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Bergamaschi, Matteo (2016) Volatile Organic Compounds in cheese production chain (VOCheese). [Ph.D. thesis]

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Abstract (english)

In recent years, consumers have become increasingly interested in the quality aspects of food. Food quality, in turn, is strongly related to the sensory characteristics such as the flavor. Several scientific studies have shown that the Volatile Organic Compounds (VOCs) released by the food are related to the flavor and can be considered as assistive markers in the production chain.
Today, the analysis of VOCs requires fast, non-invasive, and solvent free devices. It has been shown that the VOCs can be extracted, identified, and measured with a Gas Chromatography-Mass Spectrometry (GC-MS) without any pre-concentration or pre-treatment of the food.
The main objective of this PhD thesis was to investigate the presence of volatile compounds in dairy products. More precisely, this study aimed in i) qualifying and quantifying VOCs in dairy products, ii) examining their formation and iii) integrating knowledge on VOCs by tracking their release during the whole production process from the raw materials till the final dairy product. In addition, statistical analysis was applied to link VOCs with the genetic characterization of animals, dairy system and individual cow-factors (e.g. stage of lactation, order of parity and milk yield). The identification and quantification of VOCs were performed using fast and non-invasive analytical approaches (Solid Phase Micro Extraction/Gas Chromatography-Mass Spectrometry SPME/GC-MS and Proton Transfer Reaction-Time of Flight-Mass Spectrometry PTR-ToF-MS) that can monitor the evolution of VOCs. To achieve the overall goal, the research was partitioned in four interrelated subparts as described below.
The aim of the first chapter was to study the VOCs presence in the headspace of cheese. To this purpose, 150 cheeses ripened for two months were used. The cheeses were obtained through an individual model cheese-making approach using milk from individual Brown Swiss cows. Animals reared in 30 herds belonging to different dairy systems, from traditional (typical of the mountainous area) to modern ones. The study identified 55 VOCs classified in the chemical families of free fatty acids, esters, alcohols, aldehydes, ketones, lactones, terpenes, and pyrazines. We found that dairy system and individual cow characteristics (lactation stage, order of parity and daily milk yield) influenced the volatile compounds. In order, to test the instrument reproducibility and the model cheese-making procedure; data of GC analysis, order of injection of the sample into instrument, and vat were included in the statistical model. In many cases, these analytical factors did not affect the amount of VOCs released by cheese.
In the second chapter, the potential of a new spectrometric technique (PTR-ToF-MS) was investigated to study cheese quality traits on a large scale. The PTR-ToF-MS allows direct injection of the sample headspace without extraction or pre-concentration steps, has a shorter analysis time (only a few seconds per sample) and greater sensitivity that permit to monitor on-line the evolution of volatile compounds. The resulting spectral information can provide a very detailed description of samples, which is useful for characterizing food quality and typicality. In particular, we analyzed the volatile fingerprint of 1,075 model cheeses produced using individual milk of Brown Swiss cows reared in 72 herds of different dairy systems. The output of PTR (spectrum) was characterized by more than 600 spectrometric peaks (variables). After removing interfering ions and background noise a set of 240 peaks was selected. Further, based on the results of the first contribution and literature, 61 peaks were identified. These peaks represent the major part of the cheese flavor. To summarize the amount of information, a multivariate analysis (PCA) was applied associating principal components (PC) with the 240 spectrometric peaks. Following, we tried to characterize the PCs through the correlations between PCs and the spectrometric peaks. The effects of dairy system, herd within dairy system, individual cows characteristics (lactation stage, order of parity and milk yield), and vat used for the cheese-making on the PCs and on the 240 peaks were analyzed. Dairy system was correlated with PC and 57 spectrometric peaks, especially when the herds were using Total Mixer Ration (TMR) as feeding technique, including or not maize silage in the diets. Regarding the individual animal characteristics, the most significant effect was the stage of lactation (139 peaks), followed by milk yield and parity, with 31 and 21 peaks, respectively. Finally, the vat used for the cheese-making was not found to be significant, confirming the good reproducibility of the model cheese-making procedure used to study cheese quality aspects.
In the third chapter, the effect of cows’ genetics to the VOCs of ripened cheeses was assessed. Principal components and the 240 spectrometric peaks (as described above in the second contribution) were used fitting an animal model in a Bayesian framework. On average, heritability (h2) of 7% for PCs was found, which is similar to h2 of somatic cell count and much lower than the h2 of milk fat content and daily milk yield. It is interesting to note that only a small proportion of peaks showed very low h2 (<7%). The major part of them showed values similar to those found for PCs, while forty peaks presented heritability similar to that of milk yield and other milk quality traits. The variability attributed to the herd was different for the various PC. Results suggest a potential of improvement for several cheese VOCs through genetic selection in dairy cow breeding programs.
The aim of the fourth chapter was to study the effect of summer transhumance on the quality traits of dairy products. Due to the extended work, this contribution was further splitted into two parts. In the first part, the evolution of milk and cheese quality characteristics were studied, while in the second part the evolution of VOC content of dairy products was analyzed.
For the first part, chemical characteristics and technological properties of 11 dairy products obtained during summer transhumance of cows to Alpine pastures (Malga) were analyzed. Dairy products obtained throughout this period are known to give origin to high-value, healthier products, and extra tasty,. Bulk milk from 148 dairy cows reared day and night on Alpine pasture (1,860 m a.s.l.) was used. We performed 7 experimental cheese-making according to traditional mountain techniques, one every two weeks, using milk produced during the summer transhumance (from June to September). For each cheese-making we collected: milk from the evening milking (day before the cheese-making), the same milk the following morning (after natural creaming), the cream separated, the whole milk from the morning milking, the milk in vat obtained mixing the creamed evening milk with the whole morning milk, the fresh curd, the whey, the ricotta obtained from whey, and the residual scotta. Moreover, the curd was used to produce typical “Malga” cheese that was ripened for 6 and 12 months. The chemical characteristics were measured with infrared technology. Results highlighted variation in milk yield, milk chemical composition, cheese yield and curd recoveries and/or loss of nutrients in the traditional cheese-making. In particular, a reduction of milk yield, fat, protein and lactose contents of milk during summer transhumance was observed. Nevertheless, the return to lowland farming systems of the cows at the end of grazing season, positively affected milk yield and milk chemical composition. The average of cheese yield was 14.2%, while recoveries of fat, protein, total solids and energy were 85.1%, 77.8%, 49.4% and 58.1%, respectively. These results were in accordance to those found in the literature.
For the second part of this chapter, the VOCs content of sample headspace was measured through SPME/GC-MS. Forth nine VOCs belonging to the chemical families of alcohols, aldehydes, free fatty acids, ketones, esters, lactones, terpenes, phenolic, and sulphur compounds were detected. In addition, the evolution of VOCs and their chemical family across the cheese- and ricotta-making processing as well as during the cheese ripening period was tracked. The comparison between VOCs concentration of 4 types of milk (whole evening, creaming milk, whole morning, milk in vat) showed that the creaming process significantly affected about half of all the volatile organic compounds analyzed, followed by the effects of milking (evening milking vs. morning milking) and the mixing (creamed milk mixed with whole morning milk). In general, the cream, in contrast to curd and ricotta, showed higher content of free fatty acids, sulphurs and terpenes compounds. Moreover, in ricotta a higher VOC concentration was observed compared to the curd, probably due to the high temperature required during the ricotta process. The effect of the progressive nutrient depletion of milk was investigated by contrasts between VOC concentration of milk in the vat, whey, and scotta. Although milk contains a greater amount of nutrients, whey and scotta have shown a higher concentration of VOCs with the exceptions of esters, sulphurs, terpenes and phenolic compounds. Finally, the effect of ripening was tested by comparing the quantity of VOCs of curd and of aged cheeses (6 and 12 months). The release of volatile compounds increased with increasing ripening period in relation with the enzymatic and microbiological activity of cheese.
In summary, the spectrometric techniques (SPME/GC-MS and PTR-ToF-MS) used in this work demonstrated to be very efficient to characterize the volatile organic compounds of dairy products. The dairy system, and cow related factors affected the volatile fingerprint of ripened cheeses. Particularly, concerning the individual animal source of variation, lactation stage was the most important effect followed by the cow’s parity and the milk yield.
On the basis of phenotypes used in this work, the traits collected offered the potential for a genetic analysis to be carried out. The genetic analysis demonstrated the existence of an exploitable genetic variability of the volatile profile of cheese that might be useful for an (in)direct selection of dairy cows for cheese quality traits in breeding programs. Nevertheless, further research is needed in this area. In the era of genomics for e.g., it might be interesting to associate genomic regions to specific VOCs. This information might be useful for genomic breeding programs.
The evolution of volatile compounds across the production chain depends on specific technological aspects, such as the process of natural creaming, the temperature of coagulation, and the ripening period. The monitoring of volatile fingerprint permits to obtain dairy products with specific organoleptic characteristics useful to differentiate them on the market and to improve the supply chain efficiency on the basis of quality aspects.

Abstract (italian)

Negli ultimi anni, il consumatore è diventato sempre più sensibile agli aspetti qualitativi degli alimenti, i quali sono fortemente influenzati dalle caratteristiche sensoriali come l’aroma. Diversi lavori scientifici hanno dimostrato che i composti volatili (VOCs) rilasciati dall’alimento sono correlati con il suo aroma e possono essere considerati come traccianti delle filiere alimentari.
Oggi, l’analisi dei VOCs richiede strumenti rapidi, sensibili, non invasivi e che abbiano bisogno dell’impiego di pochi solventi durante la preparazione del campione. E’ stato dimostrato che i VOCs possono essere estratti, misurati e identificati con la Gas Cromatografia di Massa (GC-MS) senza pre-concentrazioni o pre-trattamenti dell’alimento da analizzare.
Gli obiettivi principali della tesi di dottorato erano di studiare la presenza di composti volatili nei prodotti lattiero-caseari. Più precisamente, questo studio aveva come obiettivi di i) qualificare e quantificare i VOCs nei prodotti lattiero-caseari, ii) esaminare la loro formazione e iii) integrare le conoscenze acquisite su questi composti attraverso tutta la filiera di produzione dalla materia prima fino al prodotto finito. Inoltre, analisi statistiche sono state utilizzate per collegare i VOCs con la caratterizzazione genetica degli animali, il sistema di allevamento e le caratteristiche individuali delle vacche (es. stadio di lattazione, ordine di parto e produzione giornaliera di latte).
L’identificazione e la quantificazione dei VOCs sono state fatte utilizzando tecniche analitiche precise, veloci e non invasive (Solid Phase Micro Extraction/Gas Chromatography-Mass Spectrometry SPME/GC-MS and Proton Transfer Reaction-Time of Flight-Mass Spectrometry PTR-ToF-MS). Per rispondere agli obiettivi generali della tesi, l’attività di ricerca è stata divisa in cinque parti connesse tra di loro.
L’obiettivo nel primo capitolo era di studiare i composti volatili presenti nello spazio di testa di campioni di formaggio. Per questo scopo, sono stati analizzati 150 formaggi stagionati per due mesi. I formaggi sono stati prodotti utilizzando una metodica di caseificazione individuale usando latte individuale di vacche di razza Bruna. Gli animali sono stati allevati in 30 aziende appartenenti a diversi sistemi di allevamento, da tradizionale (tipico della realtà montana) a moderno. In questo studio sono stati identificati 55 VOCs per ogni formaggio, classificati in diverse famiglie chimiche: acidi grassi, esteri, alcoli, aldeidi, chetoni, lattoni, terpeni e pirazine. Dai risultati emerge che il sistema di allevamento e le caratteristiche individuali delle vacche (stadio di lattazione, ordine di parto e produzione giornaliera di latte) influenzano i composti volatili. Inoltre, per testare la riproducibilità dello strumento e della metodica di caseificazione; la data di analisi cromatografica, l’ordine d’iniezione del campione nello strumento (GC), e la caldaia di caseificazione erano inclusi nel modello statistico. In molti casi, questi fattori analitici/strumentali non influenzano la quantità di VOCs rilasciata dai formaggi.
Nel secondo capitolo, il potenziale di una nuova tecnica analitica (PTR-ToF-MS) è stato approfondito per studiare, su larga scala, le caratteristiche qualitative del formaggio. Il PTR-ToF-MS dal punto di vista analitico, permette un’iniezione diretta del campione senza estrazione o pre-concentrazione, ha un breve tempo di analisi (solo pochi secondi per campione) e grande sensibilità consentendo di monitorare in tempo reale l’evoluzione dei composti volatili. L’analisi produce uno spettro molto dettagliato che può essere utile per la caratterizzazione delle qualità e della tipicità dell’alimento. In particolare, è stata analizzata l’impronta aromatica di 1,075 formaggi prodotti utilizzando latte individuale di vacche di razza Bruna allevate in 72 aziende appartenenti a diversi sistemi di allevamento. L’impronta aromatica (spettro) era caratterizzata da più di 600 picchi (variabili) per ogni formaggio. Gli spettri sono stati analizzati e dopo la rimozione degli ioni interferenti e del rumore di fondo è stato selezionato un data set costituito da 240 picchi per ogni formaggio. In seguito, basandosi sui risultati del primo contributo e sulla letteratura sono stati identificati i picchi più importanti (61) in termini quantitativi e qualitativi. Per sintetizzare la quantità di informazioni ovvero estrarre delle componenti principali (PC) è stata fatta un’analisi multivariata (PCA) a partire dai 240 picchi spettrometrici. In seguito, le PCs sono state caratterizzate sulla base delle loro correlazioni con i 240 picchi spettrometrici. Sono stati analizzati gli effetti del sistema di allevamento, dell’azienda entro sistema di allevamento, le caratteristiche individuali delle vacche (stadio di lattazione, ordine di parto e produzione di latte), e caldaia di caseificazione sulle PCs e sui 240 picchi. Dai risultati emerge che il sistema di allevamento è correlato con le PC e 57 picchi, specialmente quando le aziende come tecnica di alimentazione utilizzano il carro miscelatore (TMR) con e senza insilati nella dieta. Considerando le caratteristiche individuali delle vacche, l’effetto più significativo è lo stadio di lattazione (139 picchi), seguito dalla produzione di latte e dall’ordine di parto, con 31 e 21 picchi, rispettivamente. Infine, la caldaia di caseificazione è un effetto spesso non significativo, confermando la buona riproducibilità della micro-caseificazione utilizzata anche per lo studio di aspetti qualitativi del formaggio.
Nel terzo capitolo è stato studiato l’effetto della genetica dell’animale sui composti volatili dei formaggi. A tale scopo, sono state analizzate le componenti principali (estratte come discusso sopra nel secondo contributo) e i 240 picchi spettrometrici (PTR-ToF-MS) utilizzando un modello animale con un approccio Baesiano. Dai risultati emerge in media un’ereditabilità (h2) del 7 % per le componenti principali, la quale è simile all’h2 trovata per le cellule somatiche e leggermente più bassa di quella del contenuto di grasso nel latte e della produzione giornaliera di latte stimate in precedenza sugli stessi animali. E’ interessante osservare che solo una piccola quantità di picchi ha una bassa h2 (<7%). La maggior parte di essi presenta valori simili a quelli trovati per le PCs, mentre 40 picchi presentano ereditabilità simile a quella trovata per la produzione giornaliera di latte e ad altre caratteristiche qualitative del latte. La variabilità attribuita all'azienda è risultata diversa per le PCs. Questi risultati dimostrano che esiste un’interessante variabilità genetica di alcuni VOCs che potrebbe essere potenzialmente utilizzata nei programmi di miglioramento genetico.
L’obiettivo nel quarto capitolo era di studiare l’effetto della transumanza sulle caratteristiche qualitative di prodotti lattiero-caseari. Vista la grande mole di dati, questo contributo è stato diviso in due parti tra loro connesse. Nella prima parte è stata studiata l’evoluzione della qualità del latte e del formaggio, mentre nella seconda parte è stata analizzata l’evoluzione dei composti volatili dei prodotti lattiero-caseari nel processo di caseificazione.
Nella prima parte, sono state analizzate le proprietà fisiche, chimiche e tecnologiche di 11 prodotti lattiero-caseari raccolti durante la transumanza al pascolo Alpino (Malga) di vacche da latte. E’ risaputo che i prodotti ottenuti durante il periodo di alpeggio possono avere un valore aggiunto dovuto alle elevate proprietà nutrizionali, salutistiche e aromatiche. Per approfondire le conoscenze finora acquisite, è stata fatta questa prova in cui è stato utilizzato il latte di massa prodotto da 148 vacche allevate giorno e notte al pascolo (1,860 m s.l.m.). Durante l’esperimento, sono state fatte 7 caseificazioni seguendo tecniche tradizionali, una ogni 2 settimane, utilizzando il latte prodotto durante la transumanza (da giugno a settembre). Sono stati raccolti per ogni caseificazione: il latte della mungitura della sera (giorno prima della caseificazione), lo stesso latte il mattino successivo (dopo il processo di scrematura naturale), la panna di affioramento, il latte della mungitura del mattino, il latte in caldaia ottenuta dalla miscela tra il latte scremato della mungitura della sera con il latte della mungitura del mattino, la cagliata, il siero, la ricotta ottenuta dal siero e il residuo della lavorazione ossia la scotta. Inoltre, la cagliata è stata usata per produrre formaggi di “Malga” che sono stati stagionati per 6 e 12 mesi. Le caratteristiche chimico-fisiche sono state misurate con una tecnologia a infrarosso. I risultati dimostrano una variazione della produzione giornaliera e composizione chimica del latte, resa in formaggio e recupero/o perdita di nutrienti nel processo di caseificazione tradizionale. In particolare, si è osservata una riduzione della produzione giornaliera di latte, grasso, proteine e lattosio del latte durante la transumanza estiva. Tuttavia, si è anche osservato un effetto positivo sulla produzione e la composizione chimica del latte del ritorno delle vacche nelle aziende di fondo valle alla fine della stagione dell’alpeggio. La resa media di formaggio in questo lavoro è risultata del 14.2%, mentre i recuperi di grasso, proteine, solidi totali ed energia sono del 85.1%, 77.8%, 49.4% e 58.1%, rispettivamente. Questi risultati sono in linea con quelli trovato in letteratura.
Nella seconda parte di questo contributo, è stato misurato il contenuto di composti volatili nello spazio di testa dei campioni con la tecnica SPME/GC-MS. Dopo l’analisi, sono stati identificati 49 VOCs appartenenti alle famiglie chimiche degli alcoli, aldeidi, acidi grassi, chetoni, esteri, lattoni, terpeni e composti solforati e fenolici. Inoltre, è stata studiata l’evoluzione dei VOCs e delle loro famiglie chimiche attraverso i processi di caseificazione, di produzione della ricotta e di stagionatura del formaggio. Il confronto tra la concentrazione dei VOCs dei 4 tipi di latte (intero e scremato della sera, intero del mattino, caldaia) ha dimostrato che il processo di scrematura influenza la concentrazione di metà dei composti volatili analizzati, seguito dall’effetto della mungitura (intero della sera vs. intero del mattino) e dall’effetto del mescolamento (latte scremato della sera mescolato in parti uguali con il latte del mattino). In generale, la panna, rispetto a cagliata e ricotta, ha un maggiore contenuto di acidi grassi, terpeni e composti solforati. Inoltre, la ricotta rispetto alla cagliata ha un’elevata concentrazione di VOC, probabilmente dovuta alla maggiore temperatura utilizzata durante il processo di produzione. L’effetto del progressivo depauperamento di nutriente del latte è stato studiato attraverso il confronto tra latte in caldaia, siero e scotta. Sebbene il latte abbia un maggiore contenuto di nutrienti, il siero e la scotta hanno una maggiore concentrazione di VOC ad eccezione delle famiglie chimiche degli esteri, terpeni, composti solforati e fenolici. Infine, l’effetto della maturazione è stato valutato attraverso il confronto tra le quantità di VOC della cagliata e dei formaggi stagionati (6 e 12 mesi). Il rilascio dei composti volatili incrementa con l’aumento del periodo di maturazione probabilmente dovuto a una maggiore attività enzimatica e microbiologica nel formaggio.
In conclusione, le tecniche analitiche di spettrometria di massa utilizzate in questo lavoro (SPME/GC-MS e PTR-ToF-MS) hanno permesso di caratterizzare i composti volatili dei prodotti lattiero-caseari in maniera efficiente. Il sistema di allevamento, le caratteristiche individuali delle vacche hanno influenzato l’impronta aromatica di formaggi individuali stagionati. In particolare, riguardo alle caratteristiche individuali degli animali il principale effetto era lo stadio di lattazione seguito da ordine di parto e produzione giornaliera di latte.
Sulla base dei fenotipi raccolti in questo lavoro è stato possibile effettuare un’analisi genetica, la quale ha dimostrato l’esistenza di un’interessante variabilità genetica connessa ai composti volatili del formaggio che potrebbe essere utile per una selezione (in)diretta delle vacche da latte sulla base di aspetti qualitativi in programmi di miglioramento genetico. Tuttavia sono necessarie altre ricerche in quest’area per esempio, nell’era della genomica, sarebbe interessante associare qualche regione specifica del genoma ai composti volatili.
L’evoluzione dei composti volatile attraverso la filiera di produzione dipende da specifici aspetti tecnologici, come l’affioramento della panna, la temperatura di coagulazione e il periodo di stagionatura. Il monitoraggio dell’impronta aromatica permette di ottenere prodotti lattiero-caseari con delle specifiche caratteristiche organolettiche utili a differenziare il prodotto sul mercato e a migliorare l’efficienza dell’intera filiera produttiva sulla base di aspetti qualitativi.

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EPrint type:Ph.D. thesis
Tutor:Bittante, Giovanni
Data di deposito della tesi:31 January 2016
Anno di Pubblicazione:31 January 2016
Key Words:cheese quality; flavor; volatile organic compound; SPME/GC-MS; PTR-ToF-MS.
Settori scientifico-disciplinari MIUR:Area 07 - Scienze agrarie e veterinarie > AGR/17 Zootecnica generale e miglioramento genetico
Struttura di riferimento:Dipartimenti > Dipartimento di Agronomia Animali Alimenti Risorse Naturali e Ambiente
Codice ID:9458
Depositato il:21 Oct 2016 10:49
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