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Bellaio, Giulia (2016) Characterization of different hop (Humulus lupulus L.) cultivars: response to drought stress, chemical composition and sensory profile. [Tesi di dottorato]

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Abstract (inglese)

Hop cones, the immature inflorescences of the female plant of Humulus lupulus L., have an important role in defining flavour, bitter stability and shelf life of beer. Hop cones are characterised by a wide range of molecules, such as α- and β-acids that contribute to the bitter taste of beer, and essential oils, which are responsible for the aroma. Nowadays hop is mainly cultivated in other countries and imported in Italy as raw material. Despite hop’s cultivation is not widespread in Italy, the number of Italian microbreweries is increasing and most of them are located in Northern-Italy. Taking into accounts this geographical distribution, it is therefore interesting to determine the achievable quality of hops cultivated in the Northern-Italy area. Among factors that limit the crop production, water availability is one of the major constraint since, in Mediterranean areas, the phenological cycle of hops coincides with a climate characterized by high air temperature and drought. For this reason, the selection of hop drought-tolerant varieties is crucial for the introduction of this crop in Italy. Hop physiological, molecular and metabolic responses activated in response to drought are poorly understood. For this reason eleven hop cultivars were subjected to drought and physiological and ionomic parameters were measured throughout water stress development. A total of 8 plants for each cultivar (4 controls and 4 water stressed-plants) were used. 3-L pots were sealed to avoid water evaporation, then control plants were watered every day while stressed plants were left without irrigation till the achievement of permanent wilting (after about 30 days). Physiological and growth parameters (pots weight, leaves axes, shoots length and SPAD chlorophyll meter) were measured. Results highlight that, compared to well-watered plants, most of stressed hop varieties did not reduce their transpiration until the Fraction of Transpirable Soil Water (FTSW) ≈0.7, showing, in general, an anisohydric behaviour. The experiment allowed us to pinpoint the more drought-tolerant/susceptible hop cultivars. Besides the selection of drought-tolerant cultivars, it is important to verify the achievable quality of hops cultivated in Northern-Italy and its variability among different cultivation years. Although the effect of climatological conditions on α and β-acids formation is well-known, no information are available on the variability of hops’ volatile profiles among different growing seasons. For this reason, hop acids and volatile compounds profiles of sixteen hop varieties cultivated in the same field in Parma (Emilia-Romagna, Italy) in two different years were analysed via High Performance Liquid Chromatography (HPLC) and comprehensive two-dimensional gas chromatography (GCXGC) respectively. In general, a strong variability in the dependence of the amount of hop acids and volatile compounds on the climate was found, nevertheless, hop acids and the key compounds of aroma showed a pattern specific for each variety. Moreover, cones of ten hop varieties cultivated in 2013 were subjected to ultrasonic extraction and the extracts were used to flavour a Blond Ale beer. Each beer was then sensory analysed by a trained panel. Finally, the sensory properties of flavoured beers were evaluated in relation to hops’ volatiles profiles and correlations were pinpointed. High and significant (p< 0.05) correlation coefficients were find between specific volatile compounds and aromatic descriptors as ‘hoppy’, ‘grassy’ and ‘spicy’, while few volatiles correlated with the descriptors ‘citrusy’, ‘fruity’ and ‘floral’. A chemical and sensory characterization of volatile compounds in different hop varieties was also performed to identify the odour-active molecules of particularly citrusy hops. Indeed, although different odour impressions such as grassy, floral/fruity or spicy notes have already been described, few attempts were made to clearly correlate the citrus aroma with the chemical profile of hop oil. The essential oil of three hop varieties with a well-known ‘citrus’ character and of three ‘spicy’/’hoppy’ varieties were chemically and sensory analysed. Each essential oil was fractionated by Solid Phase Extraction (SPE) and, according to a descriptive sensory analysis of the different fractions, the 70/30 ethanol/water (v/v) SPE fraction turned out to be the most citrusy one. This fraction was then analysed via Headspace Solid Phase Microextraction (HS-SPME) and Gas Chromatography−Mass Spectrometry/Olfactometry (GC-MS/O). A total of 59 volatiles were identified, 35 of which resulted to be odour-active. In this study the character-impact odorants in the citrusy fraction of hop essential oil were outlined; hierarchical cluster analysis and Principal Component Analysis (PCA) highlighted that hops that expressed a typical citrus aroma are characterized by high levels of specific esters as methyl (E)-4-decenoate, methyl nonanoate and methyl caprate, high levels of esters of geraniol and nerol and low levels of ‘grassy/spicy’ sesquiterpenes as α-humulene and β-caryophyllene.
Summarizing, in this work different responses to drought stress were highlighted in different hop cultivars, allowing us to determine the most tolerant and drought-sensitive hop cultivars. The quality of hops cultivated in Northern-Italy (expressed as hop acids content and volatile profile) and its variability were outlined for two cultivation years and some important odour impressions in hops (especially ‘citrusy’ notes) were characterized by a molecular point of view. This project has therefore increased our understanding of hop response to drought stress and of hop aroma, providing initial practical benefits for hop growers and for the hop industry.

Abstract (italiano)

I coni di luppolo, le infiorescenze femminili della pianta Humulus lupulus L., hanno un ruolo importante nella definizione dell’aroma, della stabilità dell’amaro e della shelf-life della birra. I coni di luppolo sono caratterizzati dalla presenza di particolari sostanze, ad esempio gli α- e β-acidi che contribuiscono al sapore amaro della birra, e gli oli essenziali, responsabili dell’aroma. Attualmente il luppolo viene coltivato principalmente in altri Paesi e importato in Italia come materia prima. Nonostante la coltivazione di luppolo non sia molto diffusa in Italia, il numero di micro-birrifici è in continua crescita e la maggior parte di essi è localizzata nel Nord-Italia. Considerando questi dati, risulta interessante determinare la qualità ottenibile da luppoli coltivati in quest’area. La disponibilità d’acqua è uno dei maggiori fattori ambientali che limita la produzione e nell’area del Mediterraneo il ciclo fisiologico del luppolo coincide con un clima caratterizzato da alte temperature e periodi siccitosi. Perciò, la selezione di cultivar di luppolo più tolleranti allo stress idrico è di cruciale importanza per l’introduzione di questa coltivazione in Italia. Ciò nonostante, le riposte fisiologiche, molecolari e metaboliche attivate in luppolo in risposta allo stress idrico sono poco conosciute. Per questo motivo dieci cultivar di luppolo sono state sottoposte a stress idrico prolungato e durante la cinetica di stress sono stati misurati alcuni parametri fisiologici e la concentrazione di alcuni ioni. Per l’esperimento è stato utilizzato un totale di 8 piante per ogni cultivar (4 piante controllo e 4 piante stressate). I vasi utilizzati (di un volume di 3 L) sono stati sigillati allo scopo di evitare l’evaporazione e, mentre le piante controllo sono state irrigate ogni giorno, le piante stressate sono state lasciate senza acqua fino al raggiungimento del coefficiente di avvizzimento permanente (dopo circa 30 giorni). Sono stati misurati parametri fisiologici e di crescita (peso dei vasi, assi fogliari, lunghezza degli internodi e valori di clorofilla misurati tramite SPAD). I risultati ottenuti hanno evidenziato come, comparate alle piante controllo, le piante stressate non riducono la loro traspirazione fino al raggiungimento di una frazione di acqua traspirabile nel terreno (FTSW) ≈0.7, mostrando in generale un comportamento anisoidrico. L’esperimento ha permesso di individuare le cultivar più tolleranti/sensibili allo stress idrico. Oltre alla selezione di cultivar di luppolo tolleranti allo stress idrico, è importante verificare la qualità ottenibile da luppoli coltivati in Nord-Italia e la sua variabilità tra diverse annate di coltivazione. Nonostante l’effetto delle condizioni climatiche sulla formazione di α e β-acidi sia ben conosciuto, non ci sono invece informazioni sulla variabilità delle molecole volatili del luppolo fra diverse annate. Per questa ragione, il profilo degli acidi e dei componenti volatili di sedici cultivar di luppolo coltivate nello stesso campo sperimentale a Parma (Emilia-Romagna, Italia) in due diverse annate sono state analizzate via Cromatografia liquida ad alta prestazione (HPLC) e Gas-cromatografia bidimensionale (GCXGC) rispettivamente. È stata evidenziata, in generale, una forte variabilità degli acidi e dei componenti volatili di luppolo in rispetto all’annata. Ciò nonostante, gli acidi del luppolo e i composti volatili chiave mostrano un pattern di base tipico per ogni varietà. In aggiunta, undici varietà di luppolo coltivate nel 2013 sono state sottoposte ad estrazione ad ultrasuoni, e gli estratti ottenuti sono stati utilizzati per aromatizzare una birra stile Blond Ale. Ogni birra aromatizzata è stata successivamente analizzata sensorialmente da un panel addestrato. Infine, le proprietà sensoriali delle birre aromatizzate sono state valutate in relazione al profilo volatile delle varietà ed alcune correlazioni sono state evidenziate. Sono state evidenziate correlazioni significative (p< 0.05) fra specifici componenti volatili e i descrittori aromatici ‘luppolato’, ‘erbaceo’ e ‘speziato’, mentre poche molecole volatili sono risultate essere correlate con i descrittori ‘agrumato’, ‘fruttato’ e ‘floreale’. Una caratterizzazione chimica e sensoriale dei componenti volatili in differenti cultivar di luppolo è stata effettuata anche allo scopo di identificare le molecole odorose di luppoli particolarmente agrumati. Infatti, nonostante differenti caratteristiche odorose come note erbacee, floreali/fruttate e speziate siano già state descritte, pochi tentativi sono stati effettuati per correlare chiaramente l’aroma agrumato con il profilo chimico del luppolo. Gli oli essenziali di tre varietà di luppolo con un noto carattere ‘agrumato’ e tre varietà di luppolo ‘speziate’/’luppolate’ sono stati analizzati chimicamente e sensorialmente. Ciascun olio essenziale è stato frazionato tramite estrazione in fase solida (SPE) e, grazie all’analisi sensoriale descrittiva delle diverse frazioni, la frazione SPE 70/30 etanolo/acqua (v/v) è risultata essere la più agrumata. Questa frazione è stata successivamente analizzata tramite microestrazione in fase folida in spazio di testa (HS-SPME) e Gas Cromatografia−Spettrometria di Massa/Olfattometria (GC-MS/O). Sono state individuate 59 molecole volatili, 35 delle quali sono risultate essere molecole odorose. In questo studio sono state caratterizzate le molecole odorose della frazione agrumata dell’olio essenziale di luppolo e grazie a clusterizzazione e analisi delle componenti principali (PCA) è stato possibile evidenziare che luppoli che esprimono un tipico aroma agrumato sono caratterizzati da alti livelli di particolari esteri come ad esempio metil (E)-4-decenoato, nonanoato di metile e decanoato di metile, alti livelli di esteri del geraniolo e nerolo e bassi livelli di sesquiterpeni ‘erbacei/speziati’ come ad esempio α-umulene e β-cariofillene.
Riassumendo, in questo lavoro sono state evidenziate differenti risposte allo stress idrico in diverse cultivar di luppolo, permettendo di individuare cultivar più tolleranti e sensibili. La qualità di luppoli coltivati in Nord-Italia (espressa come concentrazione di acidi e profilo volatile) e la loro variabilità sono state delineate in due annate diverse ed alcune importanti caratteristiche organolettiche (specialmente la nota ‘agrumata’) sono state caratterizzate per la prima volta dal punto di vista molecolare. Questo progetto ha dunque aumentato la nostra conoscenza in merito alla risposta allo stress idrico in luppolo e in merito al suo aroma, fornendo informazioni basilari utili per coltivatori e per l’industria del luppolo.

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Tipo di EPrint:Tesi di dottorato
Relatore:Bona, Stefano
Dottorato (corsi e scuole):Ciclo 28 > Scuole 28 > SCIENZE DELLE PRODUZIONI VEGETALI
Data di deposito della tesi:28 Gennaio 2016
Anno di Pubblicazione:28 Gennaio 2016
Parole chiave (italiano / inglese):hop acids, volatiles, GC-MS, olfactometry, water stress, QDA test, beer
Settori scientifico-disciplinari MIUR:Area 07 - Scienze agrarie e veterinarie > AGR/02 Agronomia e coltivazioni erbacee
Struttura di riferimento:Dipartimenti > Dipartimento di Agronomia Animali Alimenti Risorse Naturali e Ambiente
Codice ID:9270
Depositato il:21 Ott 2016 10:46
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