Analytical instrumental approaches and strategies to support the sensory assessment of virgin olive oils

At the beginning, this Ph.D. project led to an overview of the most common and emerging types of fraud and possible countermeasures in the olive oil sector. Furthermore, possible weaknesses in the current conformity check system for olive oil were highlighted. Among those, despite the organoleptic assessment is a fundamental tool for establishing the virgin olive oils (VOOs) quality grade, the scientific community has evidenced some drawbacks in it. In particular, the application of instrumental screening methods to support the panel test could reduce the work of sensory panels and the cost of this analysis (e.g. for industries, distributors, public and private control laboratories), permitting the increase in the number and the efficiency of the controls. On this basis, a research line called “Quantitative Panel Test” is one of the main expected outcomes of the OLEUM project that is also partially discussed in this doctoral dissertation. In this framework, analytical activities were carried out, within this PhD project, aimed to develop and validate analytical protocols for the study of the profiles in volatile compounds (VOCs) of the VOOs headspace. Specifically, two chromatographic approaches, one targeted and one semi-targeted, to determine VOCs were investigated in this doctoral thesis. The obtained results, will allow the possible establishment of concentration limits and ranges of selected volatile markers, as related to fruitiness and defects, with the aim to support the panel test in the commercial categorization of VOOs. In parallel, a rapid instrumental screening method based on the analysis of VOCs has been investigated to assist the panel test through a fast pre-classification of VOOs samples based on a known level of probability, thus increasing the efficiency of quality control.

Kinetic and mechanistic studies on unconventional antioxidant activities of natural compounds

In chapter one, the autoxidation kinetics of natural oil substrates, including, triglyceric sunflower oil, olive oil, terpenic squalene, and p-cymene were calibrated through differential oximetry methods. Calibration allows their use as reference oxidizable substrates for further studies, e.g. for quantitative testing of antioxidants under biomimetic settings. Several essential oils samples, of different botanical species or different productions of same species were studied for their antioxidant activity in inhibited autoxidation kinetics. Their antioxidant activities were matched with their composition analyzed by GC-MS. In chapter two, the molecular mechanism of the synergy between the common phenolic antioxidants such as tocopherol and catechols with widespread essential component gamma-terpinene was studied through lipid oxidation kinetics. Wherein, gamma-terpinene was able to disclose the key intermediacy HOO·, which acted as a reducing agent regenerating the phenolic antioxidant. This counterintuitive role of HOO· radicals was further investigated in detail and allowed to rationalize for the first time the purported antioxidant behavior of PDA melanin nanoparticles. It will also open to a deeper understanding of the redox biology of quinones. Regarding melanin, its role is broadly important in living organisms and its control, including its inhibition, is of great importance with several relevant applications ranging from food preservation to control of human skin pigmentation. In chapter three, an oximetry method combined with the traditional UV-Vis spectroscopy was developed to study the tyrosinase inhibition kinetics, which allowed identifying Glabridin (from G. glabra, L.), as one of the most effective natural tyrosinase inhibitors.

Development of a colorimetric indicator label for food oxidation based on hexanal detection

In this work, a colorimetric indicator for food oxidation based on the detection of hexanal in gas-phase, has been developed. In fact, in recent years, the food packaging industry has evolved towards new generation of packaging, like active and intelligent. According to literature (Pangloli P. et al. 2002), hexanal is the main product of a fatty acid oxidation: the linoleic acid. So, it was chosen to analyse two kinds of potato chips, fried in two different oils with high concentration of linoleic acid: olive oil and sunflower oil. Five different formulas were prepared and their colour change when exposed to hexanal in gas phase was evaluated. The formulas evaluations were first conducted on filter paper labels. The next step was to select the thickener to add to the formula, in order to coat a polypropylene film, more appropriate than the filter paper for a production at industrial scale. Three kinds of thickeners were tested: a cellulose derivative, an ethylene vinyl-alcohol and a polyvinyl alcohol. To obtain the final labels with the autoadhesive layer, the polypropylene film with the selected formula and thickener was coat with a water based adhesive. For both filter paper and polypropylene labels, with and without autoadhesive layer, the detection limit and the detection time were measured. For the selected formula on filter paper labels, the stability was evaluated, when conserved on the dark or on the light, in order to determine the storage time. Both potato chips samples, stocked at the same conditions, were analysed using an optimised Headspace-Solid Phase Microextraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS) method, in order to determine the concentration of volatilized hexanal. With the aim to establish if the hexanal can be considered as an indicator of the end of potato chips shelf life, sensory evaluation was conducted each day of HS-SPME-GC-MS analysis.

Essential oils and hydrolates as potential tools for integrated prevention of bacterial diseases in plants

The present work aims to investigate the potential use of natural substances against bacterial plant pathogens. Microdilution tests were therefore carried out in vitro to identify the minimum inhibitory and bactericidal concentrations (MIC and MBC) of several EOs and Hys against selected bacterial pathogens. Commercial products based on a mixture of EOs were in addition assayed with macrodilution experiments against Erwinia amylovora (Ea-causal agent of fire blight). Subsequently, using selected EOs, Hys, and commercial products, ex vivo tests on disease incidence and Ea population dynamics were carried out; the latter experiment was followed by SEM observations. In addition, in vivo resistance induction test was carried out against bacterial leaf of tomato, caused by Xanthomonas vesicatoria (Xv). EOs and Hys showed high bactericidal activity in vitro (MBC <0.1 and <10% for the most active EOs and Hys: Origanum compactum and Thymus vulgaris EOs and Citrus aurantium var. amara Hy, respectively), but they were not effective ex vivo, while resulted very active when used in vivo as resistance inducers in the tomato-Xv pathosystem (relative protection >40%). Differently, commercial products resulted active in all tests, but not as resistance inducers against Xv. An open field trial with commercial products was carried out on strawberry plants naturally infected with Xanthomonas fragariae; the results showed discrete relative protection, concerning that provided by the conventional products based on copper; mostly, the disease severity reduction on those plants treated with EOs commercial products was significant when disease severity resulted high. The papers already published described in the present work investigate (1)the activity of Hys in comparison to EOs with respect to their active volatile content; (2) the potential use of EOs and Hys in cultural heritage; for the restoration of paintings; (3) the induction of resistance caused by plasma-activated water-based root treatments.

Advanced studies on two animal models, murine and fish. RadKI21A626T mouse model: new insights into molecular mechanisms of enteric neuro-epithelial pathology. European sea bass: study of the effects of essential oils in different diets on the gastric system.

My PhD research period was focused on the anatomical, physiological and functional study of the gastrointestinal system on two different animal models. In two different contexts, the purpose of these two lines of research was contribute to understand how a specific genetic mutation or the adoption of a particular dietary supplement can affect gastrointestinal function. Functional gastrointestinal disorders are chronic conditions characterized by symptoms for which no organic cause can be found. Although symptoms are generally mild, a small subset of cases shows severe manifestations. This subset of patients may also have recurrent intestinal sub-occlusive episodes, but in absence of mechanical causes. This condition is referred to as chronic intestinal pseudo-obstruction, a rare, intractable chronic disease. Some mutations have been associated with CIPO. A novel causative RAD21 missense mutation was identified in a large consanguineous family, segregating a recessive form of CIPO. The present thesis was aimed to elucidate the mechanisms leading to neuropathy underlying CIPO via a recently developed conditional KI mouse carrying the RAD21 mutation. The experimental studies are based on the characterization and functional analysis of the conditional KI Rad21A626T mouse model. On the other hand aquaculture is increasing the global supply of foods. The species selected and feeds used affects the nutrients available from aquaculture, with a need to improve feed efficiency, both for economic and environmental reasons, but this will require novel innovative approaches. Nutritional strategies focused on the use of botanicals have attracted interest in animal production. Previous research indicates the positive results of using essential oils (EOs) as natural feed additives for several farmed animals. Therefore, the present study was designed to compare the effects of feed EO supplementation in two different forms (natural and composed of active ingredients obtained by synthesis) on the gastric mucosa in European sea bass.

Identification of sunflower genotypes suitable for organic agriculture and development of analytical tools for their traceability

Thanks to the development and combination of molecular markers for the genetic traceability of sunflower varieties and a gas chromatographic method for the determination of the FAs composition of sunflower oil, it was possible to implement an experimental method for the verification of both the traceability and the variety of organic sunflower marketed by Agricola Grains S.p.A. The experimental activity focused on two objectives: the implementation of molecular markers for the routine control of raw material deliveries for oil extraction and the improvement and validation of a gas chromatographic method for the determination of the FAs composition of sunflower oil. With regard to variety verification and traceability, the marker systems evaluated were the following: SSR markers (12) arranged in two multiplex sets and SCAR markers for the verification of cytoplasmic male sterility (Pet1) and fertility. In addition, two objectives were pursued in order to enable a routine application in the industrial field: the development of a suitable protocol for DNA extraction from single seeds and the implementation of a semi-automatic capillary electrophoresis system for the analysis of marker fragments. The development and validation of a new GC/FID analytical method for the determination of fatty acids (FAME) in sunflower achenes to improve the quality and efficiency of the analytical flow in the control of raw and refined materials entering the Agricola Grains S.p.A. production chain. The analytical performances being validated by the newly implemented method are: linearity of response, limit of quantification, specificity, precision, intra-laboratory precision, robustness, BIAS. These parameters are used to compare the newly developed method with the one considered as reference - Commission Regulation No. 2568/91 and Commission Implementing Regulation No. 2015/1833. Using the combination of the analytical methods mentioned above, the documentary traceability of the product can be confirmed experimentally, providing relevant information for subsequent marketing.

Nanocellulose films activated with essential oils for active packaging applications

The study focused on the analysis of the state of the art of active packaging and on the development of an innovative active packaging system for food application based on the use of nanocellulose matrix embedded with essential oils. The solubility and diffusivity of thyme, cinnamon and oregano essential oils in three nanocellulose films, endowed with different carboxymethylation degree, were analysed. The antimicrobial and antioxidant activity of those films was also analyzed. Firstly, the activity against model pathogenic bacteria was tested and the minimum inhibitory concentration of each oil was determined (0.37 – 0.68 mg/mg of matrix). This initial validation was then followed by experimental settings aimed at testing the system directly on clamshell type packed raspberries. It was observed that thyme and oregano essential oils were more effective in maintaining firmness and reduce weight loss than cinnamon essential oil or controls, through 12 days storage at 1ºC. From the results obtained, it is possible to conclude that the dispersion of thyme and oregano essential oils in nanocellulose matrix is a promising technology to improve shelf-life of raspberries or other fresh fruits.

Study and characterization of low molecular weight polymer solutions for application in lubricating oils

The aim of this thesis is to evaluate the possibility of using short linear polymer chains as additives in lubricating oil applications. Through previous works, it has been seen that they are particularly resistant to mechanical degradation, which is the main reason why lubricating oils need to be changed after a while. This is the main reason why they could be proposed as alternatives in the market. The results of this work have been split into two major phases: the first concentrated on characterizing a target product obtained through thermal degradation, starting from the original long chain parent polymer, and the second focused on the technological advancement of heat exchangers. Through the studies carried out, we’ve characterized our innovative polymers and the solutions made with them and base oil at different concentrations. The most promising result is that these short random coiled polymeric chains obey to a more general universal function which express the value of specific viscosity as function of a dimensionless quantity c/c*. For the design of the unit operation, several alternatives were proposed and these all shared the same final goal: cooling the polymer without the presence of oxygen to avoid oxidation and formation of unwanted substances. We’ve analyzed the main difficulties related to the presence of these highly viscous substances and, more importantly, how to deal with this situation (e.g. by considering radial static mixer or even more complex conformations).

Doehlert Design-desirability Function Multi-criteria Optimal Separation Of 17 Phenolic Compounds From Extra-virgin Olive Oil By Capillary Zone Electrophoresis.

In Brazil, the consumption of extra-virgin olive oil (EVOO) is increasing annually, but there are no experimental studies concerning the phenolic compound contents of commercial EVOO. The aim of this work was to optimise the separation of 17 phenolic compounds already detected in EVOO. A Doehlert matrix experimental design was used, evaluating the effects of pH and electrolyte concentration. Resolution, runtime and migration time relative standard deviation values were evaluated. Derringer's desirability function was used to simultaneously optimise all 37 responses. The 17 peaks were separated in 19min using a fused-silica capillary (50μm internal diameter, 72cm of effective length) with an extended light path and 101.3mmolL(-1) of boric acid electrolyte (pH 9.15, 30kV). The method was validated and applied to 15 EVOO samples found in Brazilian supermarkets.

Microbial Lipid Production: Screening With Yeasts Grown On Brazilian Molasses.

Rhodotorula glutinis CCT 2182, Rhodosporidium toruloides CCT 0783, Rhodotorula minuta CCT 1751 and Lipomyces starkeyi DSM 70296 were evaluated for the conversion of sugars from Brazilian molasses into single-cell oil (SCO) feedstock for biodiesel. Pulsed fed-batch fermentations were performed in 1.65 l working volume bioreactors. The maximum specific growth rate (µmax), lipid productivity (Pr) and cellular lipid content were, respectively, 0.23 h(-1), 0.41 g l(-1) h(-1), and 41% for Rsp. toruloides; 0.20 h(-1), 0.27 g l(-1) h(-1), and 36% for Rta. glutinis; 0.115 h(-1), 0.135 g l(-1) h(-1), and 27 % for Rta. minuta; and 0.11 h(-1), 0.13 g l(-1) h(-1), and 32% for L. starkeyi. Based on their microbial lipid productivity, content, and profile, Rsp. toruloides and Rta. glutinis are promising candidates for biodiesel production from Brazilian molasses. All the oils from the yeasts were similar to the composition of plant oils (rapeseed and soybean) and could be used as raw material for biofuels, as well as in food and nutraceutical products.

Chemical Composition And Free Radical-scavenging, Anticancer And Anti-inflammatory Activities Of The Essential Oil From Ocimum Kilimandscharicum.

The essential oil from the leaves of Ocimum kilimandscharicum (EOOK), collected in Dourados-MS, was investigated for anticancer, anti-inflammatory and antioxidant activity and chemical composition. The essential oil was extracted by hydrodistillation, and the chemical composition was performed by gas chromatography-mass spectrometry. The essential oil was evaluated for free radical-scavenging activity using the DPPH assay and was tested in an anticancer assay against ten human cancer cell lines. The response parameter (GI50) was calculated for the cell lines tested. The anti-inflammatory activity was evaluated using carrageenan-induced pleurisy in mice. The chemical composition showed 45 components with a predominance of monoterpenes, such as camphor (51.81%), 1,8 cineole (20.13%) and limonene (11.23%). The EOOK exhibited potent free radical-scavenging activity by the DPPH assay with a GI50 of 8.31 μg/ml. The major constituents, pure camphor (IC50=12.56 μg/ml) and mixture of the limonene: 1, 8 cineole (IC50=23.25 μg/ml) displayed a potent activity. The oral administration of EOOK (at 30 and 100 mg kg(-1)), as well as the pure camphor or a mixture of 1,8 cineole with limonene, significantly inhibited the carrageenan (Cg) induced pleurisy, reducing the migration of total leukocytes in mice by 82 ± 4% (30 mg kg(-1) of EOOK), 95 ± 4% (100 mg kg(-1) of EOOK), 83 ± 9% (camphor) and 80 ± 5% (mixture of 1,8 cineole:limonene 1:1). In vitro cytotoxicity screening against a human ovarian cancer cell line displayed high selectivity and potent anticancer activity with GI50=31.90 mg ml(-1). This work describes the anti-inflammatory, anticancer and antioxidant effects of EOOK for the first time. The essential oil exhibited marked anti-inflammatory, antioxidant and anticancer effects, an effect that can be attributed the presence of majorital compounds, and the response profiles from chemical composition differed from other oils collected in different locales.

Enzymatic Biodiesel Synthesis From Yeast Oil Using Immobilized Recombinant Rhizopus Oryzae Lipase.

The recombinant Rhizopus oryzae lipase (1-3 positional selective), immobilized on Relizyme OD403, has been applied to the production of biodiesel using single cell oil from Candida sp. LEB-M3 growing on glycerol from biodiesel process. The composition of microbial oil is quite similar in terms of saponifiable lipids than olive oil, although with a higher amount of saturated fatty acids. The reaction was carried out in a solvent system, and n-hexane showed the best performance in terms of yield and easy recovery. The strategy selected for acyl acceptor addition was a stepwise methanol addition using crude and neutralized single cell oil, olive oil and oleic acid as substrates. A FAMEs yield of 40.6% was obtained with microbial oils lower than olive oil 54.3%. Finally in terms of stability, only a lost about 30% after 6 reutilizations were achieved.

Nitrogen Starvation For Lipid Accumulation In The Microalga Species Desmodesmus Sp.

Recently, to obtain lipids from microalgae has been the object of extensive research, since it is viewed as a promising feedstock for biodiesel production, especially when compared with crops such as soybean and sunflower, in terms of theoretical performance. The reduction of nutrient availability in culture media, especially nitrogen, stresses the microorganisms and affects cell growth, thus inducing lipid accumulation. This is an interesting step in biodiesel feedstock obtention from microalgae and should be better understood. In this study, four levels of nitrogen concentration in the BG-11 culture medium were evaluated in the growth of the chlorophycean microalga Desmodesmus sp. Both cell growth and lipid content were monitored over 7 days of cultivation, which yielded a final cell density of 33 × 10(6) cells mL(-1) with an initial NaNO3 concentration of 750 mg L(-1) in the medium and a maximum lipid content of 23 % with total nitrogen starvation. It was observed that the microalgae presented high lipid accumulation in the fourth day of cultivation with nitrogen starvation, although with moderate cell growth.

Titulação potenciométrica aplicada na determinação de ácidos graxos livres de óleos e gorduras comestíveis

Free fatty acids are a measure of evaluating fats and oils, submitted to abusive conditions, besides being a quality characteristic of edible vegetable oils. The official method of determination (AOCS Ca 5a-40, 2004) is based on titration, using phenolphthalein as an indicator. For crude and degummed oils, the titration end point is difficult to be observed due to the high level of pigments, resulting in dark solutions. In this case and others, such as self-life studies, in which sample quantities are limited, potentiometric titration may be a very good alternative.

Interesterificação química: alternativa para obtenção de gorduras zero trans

The function of lipids in human nutrition has been intensively debated in the last decade.This context reinforces the concern about controlling the trans fat ingestion, due to its negative implications on health. Interesterification provides an important alternative to modify the consistency of oils and fats without causing formation of trans isomers. This article reports research done towards production of zero trans fats by chemical interesterification, for different industrial purposes. Aspects related to the effect of trans fats on diet, their impact on health and modifications in Brazilian legislation are also covered.