si evince che
i positivi sono passati da 15 a 18
alveari su un totale di 21, incre-
51
level in the province of Grosseto,
using on apiary made of 31 DadantBlatt hives.
The experiment started halfway
through September 2002 and ended
at the end of November 2002.
lnitially, ·families h~ve been studied
to analyse their respective features:
strength, production~potential, pollen
collection, propolis collection, and their
aggressivity.
·_,.1•• ,
ANNO XXXIX
mentando la positività all '86%.
All'esame ispettiyo, le famiglie
infettate sperimentalmente presentano i sintomi della peste americana: covata infossata, oper~olatura
leggennente aperta, larve filamentose alla prova dello stecchino e
classico odore di putrefazione.
In Tabella 3 sono riportati i
risultati degli esami batteriologi
per la diagnosi della peste americana dopo i trattamenti effettuati con
estratto di semi di pompelmo e ~
Moreover, families' hygiene has
Sulfatiazolo. E' sorprendente nota- ~ also been ossessed because this para·
re l'efficacià dei trattamenti: solo · ~ meter is very importont in helping heal
una famiglia (trattata con polvere ~ the hive during the therapeutic treatdi estratto di semi di pompelmo)
~
risulta positiva.
~l
Discussione
Sulla base dei risultati di laboratorio e della sperimèntazione in
campo è possibile fonnulare alcune considerazioni:
Tabella 3: Risultati degli esami di laboratorio e tipo di trattamento.
Table 3: Results of the laboratory tests and freatmenf type.
Tabella 2: Risultati degli esami batteriologici o 20 e 40 giorni per lo
diagnosi di Peste americano.
Table 2: Results of the bacterio/ examinations after 20 and 40 days
fo diagnose the American foul broocl.
'
N. alveare
Hiven.
1° campion. (20 gg.)
2
Pos
Pos
Pos
Pos
Pos
5
Pos
Pos
Pos
Pos
6
Neg
7
Pos
Pos
Pos
Pos
4
8
N. alveare
Hiven.
Jst sampling (20 clays) ~ sampling(40 clays)
Neg
3
3° campionamento/3'd sampling
2° campion. (40 gg.)
1
...
~
2
3
4
5
6
7
8
Pos
9
10
Neg
Neg
Pos
11
Neg
12
Pos
Pos
Pos
Pos
13
Pos
Pos
14
Neg
15
Neg
Neg
16
Pos
Pos
17
Pos
Pos
18
19
20
Pos
Pos
Pos
Pos
Pos
Pos
19
Pos
20
Pos
Pos
Pos
21
21
22
23
24
25
26
27
28
29
30
31
Pos
Pos
Pos
-.. -·
9
10
11
12
13
14
15
16
Neg
17
18
22
23
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
ment (these results will be published
soon).
The apiary has been divided into
two groups:
· 21 families have been experimentally infected with 300,000 millions
spores per hive, spraying it on the side
of each comb (marked from n. l to n.
21)( 4, 6 ).
24
25
26
27
28
29
30
31
52
Tipo di trattamento
Treatment lype
Olio di semi pomeelmo
Gra
"s seeèls oil
Olio di semi pompelmo
Gra frutis seeils oil
Polvere semi 1>9mpelmo
Gra fruit seeds wcler
Gra frutis seeèls oil
Gra fruii seeds wJer
Non lraHata/Not treated
Sulfafiazolo/Sullathiazole
Gra
tis seeàs oil
Non trattata/ Not treatecl
Polvere semi P9111pelmo
Gra
it seeds wcler
Sulfatiazolo/Sullathiazo/e
Sulfatiazolo/Sullalhiazole
Polvere semi P9f11pelmo
Gra frvit seeds wcler
Non trattata/ Not lreated
Non trattata/Not treatecl
Polvere semi 1>9mpefm~
Gra
it seeds wrler
Grapefruit seeds powder
Oliodisemi~mo
lrvtis
s oil
Grapefruit seeds powcler
Sulfatiazolo/Sulfrzthiazole
ono di semi pomeeJmo
Gra
tis seeils oil
Sulfatiazofo Sullathiazole
Gra Fruit seeds wder
Gra frvit seeds wc/ef
Sulfatiazolo Sullathiazole
Gra ruit seeds wcler
Gra frutis séeiJs oil
Olio di semi p0rrieefm0
Gm
tis seeàs oil
Sulfatiazolo/Sulfathiazole
Gra
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
N
Neg
Neg
Pos
Neg
~
Neg
Neg
i
·/
·. i...ANGSLLA E ALTRl
- Nessuna famiglia è morta' di
peste americana. ...
- Al tennine della sperimentazione è stata valutata di nuovo la
forza delle famiglie. Tutte hanno
mostrato uno stato generale buono
con api su 4n telai.Iii, hanno avuto
buona scorta di polline e la covata
non ha mostrato nessun segno di
patolo~a riferibile a peste.
- Dovrà essere approfondito e
meglio valutato il rapporto tra il
comportamento igienico della
famiglia e l'efficacia dimostrata
dal prodotto sperimentato.
- Nonostante I'elevato nwnero
di spore somministrato (300
miliardi per alveare) non tutte le
famiglie si sono infettate.
- L'olio di estratto di pompelmo somministrato con candito o
soluzione zuccherina si è dimostrato poco appetibile per le api.
- Il prodotto sembra che manifesti la stessa efficacia sia negli
alveari infettati sperimentalmente
sia in quelli con infezione naturale.
- Mettendo in relazione l'unica
positività avuta e la capacità inibente dimostrata "in vitro" sembrerebbe esserci una differenza tra
la forma fisica del prodotto, indicando una maggiore efficacia di
quella o~eosa rispetto a quella in
polvere (dato da verificare).
.Non sembra opportuno trarre
delle conclusioni generalizzate da
questo studio pilota.
E' meglio pensare che la sperimentazione effettuata possa essere
una traccia importante per lo sviluppo di un progetto interregionale
che comprenda un numero cospicuo di alveari, l'integrazione con
altri eventuali fattori come la capacità igienica della famiglia, tempi e
situazioni climatiche delle diverse
regioni e controllo della linea
genetica delle regine.
Solo attraverso uno studio articolato si potranno avere risultati di
ausilio nel trattamento della peste
americana nell'ambito di una api-
VETERINARIA ITALIANA
col tura biologica al fine di commercializzare prodotti di qualità. .
Ringraziamenti
Si ringrazia il dottor J. Vitalis
per la disponibilità del prodotto, il
dottor A. Di Marco, il sig. S.
Mancini per la sperimentazione e
i tecnici di "Apicoltura Pasini"
per la conduzione dell'apiario.
Bibliografia/References
1. Anonimo 2001. L'extrait de pèpins de
pamplemousse. Info-Reines, 55: 4-6.
2. Bryant T.G. 1980. Why we should not
use drugs to control Bacillus larvae.
Apiarist, 14 (3).
3. Cremasco S., F. Mutinelli e A. Irsara
1994. Attività degli oli essenzi~li nelle
malattie delle api. Ape nostra amica, 16
(2): 4-10.
4. Gochnauer T.A. 1981. The distribution
of Bacillus larvae spores in the environs of
colonies infected with American foulbrood
disease. America bee J.,121 (5): 332-335.
5. Heggers J.P., J. Cottingham, J.
Gusman, L. Reagor, L. McCoy, E.
Carino, R. Cox, J.G. Zhao and L. Reagor 2002. The effectiveness of processed
grapefruit-seed extract as an antibacterial
agent: II. Mechanism of action and "in
vitro" toxicity. J Altern Complement
Med., 8 (3): 333-340.
6. Hornitzky M.A.Z. 1997. The spread
of Paenibacillus larvae subsp. larvae
infections in aìi apiary. J. of Apicultural
Research, 37 (4): 261-265.
7. Hornitzky M.A.Z. 1998. The pathogenicity of Paenibacillus larvae subsp. larvae spores and vegetative cells to honey
bee (Apis mellifera) colonies and their
susceptibility to royal jelly. J. of Apicultural Research, 37 (4): 267-271.
8. Reagor L., J. Gusman, L. McCoy, E.
Carino and J .P. Heggers 2002. The
effectiveness of processed grapefruitseed extract as an antibacterial agent: I.
An ''in vitro" agar assay. J Altero Complement Med., 8 (3): 325-332.
9. Sakogawa T ., S. Kataoka, A. Okayama 1999. A scientific note on "in vitro"
experimental infection of American Foulbrood in honeybees Apis mellifera. L.
Apidologie, 30 (1): 75.
10. Von Woedtke T., B. Schluter, P.
Pflegel, U. Lindequist and W.D. Julich
1999. Aspects of the antimicrobial efficacy of grapefruit seed extract and its
relation to preservative substances contained. Phannazie, 54 (6): 452-456.
53
R
- l O fomilies naturally infected and
diagnosed through clinica! inspection
(marked from n. 22 to n.31)
To verify the stage of the infection,
samples have been taken from the
brood combs 20 and 40 days after the
start of the experiment.
Experimental tests
in the apiary: trealments
Before the experimental treatment,
some preliminary tests have been carried out to determina the optimal concentration of the therapeutic agent, possible toxic effects on bees, and its
appeal related to the way it was admin istered (candied, sugar solution,
water).
Three groups have been randomly
created:
First group: 1O hives, 7 experimentally infected and 3 naturally infected,
have been treated with grapefruit seeds
extract oil (2 g in 50 ml sugar solution
at 50% per hive, 3 times in ·7 days).
This suspension has been sprayed on
the combs covered by bees.
Second group: 1O hives, 7 ~xperi
mentally infected and 3 naturally infected, have been treated with grapefruit
seeds extract powder (2 g in 50 ml
sugar solution at 50% per hive, 3 times
in 7 days). This suspension has been
sprayed on the combs covered by
bees.
Third group: 7 hives, 3 experimentally infected and 4 naturally infected,
have been treated with Sulfathiazole (1
g in 300 ml sugar solution at 50% per
hive, 3 times in 7 days) .
Four hives have been excluded
from the treatment because one of the
families became orphan while the
other three always tested negative to
the bacterial examination in the first
1wo samplings. At the end of the three
treatments, portions of brood combs
were taken, on different frames, to
look for Paenibacillus larvae subsp larvae. In hives with no brood the search
has been carri ed out on honey.
Results
Table 1 displays the results of the
grapefruit extract inhibition test in vitro.
As we can see, the inhibiting àbility of
the product, both in oil and in powder,
against Paenibacillus larvae subsp larvae is higher than that of other strains.
"
As a mafter of fact, the diluted at'ld simple product sho~ a double inhibition
ability compored to thot of other
germs; in particulor, the 1:80 diluted
oily product show a inhibiting halo
with a diameter of 8 mm whereas other
strains have only 3 mm.
Table 2 displays the results of the
fi~st sampling, carried out 20 days
after the infection to verify the infecting ability of the spores.
Obviously, oll the naturally infected hives tested positive to the bacterio! examination whereas, out of the
21 experimentally infected hives, 15
tested positive and 6 negative, with a
positivity percentage of 71 %.
From the results of the second sampling, carried out 40 days after the
infection, we con see that the positive
hives went from 15 to l 8 out of 21
increasing the positivity to 86%.
On the inspective test, experimentally infected families showed the
symptoms of the Am~rican foul brood:
sunken brood, slightly open operculum, thready larvae and the typical
putrefaction odour.
Table 3 shows the results of the
bacterial examinations to diagnose
American foul brood after the treotments with grapefruit seeds extract
and Sulfathiazole. lt is surprising to
I
see the efficiency of the treotments:
only 011e Family (treated with grapefruit seeds extract powder) tested positive.
Discussion
On the basis of the laboratory
results and the experiments it is possible to formulate some remorks:
• No family died of American foul
brood.
- At the end of the experiment,
families' strength has been assessed
ogain: they ali showed good generai
conditions hoving bees on 4/7 frames, they had a good pollen provision and the brood didn't show any
sign of foul brood related pathology.
- The relationship between the
hygienic behaviour of the fomily and
the efficiency of the te.sted product
should be studied and verified with
more attention.
- Not all the Families were infected
in spite of the high number of spores
used (300,000 millions per hive).
- Grapefruit extract oil given with
candy or sugar solution had little
appeal on bees.
- The product seems to be as efficient on the experimentally infected
hives as on the naturolly infected
ones.
- Comparing the only positive case
to the inhibiting ability showed in
vitro, we con see o difference
between the two ways to give the
treatment and a higher efficiency of
the oil compared to the powder {to be
verified).
lt is not advisable to make generai
conclusions from this pilot study.
lt is better to consider this experime nt as on important step For the
development of on interregional
project involving a large number of
hives and integrated with other factors
such as the hygienic ability of the
Family, weather conditions of the
various regions, and gene line contrai
of the queen bees.
Only with a careful study will it be
possible to have useful results to fight
American foul brood within organic
apiculture, in arder to sell quality products.
Acknowledgments
Thank you to Dr. J. Vitalis for the product availability, Dr. Agostino di Marco and Mr. Sebastiano Mancini for
their time dedicated to the experiment
and all the workers at "Apicoltura
Pasini" for managing the apiary
•. !·
i
j
54
j
DICHIARAZIONE SOSTITUTIVA DI CERTIFICAZIONE
(Art. 46 del D.P.R. 28 dicembre 2000, n. 445)
li sottoscritto Di Fabio Federico nato a
il
residente in
con riferimento all'istanza di partecipazione
Via
all'avviso di pubblica selezione, per titoli e colloquio, finalizzato alla formulazione di
una graduatoria di merito per il conferimento della borsa di studio per N. 2
laureato in SCIENZE E TECNOLOGIE ALIMENTARI NELL'AMBITO NELL'AMBITO DEL
PROGETTO LETTERA "A" MIGLIORAMENTO DELLE ACQUE DESTINATE AL CONSUMO
UMANO; ai sensi e per gli effetti dell'art. 46 del D.P.R. n. 445 del 28 dicembre 2000,
sotto la propria responsabilità e consapevole delle sanzioni penali richiamate
dall'art. 76 in caso di dichiarazioni mendaci e della decadenza dei benefici
eventualmente conseguenti al provvedimento emanato sulla base di dichiarazioni
non veritiere di cui all'art. 75 del succitato D.P.R., informato/a su quanto previsto
dal D.Lgs. 30 giugno 2003, n. 196
DICHIARA
o
di essere in possesso della Laurea in Scienze e Tecnologie Alimentari conseguito
in data 20 marzo 2000 presso l'Università degli studi d i Bologna sede di Cesena
(FC)
o di essere in possesso dell'ulteriore titolo di studio:
_.,;
Dottorato di Ricerca in Scienze degli Alimenti conseguito presso Università degli
Studi di Teramo nell 'anno 2009.
o di essere in possesso dei seguenti titoli valutabili:
;; Master di Perfezionamento in Legislazione Nazionale e Comunitaria degli
Alimenti presso Università degli Studi di Teramo nell 'anno 2003;
- Esame di Stato per l' abilitazione alla professione di Tec nologo Alimentare
conseguita presso Università degli Studi Di Bologna sede di Cesena (FC) anno
2001
- Borsa di Studio SOCRATES/ERASMUS presso UNIVERSIDADE TECNICA DE LISBOA,
ISTITUTO SUPERIOR DE AGRONOMIA anno accademico 1997 /1998.
o di essere in possesso de seguenti attestati di partecipazione a congressi,
convegni, aggiornamento, diplomi di specializzazione, formazione, qualificazione
tecnica, ecc.
~ Attestato di partecipazione "Q-DA Y fare qualità oggi nel laboratorio di analisi
delle acque" HACH-LANGE. Park Hotel Villaferrata 20 settembre 2011.
- Corso di Formazione: "Corso Avanzato di Software Chromeleon
ottimizzazione della gestione dei dati Cromatografici" presso ART A Abruzzo 23 febbraio 201 O.
- Seminario: Applicazioni di spettrometria di massa nella sicurezza alimentare ed
ambientale: soluzioni analitiche e nuove soluzioni. Presso Università degli Studi
di Camerino, Centro Universitario di Ricerca per lo sviluppo e la gestione delle
risorse dell'ambiente marino costiero San Benedetto del Tronto (AP) 5 maggio
2010.
Dichiarazione sosti1utiva di certific azione
1/3
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