Due to their recognized health-promoting effects, concentrates of PolyUnsaturated Fatty Acids (PUFA) are considered both valuable dietary supplements and ingredients to enrich foodstuff or to produce functional food. Moreover, the interest in mono- and diacylglycerols (MAG and DAG, respectively) of PUFA as non-ionic emulsifiers and stabilizers in food, cosmetic and pharmaceutical industries is rapidly increasing. Hempseed oil has an high content of unsaturated fatty acids, such as linoleic acid (LA, 59%) and α-linolenic acid (ALA, 18%), that constitute 77% of the total content of fatty acids in the oil, oleic acid (OA, 13%) and a minor amount of saturated fatty acids (7% of palmitic acid, PA; 3% of stearic acid, SA). Particularly, the ω-6/ω-3 ratio in hempseed oil (3.3:1) is considered highly beneficial from a nutritional point of view. Recently, the lipase-catalyzed hydrolysis of hempseed oil in a homogeneous medium based on oil and t-BuOH/water was developed with the aim to obtain an oil devoid of saturated fatty acids and enriched in PUFA (as MAG and DAG) for nutraceutical/food applications. Upon an enzymatic screening, the lipase from Pseudomonas cepacia (PCL) was selected as the biocatalyst. The overall purified glyceride component (MAG and DAG) obtained from the hydrolysis catalyzed by PCL accounted for 82% of PUFA (LA+ALA, yield 29%) [5]. This procedure has many advantages as it occurs under mild conditions, it is easily scalable and it relies on a standard chromatography purification step. In this work, carried out in collaboration with the Tuscia University, PCL was covalently immobilized and used to set up a reaction system suitable to be transferred to a continuous flow mode in a Packed-Bed Reactor (PBR). To this aim, different supports and binding chemistry were screened in the immobilization trials. Optimization of the reaction conditions was carried out using a 2^3 full factorial design including the type of organic solvent, the percentage of water and temperature as variables. The model derived from the experimental design showed that none of the variables considered and none interaction between them significatively affected the response (i.e. the percentage of PUFA as acylglycerols). Indeed, the experimental conditions previously used in the batch hydrolysis catalyzed by non immobilized PCL were applied to the biotransformation performed by PBR. The hydrolysis of hempseed oil was carried out by feeding the reaction mixture upwards the PBR (containing the immobilized enzymes) at varying flow rates through a peristaltic pump. The system was coupled in-line with a second glass column filled with an ionic exchange resin to retain the hydrolyzed free fatty acids (FFA), thus allowing the straightforward purification of the glyceride component. A mixture of MAG and DAG (yield 40%) composed of LA and ALA (82%) and OA (16%) was obtained in less than 2 hours. The PCL-based PBR was successfully used for 10 cycles without any decrease of activity. In the view of applying this technology to rice bran oil (RBO), a preliminary screening of nine commercial lipases was carried out under different conditions. The final goal of this work will be the achievement of the total hydrolysis of RBO to FFA that will be used as precursors for the chemical synthesis of an array of high-added value products.
Per i riconosciuti effetti benefici sulla salute, gli acidi grassi polinsaturi (PUFA) sono utilizzati sia come integratori alimentari, sia come ingredienti di cibi funzionali (functional food). Mono- e diacilgliceroli (MAG e DAG) di PUFA sono allo studio anche come emulsionanti non ionici e stabilizzanti in campo alimentare, cosmetico e farmaceutico in alternativa ai corrispondenti derivati a base di acidi grassi saturi. L’olio di canapa è caratterizzato da un elevato contenuto di acidi grassi insaturi, in particolare acido linoleico (LA, 59%) e acido α-linolenico (ALA, 18%), che costituiscono il 77% degli acidi grassi totali presenti nell‘olio, da acido oleico (OA, 13%) e da una quantità minore di acidi grassi saturi (7% di acido palmitico, PA; 3% di acido stearico, SA). Non solo: il rapporto tra acidi grassi ω-6 e ω-3 che caratterizza l’olio di canapa (3.3:1) è considerato “ideale” dal punto di vista nutrizionale. Recentemente è stata messa a punto l’idrolisi enzimatica di olio di canapa in un medium omogeneo costituito da olio e t-BuOH/acqua allo scopo di eliminare dall’olio gli acidi grassi saturi e di arricchirlo in PUFA (sottoforma di MAG e DAG) per applicazioni alimentari e nutraceutiche. Dallo screening di sette lipasi commerciali è stata selezionata la lipasi da Pseudomonas cepacia (PCL). La componente glicerica totale purificata (MAG e DAG) conteneva 82% di PUFA (LA+ALA, resa=29% dopo 24 ore). L’approccio biocatalitico presenta molti vantaggi: avviene in condizioni blande, è facilmente scalabile e richiede una purificazione cromatografica standard. In questa tesi, svolta in collaborazione con l’Università della Tuscia, PCL è stata immobilizzata covalentemente e utilizzata per la messa a punto di un sistema di reazione trasferibile in modalità a flusso continuo in un Packed-Bed Reactor (PBR). A questo scopo, per l’immobilizzazione sono stati valutati diversi supporti e binding chemistry. Successivamente, è stata eseguita l’ottimizzazione delle condizioni di reazione mediante un approccio chemometrico basato su un disegno fattoriale completo (2^3) comprendente come fattori il tipo di solvente organico, la percentuale di acqua e la temperatura. Il modello ha evidenziato che né le variabili studiate, né le interazioni tra di esse sono significative ai fini della risposta (definita come percentuale di PUFA legati al glicerolo dopo l’idrolisi enzimatica). L’idrolisi dell’olio di canapa è stata quindi effettuata nelle medesime condizioni sperimentali utilizzate nella sintesi in batch, facendo fluire la miscela di reazione nel PBR (contenente l’enzima immobilizzato). Il PBR è stato collegato in serie a una seconda colonna contenente una resina a scambio ionico in grado di trattenere gli acidi grassi idrolizzati, consentendo quindi la separazione in-line degli acidi grassi liberi (FFA) dai MAG e DAG costituiti esclusivamente dagli acidi grassi insaturi. La miscela di MAG e DAG composta da LA e ALA (82%) e OA (16%) è stata ottenuta in meno di 2 ore con una resa del 40%. Il PBR è stato utilizzato per 10 cicli di reazione senza perdita di attività. In vista dell’utilizzo di questa tecnologia per il trattamento dell’olio di pula di riso (RBO), è stato effettuato uno screening di nove lipasi commerciali in diverse condizioni. Obiettivo di questo lavoro è la messa a punto dell’idrolisi enzimatica totale di RBO a FFA, che saranno utilizzati come precursori per la sintesi chimica di diversi composti ad elevato valore aggiunto.
“From batch to flow reactions”: produzione di concentrati di acidi grassi polinsaturi mediante idrolisi enzimatica di olio di canapa in Packed-Bed Reactor
QUAGLIA, MIRIAM
2014/2015
Abstract
Due to their recognized health-promoting effects, concentrates of PolyUnsaturated Fatty Acids (PUFA) are considered both valuable dietary supplements and ingredients to enrich foodstuff or to produce functional food. Moreover, the interest in mono- and diacylglycerols (MAG and DAG, respectively) of PUFA as non-ionic emulsifiers and stabilizers in food, cosmetic and pharmaceutical industries is rapidly increasing. Hempseed oil has an high content of unsaturated fatty acids, such as linoleic acid (LA, 59%) and α-linolenic acid (ALA, 18%), that constitute 77% of the total content of fatty acids in the oil, oleic acid (OA, 13%) and a minor amount of saturated fatty acids (7% of palmitic acid, PA; 3% of stearic acid, SA). Particularly, the ω-6/ω-3 ratio in hempseed oil (3.3:1) is considered highly beneficial from a nutritional point of view. Recently, the lipase-catalyzed hydrolysis of hempseed oil in a homogeneous medium based on oil and t-BuOH/water was developed with the aim to obtain an oil devoid of saturated fatty acids and enriched in PUFA (as MAG and DAG) for nutraceutical/food applications. Upon an enzymatic screening, the lipase from Pseudomonas cepacia (PCL) was selected as the biocatalyst. The overall purified glyceride component (MAG and DAG) obtained from the hydrolysis catalyzed by PCL accounted for 82% of PUFA (LA+ALA, yield 29%) [5]. This procedure has many advantages as it occurs under mild conditions, it is easily scalable and it relies on a standard chromatography purification step. In this work, carried out in collaboration with the Tuscia University, PCL was covalently immobilized and used to set up a reaction system suitable to be transferred to a continuous flow mode in a Packed-Bed Reactor (PBR). To this aim, different supports and binding chemistry were screened in the immobilization trials. Optimization of the reaction conditions was carried out using a 2^3 full factorial design including the type of organic solvent, the percentage of water and temperature as variables. The model derived from the experimental design showed that none of the variables considered and none interaction between them significatively affected the response (i.e. the percentage of PUFA as acylglycerols). Indeed, the experimental conditions previously used in the batch hydrolysis catalyzed by non immobilized PCL were applied to the biotransformation performed by PBR. The hydrolysis of hempseed oil was carried out by feeding the reaction mixture upwards the PBR (containing the immobilized enzymes) at varying flow rates through a peristaltic pump. The system was coupled in-line with a second glass column filled with an ionic exchange resin to retain the hydrolyzed free fatty acids (FFA), thus allowing the straightforward purification of the glyceride component. A mixture of MAG and DAG (yield 40%) composed of LA and ALA (82%) and OA (16%) was obtained in less than 2 hours. The PCL-based PBR was successfully used for 10 cycles without any decrease of activity. In the view of applying this technology to rice bran oil (RBO), a preliminary screening of nine commercial lipases was carried out under different conditions. The final goal of this work will be the achievement of the total hydrolysis of RBO to FFA that will be used as precursors for the chemical synthesis of an array of high-added value products.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/22881