The Contribution of Theoretical Prediction Studies to the Antioxidant Activity Assessment of the Bioactive Secoiridoids Encountered in Olive Tree Products and By-Products.

Assessment of the antioxidant activity of different types of natural compounds is a complex research area that encompasses various in vitro tests and in vivo studies. Sophisticated modern analytical tools permit the unambiguous characterization of the compounds present in a matrix. The contemporary researcher, knowing the chemical structure of the compounds present, can carry out quantum chemical calculations that provide important physicochemical information assisting the prediction of antioxidant potential and the mechanism behind the activity of target compounds before further experimentation. The efficiency of calculations is steadily improved due to the rapid evolution of both hardware and software. It is possible, consequently, to study compounds of medium or even larger size, incorporating also models that simulate the liquid phase (solution). This review contributes to the establishment of theoretical calculations as an inherent part of the antioxidant activity assessment process, having as a case study the complex mixtures of olive bioactive secoiridoids (oleuropein, ligstroside, and related compounds). The literature indicates great variability in theoretical approaches and models used so far for only a limited number of this group of phenolic compounds. Proposals are made for standardization of methodology (reference compounds, DFT functional, basis set size, and solvation model) to facilitate comparisons and communication of findings.

On the Squalene Content of CV Chondrolia Chalkidikis and Chalkidiki (Greece) Virgin Olive Oil.

This work is a continuation of efforts to establish the nutritional profile of virgin olive oil (VOO) from cv. Chondrolia Chalkidikis and Chalkidiki and to strengthen its positioning in the global VOO landscape. VOOs produced at an industrial scale in different olive mills of the Chalkidiki (Greece) regional unit as well as VOOs obtained at the laboratory scale from drupes of different maturity stages for four consecutive harvesting years were examined for their squalene (SQ) content using both HPLC and GC procedures. The mean values of SQ were found to be 4228 (HPLC) and 4865 (GC) mg/kg oil (n = 15) and were of the same magnitude as that in VOOs from cv Koroneiki (4134 mg/kg, n = 23). Storage of VOOs in the dark at room temperature for 18 months indicated an insignificant mean SQ content loss (~2%) in comparison to a mean loss of 26% for alpha-tocopherol content. This finding strengthens our view that SQ does not act as a radical scavenger that donates hydrogen atoms to the latter. The four consecutive harvest years studied indicated a clear declining trend in VOO SQ concentration upon olive ripening. To our knowledge, this is the first systematic work concerning the SQ content of Chondrolia Chalkidikis and Chalkidiki VOOs.

The Potential of Virgin Olive Oil from cv. Chondrolia Chalkidikis and Chalkidiki (Greece) to Bear Health Claims according to the European Legislation.

The European food legislation authorizes the use of certain health claims based on a scientific basis. This study aimed to evaluate the fatty acid, tocopherol, and polar phenol composition of virgin olive oil (VOO) from cv. Chondrolia Chalkidikis and Chalkidiki regarding the fulfillment of official requirements for the health claims of 'oleic acid', 'vitamin E', and 'olive oil polyphenols'. The examination of representative industrial VOOs from 15 olive mills of the Chalkidiki regional unit showed that the two cultivars yield oils contained the necessary concentrations of the responsible bioactive compounds. This evidence was further substantiated by a four harvest study whereby olives from different maturity stages were sampled from three olive groves. Oils were extracted at a laboratory scale and examined for their content in the above-mentioned three categories of constituents. Oils produced at industrial scale from olives harvested on the 'technological optimum' stage according to the olive grove proprietor were also analyzed. Extra virgin olive oil of the studied cultivars can safely bear the generic claims for 'oleic acid' and 'vitamin E'. The cultivars present great potential regarding the total hydroxytyrosol and tyrosol content of the extracted oil required to attain the third health claim that may be influenced negatively by manufacturing practices.

Suggestions on the Contribution of Methyl Eugenol and Eugenol to Bay Laurel (Laurus nobilis L.) Essential Oil Preservative Activity through Radical Scavenging.

The present study examined the radical scavenging potential of the two benzene derivatives found in the bay laurel essential oil (EO), namely methyl eugenol (MEug) and eugenol (Eug), theoretically and experimentally to make suggestions on their contribution to the EO preservative activity through such a mechanism. Calculation of appropriate molecular indices widely used to characterize chain-breaking antioxidants was carried out in the gas and liquid phases (n-hexane, n-octanol, methanol, water). Experimental evidence was based on the DPPH• scavenging assay applied to pure compounds and a set of bay laurel EOs chemically characterized with GC-MS/FID. Theoretical calculations suggested that the preservative properties of both compounds could be exerted through a radical scavenging mechanism via hydrogen atom donation. Eug was predicted to be of superior efficiency in line with experimental findings. Pearson correlation and partial least square regression analyses of the EO antioxidant activity values vs. % composition of individual volatiles indicated the positive contribution of both compounds to the radical scavenging activity of bay laurel EOs. Eug, despite its low content in bay laurel EOs, was found to influence the most the radical scavenging activity of the latter.

Diagnostic Potential of FT-IR Fingerprinting in Botanical Origin Evaluation of Laurus nobilis L. Essential Oil is Supported by GC-FID-MS Data.

The last years, non-targeted fingerprinting by Fourier-transform infrared (FT-IR) spectroscopy has gained popularity as an alternative to classical gas chromatography (GC)-based methods because it may allow fast, green, non-destructive and cost-effective assessment of quality of essential oils (EOs) from single plant species. As the relevant studies for Laurus nobilis L. (bay laurel) EO are limited, the present one aimed at exploring the diagnostic potential of FT-IR fingerprinting for the identification of its botanical integrity. A reference spectroscopic dataset of 97 bay laurel EOs containing meaningful information about the intra-species variation was developed via principal component analysis (PCA). This dataset was used to train a one-class model via soft independent modelling class analogy (SIMCA). The model was challenged against commercial bay laurel and non-bay laurel EOs of non-traceable production history. Overall, the diagnostic importance of spectral bands at 3060, 1380-1360, 1150 and 1138 cm-1 was assessed using GC-FID-MS data. The findings support the introduction of FT-IR as a green analytical technique in the quality control of these often mislabeled and/or adulterated precious products. Continuous evaluation of the model performance against newly acquired authentic EOs from all producing regions is needed to ensure validity over time.

In House Validated UHPLC Protocol for the Determination of the Total Hydroxytyrosol and Tyrosol Content in Virgin Olive Oil Fit for the Purpose of the Health Claim Introduced by the EC Regulation 432/2012 for "Olive Oil Polyphenols".

An ongoing challenge in olive oil analytics is the development of a reliable procedure that can draw the consensus of all interested parties regarding the quantification of concentrations above the required minimum value of 5 mg of bioactive "olive oil polyphenols" per 20 g of the oil, to fulfill the health claim introduced by the European Commission (EC) Regulation 432/2012. An in-house validated ultra-high performance liquid chromatography (UHPLC) protocol fit for this purpose is proposed. It relies on quantification of the total hydroxytyrsol (Htyr) and tyrosol (Tyr) content in the virgin olive oil (VOO) polar fraction (PF) before and after acidic hydrolysis of their bound forms. PF extraction and hydrolysis conditions were as previously reported. The chromatographic run lasts ~1/3 of the time needed under high performance liquid chromatography (HPLC) conditions, this was also examined. Eluent consumption for the same piece of information was 6-fold less. Apart from being cost effective, a larger number of samples can be analyzed daily with less environmental impact. Two external curves, detection at 280 nm and correction factors for molecular weight difference are proposed. The method, which is fit for purpose, is selective, robust with satisfactory precision (percentage relative standard deviation (%RSD) values < 11%) and recoveries higher than 87.6% for the target analytes (Htyr, Tyr). Standard operational procedures are easy to apply in the olive oil sector.

Toward a Harmonized and Standardized Protocol for the Determination of Total Hydroxytyrosol and Tyrosol Content in Virgin Olive Oil (VOO). The Pros of a Fit for the Purpose Ultra High Performance Liquid Chromatography (UHPLC) Procedure.

Τoward a harmonized and standardized procedure for the determination of total hydroxytyrosol and tyrosol content in virgin olive oil (VOO), the pros of a recently published in house validated ultra high performance liquid chromatography (UHPLC) protocol are discussed comparatively with those of other procedures that determine directly or indirectly the compounds hosted under the health claim on "olive oil polyphenols" (EC regulation 432/2012). Authentic VOOs were analyzed with five different liquid chromatographic separation protocols and 1H-NMR one in five different laboratories with expertise in VOO phenol analysis within three months. Data comparison indicated differences in absolute values. Method comparison using appropriate tools (Passing-Bablok regression and Bland Altman analyses) for all protocols vs. the UHPLC one indicated slight or statistically significant differences. The results were also discussed in terms of cost effectiveness, detection means, standard requirements and ways to calculate the total hydroxytyrosol and tyrosol content. Findings point out that the in-house validated fit for the purpose UHPLC protocol presents certain pros that should be exploited by the interested parties. These are the simplicity of sample preparation, fast elution time that increase the number of samples analyzed per day and integration of well-resolved peaks with the aid of only two commercially available external standards. Importance of correction factors in the calculations is stressed.

On the Traceability of Commercial Saffron Samples Using ¹H-NMR and FT-IR Metabolomics.

In previous works on authentic samples of saffron of known history (harvest and processing year, storage conditions, and length of time) some biomarkers were proposed using both FT-IR and NMR metabolomics regarding the shelf life of the product. This work addresses the difficulties to trace back the "age" of commercial saffron samples of unknown history, sets a limit value above which these products can be considered substandard, and offers a useful tool to combat saffron mislabeling and fraud with low-quality saffron material. Investigations of authentic and commercial saffron samples of different origin and harvest year, which had been stored under controlled conditions for different lengths of time, allowed a clear-cut clustering of samples in two groups according to the storage period irrespectively of the provenience. In this respect, the four-year cut off point proposed in our previous work assisted to trace back the "age" of unknown samples and to check for possible mislabeling practices.

Insight of Saffron Proteome by Gel-Electrophoresis.

Saffron is a spice comprised of the dried stigmas and styles of Crocus sativus L. flowers and, since it is very expensive, it is frequently adulterated. So far, proteomic tools have never been applied to characterize the proteome of saffron or identify possible cases of fraud. In this study, 1D-Gel Electrophoresis was carried out to characterize the protein profile of (i) fresh stigmas and styles of the plant; (ii) dried stigmas and styles from different geographical origins (Spanish, Italian, Greek and Iranian) that had been stored for various periods of time after their processing; and (iii) two common plant adulterants, dried petals of Carthamus tinctorius L. and dried fruits of Gardenia jasminoides Ellis. A selective protein extraction protocol was applied to avoid interference from colored saffron metabolites, such as crocins, during electrophoretic analyses of saffron. We succeeded in separating and assigning the molecular weights to more than 20 proteins. In spite of the unavailability of the genome of saffron, we were able to identify five proteins by Peptide Mass Fingerprinting: phosphoenolpyruvate carboxylase 3, heat shock cognate 70 KDa protein, crocetin glucosyltransferase 2, α-1,4-glucan-protein synthase and glyceraldehydes-3-phosphate dehydrogenase-2. Our findings indicate that (i) few bands are present in all saffron samples independently of origin and storage time, with amounts that significantly vary among samples and (ii) aging during saffron storage is associated with a reduction in the number of detectable bands, suggesting that proteases are still active. The protein pattern of saffron was quite distinct from those of two common adulterants, such as the dried petals of Carthamus tinctorius and the dried fruits of Gardenia jasminoides indicating that proteomic analyses could be exploited for detecting possible frauds.

Implementing principles of traditional concentrated grape must fermentation to the production of new generation balsamic vinegars. Starter selection and effectiveness.

In an effort to implement principles of traditional concentrated grape must fermentation to the production of new generation balsamic vinegars (BVs), the specific goals of the study were the isolation and molecular identification of the predominant yeasts in concentrated grape must (cv. Xinomavro), their technological characterization and the evaluation of the fermentative aptitude of the selected strains. Tolerance against 5-hydroxymethyl-furfural (HMF) and furfural, acetic acid and glucose concentration was examined by appropriate methods and tests. The enological characteristics studied were acetic acid and H2S production, foaming and flocculation ability and key enzymatic activity. PCR-RFLP analysis revealed only the presence of Saccharomyces cerevisiae and Hanseniaspora uvarum among the 14 predominant osmophilic yeast isolates. Tolerance to both HMF and furfural was found strain- and dose-dependent and was suggested as a critical factor in the pre-selection of yeast starters. The most tolerant yeasts to these stress factors, a S. cerevisiae and a non-Saccharomyces strains, showed satisfactory growth in the presence of high glucose and acetic acid content (up to 600 g/L and 2 % w/w, respectively) and desirable enological characteristics. Results from the comparative evaluation of the fermentative aptitude of these strains with a commercial wine strain highlighted that the isolates had glucophilic behaviour and ability to produce desirable amounts of ethanol (100-120 g/kg) in short time (~20 d). The key volatiles useful for varietal discrimination and differentiation between the BVs and the traditional ones were also evaluated.

Enhanced Bioaccessibility of Crocetin Sugar Esters from Saffron in Infusions Rich in Natural Phenolic Antioxidants.

The present study aims to examine whether and to what extent the bioaccessibility of the major saffron apocarotenoids, namely crocetin sugar esters (CRTSEs), is affected by the presence of strong water-soluble antioxidants, ingredients of the herbs found in commercial tea blends with saffron. An in vitro digestion model was applied to infusions from these products to investigate the possible changes. All of the studied infusions were rich in total phenols (9.9-22.5 mg caffeic acid equivalents/100 mg dry infusion) and presented strong DPPH radical scavenging activity regardless of the composition of the corresponding herbal blends. RP-HPLC-DAD and LC-MS analysis enabled the grouping of the infusions into hydroxycinnamic acid-rich and in flavan-3-ol-rich ones. CRTSEs in herbal tea infusions were found to be significantly more bioaccessible (66.3%-88.6%) than those in the reference saffron infusion (60.9%). The positive role of strong phenolic antioxidants (caffeic acid, rosmarinic acid) on the stability of CRTSEs was also evidenced in model binary mixtures. On the contrary, cinnamic acid, exerting no antioxidant activity, did not have such an effect. Our findings suggest that strong radical scavengers may protect the crocetin sugar esters from oxidation during digestion when present in excess.

On the Importance of the Starting Material Choice and Analytical Procedures Adopted When Developing a Strategy for the Nanoencapsulation of Saffron (Crocus sativus L.) Bioactive Antioxidants.

Saffron is known as the most expensive spice in the world. It is comprised of the dried stigmas of the pistil of the Crocus sativus L., which is a cultivated, sterile crocus plant. This plant material is now recognized as the unique edible source of certain bioactive apocarotenoids for which in-vivo antioxidant properties have been reported. Among the latter, crocins, red-orange natural colorants, and their parent molecule crocetin prevail in bioactivity significance. This review is focused on the strategies developed so far for their nanoencapsulation in relation to the characteristics of the starting material, extraction procedures of the bioactive antioxidants and analytical methods applied for their characterization and quantification throughout the process. The literature so far points out gaps that lead to publishable data, on one hand, but not necessarily to repeatable and meaningful processes due to incomplete characterization of the starting and the released material in efficiency and stability studies of the nanoencapsulates. Accurate terminology and quantitative chromatographic or spectrophotometric procedures for the determination of the core compounds are needed. Authenticity control and quality of saffron samples, and the verification of the concentrations of compounds in commercial preparations labeled as 'crocin,' are prerequisites in any experimental design setup.

Minor bioactive lipids.

Bioactive lipids-major and minor-comprise an array of compounds belonging to different chemical categories. Among the minor bioactive lipids carotenoids, sterols and tocochromanols attract continuously the interest of food scientists, nutritionists and medical doctors for their importance in food processing, preservation and for their health properties. Provitamin A and non-provitamin A carotenoids are found in various food sources of plant and animal origin and are added to foods as colorants. Their interactions with other food ingredients are critical because of their role against reactive oxygen species. The role of cholesterol through the diet after decades of disputes is better justified whereas at the same time emphasis is given to the technological and health aspects of phytosterols, which became very efficiently part of the daily diet for many population groups. Last but not least the importance of vitamin E is in a continuous debate for over 100years whereas studies on tocotrienols are intensified as a result of a transient to palm oil product consumption globally. Chemistry, natural occurrence, absorption and metabolism, dietary intake and dietary recommendations, major health impacts and key technological issues are updated and discussed with the support of recent findings.

Building a novel strategic research agenda for METROFOOD-RI: design process and multi-stakeholder engagement towards thematic prioritization.

Introduction: The European research landscape suffers widely from fragmentation and little cross-border research collaboration. Efforts are underway to bring the European Research Area to a higher level of performance and capacity in cutting-edge science, with high anticipations for the promotion of multidisciplinary research infrastructures of transnational engagement. A European distributed research infrastructure active in this framework is METROFOOD-RI, committed to promoting metrology in food and nutrition with particular focus on measurement research related to agrifood systems. Methods: For research infrastructures, streamlining resources among partner organizations and establishing priorities around specific topics is critical for ensuring smooth operation. Similarly, METROFOOD-RI faced the challenge of exploring its strategic direction and research priorities as revealed in its first Strategic Research and Innovation Agenda (SRIA). This report details how the internal process of topic identification and prioritization progressed within the METROFOOD-RI SRIA and what obstacles were encountered along the way. A dual-track strategy was taken for locating future SRIA topics, applying a top-down and bottom-up approach, followed by internal consultation with METROFOOD-RI experts. The topic prioritization drew on a vote among the METROFOOD-RI Management Committee employing a custom-designed numerical rating scale questionnaire. Based on the maximum scores obtained for each topic, appropriate thresholds were introduced for classifying individual topics into high, medium, low, and very low priority ones. Results: A total of 80 topics categorized into eight major clusters of challenges were located as potential SRIA candidates. Upon prioritization, 9 topics of very high priority and 16 topics of medium priority were identified as key research thematic areas of the newly developed SRIA. Discussion: As a strategic framework, the SRIA occupies a central position and sets not only the scientific focus of the research infrastructure in the coming years, but also contributes to realizing the full potential and excellence of METROFOOD-RI, selectively expanding the existing portfolio and thus contributing to maximum efficiency and sustainability. It is anticipated that the lessons learned by METROFOOD-RI and its experiences shared are a valuable stimulus and guide for those who are taking on the challenge of setting-up a SRIA and are looking for edifying and constructive information on how to do so.

Edible oils from olive drupes as a source of bioactive pentacyclic triterpenes. Is there a prospect for a health claim authorization?

Virgin olive oil and olive-pomace oil constitute high nutritional value edible oils due to the presence of oleic acid and a variety of bioactives. Among the latter, the group of pentacyclic triterpenes (PcTr) is the least studied. This review provides an insight into the biosynthesis of PcTr in the olive fruit, mainly of oleanane-type, and the factors influencing their transfer to the oil. Particular attention is given to the extraction methods along with the liquid and gas chromatography coupled to mass spectrometry protocols used for the discrimination and determination of PcTr. The in vivo bioactive properties of PcTr through the intake of these oils against cardiovascular diseases, liver dysfunction, obesity and diabetes are presented with a prospect of a future health claim authorization. Gaps in literature are pointed out to support this goal.

Bay Laurel (Laurus nobilis L.) Essential Oil as a Food Preservative Source: Chemistry, Quality Control, Activity Assessment, and Applications to Olive Industry Products.

Essential oils (EOs) find application as flavoring agents in the food industry and are also desirable ingredients as they possess preservative properties. The Mediterranean diet involves the use of a lot of herbs and spices and their products (infusions, EOs) as condiments and for the preservation of foods. Application of EOs has the advantage of homogeneous dispersion in comparison with dry leaf use in small pieces or powder. Among them, Laurus nobilis (bay laurel) L. EO is an interesting source of volatiles, such as 1,8-cineole and eugenol, which are known for their preservative properties. Its flavor suits cooked red meat, poultry, and fish, as well as vegetarian dishes, according to Mediterranean recipes. The review is focused on its chemistry, quality control aspects, and recent trends in methods of analysis and activity assessment with a focus on potential antioxidant activity and applications to olive industry products. Findings indicate that this EO is not extensively studied in comparison with those from other Mediterranean plants, such as oregano EO. More work is needed to establish authenticity and activity methods, whereas the interest for using it for the preparation of flavored olive oil or for the aromatization and preservation of table oils must be further encouraged.

Strategic Priorities of the Scientific Plan of the European Research Infrastructure METROFOOD-RI for Promoting Metrology in Food and Nutrition.

The pan-European distributed Research Infrastructure for Promoting Metrology in Food and Nutrition (METROFOOD-RI) has evolved in the frame of the European Strategy Forum on Research Infrastructures (ESFRI) to promote high-quality metrology services across the food chain. The METROFOOD-RI comprises physical facilities and electronic facilities. The former includes Reference Material plants and analytical laboratories (the 'Metro' side) and also experimental fields/farms, processing/storage plants and kitchen-labs (the 'Food' side). The RI is currently prepared to apply for receiving the European Research Infrastructure Consortium (ERIC) legal status and is organised to fulfil the requirements for operation at the national, European Union (EU) and international level. In this view, the METROFOOD-RI partners have recently reviewed the scientific plan and elaborated strategic priorities on key thematic areas of research in the food and nutrition domain to which they have expertise to contribute to meet global societal challenges and face unexpected emergencies. The present review summarises the methodology and main outcomes of the research study that helped to identify the key thematic areas from a metrological standpoint, to articulate critical and emerging issues and demands and to structure how the integrated facilities of the RI can operate in the first five years of operation as ERIC.

Quality aspects of European virgin olive oils with registered geographical indications: Emphasis on nutrient and non-nutrient bioactives.

European virgin olive oil with geographical indications are strictly regulated and are of importance for the producing member states, Spain, Italy, Greece, Portugal, France, Slovenia and Croatia. These products are consumed locally, or within the European Union but are also exported worldwide. The chapter stresses on the importance of combining origin indications with other certifications or opportunities raising from European legislation in the agri-food sector so that to tighten consumer loyalty for this category of products. Emphasis is given to the richness of virgin olive oil in bioactive compounds that are already covered by nutritional and health claims (oleic acid, vitamin E, "polyphenols") and to those compounds that can be exploited in the future toward the same direction (squalene, oleanolic and maslinic acids).

Flavored Olive Oil as a Preservation Means of Reduced Salt Spanish Style Green Table Olives (cv. Chalkidiki).

Reformulation of products fermented in brine is a challenging area of research. Continuing the efforts toward the establishment of table olives as a healthy food for all population groups, this study aimed at examining whether olive oil flavored with essential oils can be used as a preservation means for reduced salt Spanish style green table olives (cv. Chalkidiki). Response surface methodology was applied to organize experimentation and assess data. As independent factors, concentrations of the essential oils used (oregano, lemon balm and bay laurel) and time of storage under vacuum were set. Microbiological parameters (pathogens and fermentation-related microbes), color and firmness attributes were used as responses. Models indicated that each essential oil exerted a preservative role to maintain microbiological quality of reduced salt table olives. Concurrently, appearance attributes of the latter were retained at desirable values. Oregano essential oil had a profound role against pathogens. Lemon balm and bay laurel essential oils were found to be important for yeast population control. The results are promising toward the use of flavored olive oil as a preservation means for tailor-made reduced salt table olives, a practice that may enhance local industry innovative activity in a practical and effective way.

Observations on squalene accumulation in Saccharomyces cerevisiae due to the manipulation of HMG2 and ERG6.

The constructed strains AM63, having an extra copy of the HMG2 gene with a K6R stabilizing mutation in Hmg2p expressed under the control of the inducible galactose promoter and stably integrated into the chromosomal HO locus, and AM64, a derivative of AM63 with an additional deletion of the ERG6 gene, were used as tools to test the squalene accumulation capacity of Saccharomyces cerevisiae. Kinetic data indicated high squalene levels in the early stages of semi-anaerobic cultivation of these strains. The stable Hmg2p induced a strong increase in the squalene pool and a smaller increase in the lanosterol pool. In AM63, the squalene content was approximately 20-fold higher than in the wild-type EGY48 parental strain. In AM64, the combined Hmg2p stabilization and ERG6 deletion did not further enhance squalene accumulation, as lack of ergosterol feedback inhibition led to an elevated transfer of surplus squalene into C27 sterols. The obtained maximum capacity of the selected strains to accumulate squalene and our observations provide a further understanding of the regulation of ergosterol pathway and may also be used as a reference value for its production using food-grade strains of S. cerevisiae.