Phenolic compounds from olive mill wastes: Health effects, analytical approach and application as food antioxidants

Background Over the years, food industry wastes have been the focus of a growing interest due to their content in high added-value compounds. A good example are the olive oil by-products (OOBP), which retain a great amount of phenolic compounds during olive oil production. Their structure and biological properties justify their potential use as antioxidants in other food products. The efficient recovery of phenolic compounds has been extensively studied and optimized in order to maximize their reintroduction in the food chain and contribute to a higher valorization and better management of wastes from olive oil industry. Scope and approach This paper reviews the most representative phenolic compounds described in OOBP and their biological properties. New extraction procedures to efficiently recover these compounds and the most advanced chromatographic techniques that have been used for a better understanding of the phenolic profile of these complex matrices are also referred. Finally, this paper reports the main applications of OOBP, with emphasis on their phenolic content as natural antioxidants for food applications. Key findings and conclusions Besides their antioxidant activity, phenolic compounds from OOBP have also shown antimicrobial and antitumoral properties. Their application as food antioxidants requires new extraction techniques, including the use of non-toxic solvents and, in a pilot scale, the use of filters and adsorbent resins. The inclusion of phenolic compounds from OOBP in some food matrices have improved not only their antioxidant capacity but also their sensory attributes.

Combined bioremediation and enzyme production by Aspergillus sp. in olive mill and winery wastewaters

Olive mill wastewaters (OMW) and vinasses (VS) are effluents produced respectively by olive mills and wineries, both sectors are of great economic importance in Mediterranean countries. These effluents cause a large environmental impact, when not properly processed, due to their high concentration of phenolic compounds, COD and colour. OMW may be treated by biological processes but, in this case, a dilution is necessary, increasing water consumption. The approach here in proposed consists on the bioremediation of OMW and VS by filamentous fungi. In a screening stage, three fungi (Aspergillus ibericus, Aspergillus uvarum, Aspergillus niger) were selected to bioremediate undiluted OMW, two-fold diluted OMW supplemented with nutrients, and a mixture of OMW and VS in the proportion 1:1 (v/v). Higher reductions of phenolic compounds, colour and COD were achieved mixing both residues; with A. uvarum providing the best results. In addition, the production of enzymes was also evaluated during this bioremediation process, detecting in all cases lipolytic, proteolytic and tannase activities. A. ibericus, A. uvarum and A. niger achieved the highest value of lipase (1253.7 ± 161.2 U/L), protease (3700 ± 124.3 U/L) and tannase (284.4 ± 12.1 U/L) activities, respectively. Consequently, this process is an interesting alternative to traditional processes to manage these residues, providing simultaneously high economic products, which can be employed in the same industries.

Identification and characterisation of phenolic compounds extracted from Moroccan olive mill wastewater

Olive mill wastewater, hereafter noted as OMWW was tested for its composition in phenolic compounds according to geographical areas of olive tree, i.e. the plain and the mountainous areas of Tadla-Azilal region (central Morocco). Biophenols extraction with ethyl acetate was efficient and the phenolic extract from the mountainous areas had the highest concentration of total phenols' content. Fourier-Transform-Middle Infrared (FT-MIR) spectroscopy of the extracts revealed vibration bands corresponding to acid, alcohol and ketone functions. Additionally, HPLC-ESI-MS analyses showed that phenolic alcohols, phenolic acids, flavonoids, secoiridoids and derivatives and lignans represent the most abundant phenolic compounds. Nüzhenide, naringenin and long chain polymeric substances were also detected. Mountainous areas also presented the most effective DPPH scavenging potential compared to plain areas; IC50 values were 11.7 ± 5.6 µg/ml and 30.7 ± 4.4 µg/ml, respectively. OMWW was confirmed as a rich source of natural phenolic antioxidant agents.

Development of a biorefinery scheme for the valorization of olive mill wastewaters and grape pomaces

In the Mediterranean area, olive mill wastewater (OMW) and grape pomace (GP) are among the major agro-industrial wastes produced. These two wastes have a high organic load and high phytotoxicity. Thus, their disposal in the environment can lead to negative effects. Second-generation biorefineries are dedicated to the valorization of biowaste by the production of goods from such residual biomasses. This approach can combine bioremediation approaches to the generation of noble molecules, biomaterials and energy. The main aim of this thesis work was to study the anaerobic digestion of OMW and GP under different operational conditions to produce volatile fatti acids (VFAs) (first stage aim) and CH4 (second stage aim). To this end, a packed-bed biofilm reactor (PBBR) was set up to perform the anaerobic acidogenic digestion of the liquid dephenolized stream of OMW (OMWdeph). In parallel, the solid stream of OMW (OMWsolid), previously separated in order to allow the solid phase extraction of polyphenols, was addressed to anaerobic methanogenic digestion to obtain CH4. The latter experiment was performed in 100ml Pyrex bottles which were maintained at different temperatures (55-45-37°C). Together with previous experiments, the anaerobic acidogenic digestion of fermented GP (GPfreshacid) and dephenolized and fermented GP (GPdephacid) was performed in 100ml Pyrex bottles to estimate the concentration of VFAs achievable from each aforementioned GPs. Finally, the same matrices of GP and not pre-treated GP (GPfresh) were digested under anaerobic methanogenic condition to produce CH4. Anaerobic acidogenic and methanogenic digestion processes of GPs lasted about 33 days. Instead, the anaerobic acidogenic and methanogenic digestion process of OMWs lasted about 121 and 60 days, respectively. Each experiment was periodically monitored by analysing volume and composition of produced biogas and VFA concentration. Results showed that VFAs were produced in higher concentrations in GP compared to OMWdeph. The overall concentration of VFAs from GPfreshacid was approximately 39.5 gCOD L-1, 29 gCOD L-1 from GPdephacid, and 8.7 gCOD L-1 from OMWdeph. Concerning the CH4 production, the OMWsolid reached a high biochemical methane potential (BMP) at a thermophilic temperature (55°) than at mesophlic ones (37-45°C). The value reached was about 358.7 mlCH4 gSVsub-1. In contrast, GPfresh got a high BMP but at a mesophilic temperature. The BMP was about 207.3 mlCH4 gSVsub-1, followed by GPfreshacid with about 192.6 mlCH4 gSVsub-1 and lastly GPdephacid with about 102.2 mlCH4 gSVsub-1. In summary, based on the gathered results, GP seems to be a better carbon source for acidogenic and methanogenic microrganism compared to OMW, because higher amount of VFAs and CH4 were produced in AD of GP than OMW. In addition to these products, polyphenols were extracted by means of a solid phase extraction (SPE) procedure by another research group, and VFAs were utilised for biopolymers production, in particular polyhydroxyalkanoates (PHAs), by the same research group in which I was involved.

Economic feasibility analysis of the implementation of a high value-added molecule extraction system in an olive oil mill

The production of olive oil generates several by-products that can be seen as an additional business opportunity. Among them are the olive pits, already used for heat and/or electricity generation in some mills. They contain compounds that are commercially very interesting and, if recovered, contribute to the sustainability of the olive mills. The work presented in this paper is a preliminary evaluation of the economic feasibility of implementing a system based on a batch prototype with 1 m3 for the extraction of high value-added bioactive molecules from olive pits that are separated during the production of virgin olive oil. For the analysis, a small representative olive mill in Portugal was considered and the traditional Discounted Cash Flow Method was applied. Based on the assumptions made, the simple payback for implementation a system for the extraction of value-added molecules from the olive pits is around 7 years.

Olive pomace pretreatments to enhance its valorisation by solid-state fermentation

Dissertação de mestrado em Bioengenharia

Soil quality assessment based on carbon stratification index in different olive grove management practices in Mediterranean areas

In Mediterranean areas, conventional tillage increases soil organic matter losses, reduces soil quality, and contributes to climate change due to increased CO2 emissions. CO2 sequestration rates in soil may be enhanced by appropriate agricultural soil management and increasing soil organic matter content. This study analyzes the stratification ratio (SR) index of soil organic carbon (SOC), nitrogen (N) and C:N ratio under different management practices in an olive grove (OG) in Mediterranean areas (Andalusia, southern Spain). Management practices considered in this study are conventional tillage (CT) and no tillage (NT). In the first case, CT treatments included addition of alperujo (A) and olive leaves (L). A control plot with no addition of olive mill waste was considered (CP). In the second case, NT treatments included addition of chipped pruned branches (NT1) and chipped pruned branches and weeds (NT2). The SRs of SOC increased with depth for all treatments. The SR of SOC was always higher in NT compared to CT treatments, with the highest SR of SOC observed under NT2. The SR of N increased with depth in all cases, ranging between 0.89 (L-SR1) and 39.11 (L-SR3 and L-SR4).The SR of C:N ratio was characterized by low values, ranging from 0.08 (L-SR3) to 1.58 (NT1-SR2) and generally showing higher values in SR1 and SR2 compared to those obtained in SR3 and SR4. This study has evaluated several limitations to the SR index such as the fact that it is descriptive but does not analyze the behavior of the variable over time. In addition, basing the assessment of soil quality on a single variable could lead to an oversimplification of the assessment. Some of these limitations were experienced in the assessment of L, where SR1 of SOC was the lowest of the studied soils. In this case, the higher content in the second depth interval compared to the first was caused by the intrinsic characteristics of this soil's formation process rather than by degradation. Despite the limitations obtained SRs demonstrate that NT with the addition of organic material improves soil quality.

Optimization of lipase production from yeasts strains isolated from olive mil wastewater

Dissertação submetida à Universidade de Lisboa, Faculdade de Ciências para a obtenção do Grau de Mestre em Microbiologia Aplicada.

Valorization of by-products from olive oil industry and their utilization for innovative food formulation

The purpose of this thesis work was the valorization of the main by-products obtained from olive oil production chain (wastewater and pomace) and their utilization in innovative food formulation. In the first part of the thesis, an olive mill wastewater extract rich in phenols were used in the formulation of 3 innovative meat products: beef hamburgers, cooked ham and würstels. These studies confirms that olive mill wastewaters extract rich in phenols could be an alternative for the reduction/total replacement of additives (i.e., nitrites) in ground and cooked meat preparations, which would promote the formulation of healthier clean label products and improve the sustainability of the olive oil industry with a circular economy approach, by further valorizing this olive by-product. In the second part of the thesis, the lipid composition and oxidative stability of a spreadable product obtained from a fermented and biologically de-bittered olive pomace, was assessed during a shelf-life study. This study confirmed that olive pomace represents an excellent ingredient for the formulation of functional foods In the third and last part of the thesis, carried out at the Universidad de Navarra (Pamplona, Spain), during a period abroad (3 months), three extracts obtained from purification of olive mill wastewaters, were subjected to in-vitro digestion and characterized. From the analysis of the three phenolic extracts, it emerged that the most promising extract to be used in the food field is the spry-dried one. Thanks to its formulation containing maltodextrins it manages to maintain its antioxidant capacity even after being underwent to in-vitro digestion. This thesis work is a part of the PRIN 2015 project (PROT: 20152LFKAT) "Olive phenols as multifunctional bioactives for healthier food: evaluation of simplified formulation to obtain safe meat products and new foods with higher functionality", coordinated by University of Perugia.

Processo biotecnologico a cellule immobilizzate per la produzione di acidi grassi volatili da acque di vegetazione

Le acque di vegetazione (AV) costituiscono un serio problema di carattere ambientale, sia a causa della loro elevata produzione sia per l’ elevato contenuto di COD che oscilla fra 50 e 150 g/l. Le AV sono considerate un refluo a tasso inquinante fra i più elevati nell’ambito dell’industria agroalimentare e la loro tossicità è determinata in massima parte dalla componente fenolica. Il presente lavoro si propone di studiare e ottimizzare un processo non solo di smaltimento di tale refluo ma anche di una sua valorizzazione, utlizzandolo come materia prima per la produzione di acidi grassi e quindi di PHA, polimeri biodegradabili utilizzabili in varie applicazioni. A tale scopo sono stati utilizzati due bioreattori anaerobici a biomassa adesa, di identica configurazione, con cui si sono condotti due esperimenti in continuo a diverse temperature e carichi organici al fine di studiare l’influenza di tali parametri sul processo. Il primo esperimento è stato condotto a 35°C e carico organico pari a 12,39 g/Ld, il secondo a 25°C e carico organico pari a 8,40 g/Ld. Si è scelto di allestire e mettere in opera un processo a cellule immobilizzate in quanto questa tecnologia si è rivelata vantaggiosa nel trattamento continuo di reflui ad alto contenuto di COD e carichi variabili. Inoltre si è scelto di lavorare in continuo poiché tale condizione, per debiti tempi di ritenzione idraulica, consente di minimizzare la metanogenesi, mediata da microrganismi con basse velocità specifiche di crescita. Per costituire il letto fisso dei due reattori si sono utilizzati due diversi tipi di supporto, in modo da poter studiare anche l’influenza di tale parametro, in particolare si è fatto uso di carbone attivo granulare (GAC) e filtri ceramici Vukopor S10 (VS). Confrontando i risultati si è visto che la massima quantità di VFA prodotta nell’ambito del presente studio si ha nel VS mantenuto a 25°C: in tale condizione si arriva infatti ad un valore di VFA prodotti pari a 524,668 mgCOD/L. Inoltre l’effluente in uscita risulta più concentrato in termini di VFA rispetto a quello in entrata: nell’alimentazione la percentuale di materiale organico presente sottoforma di acidi grassi volatili era del 54 % e tale percentuale, in uscita dai reattori, ha raggiunto il 59 %. Il VS25 rappresenta anche la condizione in cui il COD degradato si è trasformato in percentuale minore a metano (2,35 %) e questo a prova del fatto che l’acidogenesi ha prevalso sulla metanogenesi. Anche nella condizione più favorevole alla produzione di VFA però, si è riusciti ad ottenere una loro concentrazione in uscita (3,43 g/L) inferiore rispetto a quella di tentativo (8,5 g/L di VFA) per il processo di produzione di PHA, sviluppato da un gruppo di ricerca dell’università “La Sapienza” di Roma, relativa ad un medium sintetico. Si può constatare che la modesta produzione di VFA non è dovuta all’eccessiva degradazione del COD, essendo questa nel VS25 appena pari al 6,23%, ma piuttosto è dovuta a una scarsa concentrazione di VFA in uscita. Questo è di buon auspicio nell’ottica di ottimizzare il processo migliorandone le prestazioni, poiché è possibile aumentare tale concentrazione aumentando la conversione di COD in VFA che nel VS25 è pari a solo 5,87%. Per aumentare tale valore si può agire su vari parametri, quali la temperatura e il carico organico. Si è visto che il processo di acidogenesi è favorito, per il VS, per basse temperature e alti carichi organici. Per quanto riguarda il reattore impaccato con carbone attivo la produzione di VFA è molto ridotta per tutti i valori di temperatura e carichi organici utilizzati. Si può quindi pensare a un’applicazione diversa di tale tipo di reattore, ad esempio per la produzione di metano e quindi di energia.

Biometanazione della frazione organica di rifiuti solidi urbani da raccolta indifferenziata mediante codigestione con refluo zootecnico o addizione di solfuro di sodio

Il presente elaborato è stato finalizzato allo sviluppo di un processo di digestione anaerobica della frazione organica dei rifiuti solidi urbani (FORSU oppure, in lingua inglese OFMSW, Organic Fraction of Municipal Solid Waste) provenienti da raccolta indifferenziata e conseguente produzione di biogas da impiegarsi per il recupero energetico. Questo lavoro rientra nell’ambito di un progetto, cofinanziato dalla Regione Emilia Romagna attraverso il Programma Regionale per la Ricerca Industriale, l’Innovazione e il Trasferimento Tecnologico (PRRIITT), sviluppato dal Dipartimento di Chimica Applicata e Scienza dei Materiali (DICASM) dell’Università di Bologna in collaborazione con la Facoltà di Ingegneria dell’Università di Ferrara e con la società Recupera s.r.l. che applicherà il processo nell’impianto pilota realizzato presso il proprio sito di biostabilizzazione e compostaggio ad Ostellato (FE). L’obiettivo è stato la verifica della possibilità di impiegare la frazione organica dei rifiuti indifferenziati per la produzione di biogas, e in particolare di metano, attraverso un processo di digestione anaerobica previo trattamento chimico oppure in codigestione con altri substrati organici facilmente fermentabili. E’ stata inoltre studiata la possibilità di impiego di reattori con biomassa adesa per migliorare la produzione specifica di metano e diminuire la lag phase. Dalla sperimentazione si può concludere che è possibile giungere allo sviluppo di metano dalla purea codigerendola assieme a refluo zootecnico. Per ottenere però produzioni significative la quantità di solidi volatili apportati dal rifiuto non deve superare il 50% dei solidi volatili complessivi. Viceversa, l’addizione di solfuri alla sola purea si è dimostrata ininfluente nel tentativo di sottrarre gli agenti inibitori della metanogenesi. Inoltre, l’impiego di supporti di riempimento lavorando attraverso processi batch sequenziali permette di eliminare, nei cicli successivi al primo, la lag phase dei batteri metanogeni ed incrementare la produzione specifica di metano.

Development of instrumental and sensory analytical methods of food obtained by traditional and emerging technologies

The consumer demand for natural, minimally processed, fresh like and functional food has lead to an increasing interest in emerging technologies. The aim of this PhD project was to study three innovative food processing technologies currently used in the food sector. Ultrasound-assisted freezing, vacuum impregnation and pulsed electric field have been investigated through laboratory scale systems and semi-industrial pilot plants. Furthermore, analytical and sensory techniques have been developed to evaluate the quality of food and vegetable matrix obtained by traditional and emerging processes. Ultrasound was found to be a valuable technique to improve the freezing process of potatoes, anticipating the beginning of the nucleation process, mainly when applied during the supercooling phase. A study of the effects of pulsed electric fields on phenol and enzymatic profile of melon juice has been realized and the statistical treatment of data was carried out through a response surface method. Next, flavour enrichment of apple sticks has been realized applying different techniques, as atmospheric, vacuum, ultrasound technologies and their combinations. The second section of the thesis deals with the development of analytical methods for the discrimination and quantification of phenol compounds in vegetable matrix, as chestnut bark extracts and olive mill waste water. The management of waste disposal in mill sector has been approached with the aim of reducing the amount of waste, and at the same time recovering valuable by-products, to be used in different industrial sectors. Finally, the sensory analysis of boiled potatoes has been carried out through the development of a quantitative descriptive procedure for the study of Italian and Mexican potato varieties. An update on flavour development in fresh and cooked potatoes has been realized and a sensory glossary, including general and specific definitions related to organic products, used in the European project Ecropolis, has been drafted.

Valorizzazione di acque di vegetazione provenienti da oleifici tramite recupero di polifenoli per adsorbimento

The environmental problems associated to the disposal of the olive oil wastewater (OMW, Olive Mill Wastewaters), and the difficulty in the recycling because of their polyphenolic content, led to propose the separation of their polyphenols. The recovery of polyphenols from olive mill wastewaters (OMWs) provides the double opportunity to obtain high-added value biomolecules and to reduce the phytotoxicity of the effluent. The separation can be obtained by a continuous flow extraction process of adsorption and desorption, this process is characterized by the possibility to recycling the adsorbing phase and the extraction solvent. The present work is part of a project aimed to the development of pilot scale process based on the use of the AMBERLITE XAD16 as adsorption resin. The adsorption stage is preceded by a wastewater filtration pretreatment, and a final desorption process is added to recover the polyphenols using acidified ethanol as the solvent.

Arsenic and selenium mobilisation from organic matter treated mine spoil with and without inorganic fertilisation

Organic matter amendments are applied to contaminated soil to provide a better habitat for re-vegetation and remediation, and olive mill waste compost (OMWC) has been described as a promising material for this aim. We report here the results of an incubation experiment carried out in flooded conditions to study its influence in As and metal solubility in a trace elements contaminated soil. NPK fertilisation and especially organic amendment application resulted in increased As, Se and Cu concentrations in pore water. Independent of the amendment, dimethylarsenic acid (DMA) was the most abundant As species in solution. The application of OMWC increased pore water dissolved organic-carbon (DOC) concentrations, which may explain the observed mobilisation of As, Cu and Se; phosphate added in NPK could also be in part responsible of the mobilisation caused in As. Therefore, the application of soil amendments in mine soils may be particularly problematic in flooded systems.

Anaerobic digestion process for biogas and biomolecules production: microflora identification and characterization

The anaerobic process was efficient in organic matter removal. During the process, an interesting compound as quercetin was produced inside of reactor. Phylogenetic analysis showed the presence of phylotypes affiliated with gamma-Proteobacteria, Choroflexi, and Bacteroidetes. Archaea were represented by phylotypes belonging to the genus Methanosarcina and Methanosaeta.