The Effect of Drying Methods and Extraction Techniques on Oleuropein Content in Olive Leaves.

Increased demand for olive oil has caused higher quantities of byproducts in olive processing, such as olive leaves, olive skins, and vegetation water. It is well known that olive leaves contain several phenolic compounds, including secoiridoids. Oleuropein is the major secoiridoid in olive leaves. Oleuropein has been found to exhibit antioxidative, antimicrobial, antiviral, and antiatherogenic activities. We studied the effect of extraction techniques and drying methods on oleuropein content in olive leaves of Istrska belica and Lecino cultivar. Three different procedures of drying were used: at room temperature, at 105 °C, and freeze drying. Ethanol-modified supercritical extraction with carbon dioxide, conventional methanol extraction, and ultrasonic extraction with deep eutectic solvent were performed. Antioxidant activity was determined, as well as methanolic and supercritical extracts. The presence of olive polyphenols was confirmed by the HPLC method.

The Effect of Olive Fruit Fly Bactrocera oleae (Rossi) Infestation on Certain Chemical Parameters of Produced Olive Oils.

Olives affected by active and damaging infestation (olive fruit fly Bactrocera oleae (Rossi)) were assayed for their chemical composition. Biophenols were determined by HPLC, sterols, triterpenic dialcohols, and fatty acids by gas chromatography analysis. The acquired data were statistically analyzed. Oils produced from "Istrska belica" fruit affected by active infestation compared to the oils made from fruit affected by damaging infestation showed higher amounts of total oleuropein biofenols (377.3 versus (vs.) 106.6 mg/kg), total biophenols (755 vs. 377 mg/kg), lignans (85.3 vs. 32.9 mg/kg), the dialdehydic form of decarboxymethyl oleuropein aglycone (DMO-Agl-dA) (148.3 vs. 49.0 mg/kg), its oxidized form (DMO-Agl-dA)ox (35.2 vs. 8.5 mg/kg), the dialdehydic form of oleuropein aglycone (O-Agl-dA) (61.1 vs. 8.0 mg/kg), the dialdehydic form of ligstroside aglycone (L-Agl-dA) (63.5 vs. 28.0 mg/kg), the aldehydic form of oleuropein aglycone (O-Agl-A) (40.6 vs. 8.4 mg/kg), and lower amounts of tyrosol (Tyr) (6.0 vs. 13. 9 mg/kg) and the aldehydic form of ligstroside aglycone (L-Agl-A) (13.8 vs. 40.3 mg/kg). Higher values of stigmasterol (2.99%) and lower values of campesterol (2.25%) were determined in oils affected by damaging infestation; an increase in triterpenic dialcohols was also observed (3.04% for damaging and 1.62% for active infestation). Oils affected by damaging infestation, compared to active infestation, showed lower amounts of oleic acid (73.89 vs. 75.15%) and higher amounts of myristic (0.013 vs. 0.011%), linoleic (7.27 vs. 6.48%), and linolenic (0.74 vs. 0.61%) acids.

An Integrated Characterization of Jujube (Ziziphus jujuba Mill.) Grown in the North Adriatic Region.

Jujube (Ziziphus jujuba Mill.) has favourable horticultural properties including adaptation to arid conditions, abiotic and biotic stresses, as well as positive impact on human health. The present study describes the characterization of genetic diversity of the germplasm of jujube from the Istrian peninsula, the determination of important chemical compounds, antioxidative properties in relation to antibacterial and antifungal activities of jujube fruit extracts, and the determination of nutritional properties of jujube fruit. The results of the genetic analysis showed that most of the samples from the Istrian peninsula belong to two recently introduced varieties, 'Li' and 'Lang', and the most widespread local variety 'Navadna zizola'. The local variety has smaller fruit than the 'Li' and 'Lang' varieties, with thick and fleshy mesocarp. Chemical analysis indicated that fruits of the local variety contained a valuable source of dietary fibre ((9.7±0.6) g/100 g) and were rich in minerals such as (in g/100 g dry mass): potassium (829±51), calcium (177±11) and phosphorus (129±19). Aqueous extracts showed slight antibacterial activity, while ethanol extracts had higher mass fractions of phenolic compounds (expressed as gallic acid equivalents (GAE), 5.8-8.7 mg/g) than the aqueous extracts, but did not show antimicrobial activity. Compounds other than phenolic compounds in jujube fruit may be more biologically active. Based on the results of these analyses, the local Istrian jujube variety is a promising candidate for cultivation potential.

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.

Genetic, Biochemical, Nutritional and Antimicrobial Characteristics of Pomegranate (Punica granatum L.)Grown in Istria.

This study characterises the genetic variability of local pomegranate (Punica granatum L.) germplasm from the Slovenian and Croatian areas of Istria. The bioactive components and antioxidant and antimicrobial properties of ethanol and water extracts of different parts of pomegranate fruit were also determined, along with their preliminary nutritional characterisation. Twenty-six different genotypes identified with microsatellite analysis indicate the great diversity of pomegranate in Istria. The pomegranate fruit ethanol extracts represent rich sources of phenolic compounds (mean value of the mass fraction in exocarp and mesocarp expressed as gallic acid is 23 and 16 mg/g, respectively). The ethanol extracts of pomegranate exocarp and mesocarp showed the greatest antimicrobial activity against Candida albicans, Candida parapsilosis, Rhodotorula mucilaginosa, Exophiala dermatitidis and Staphylococcus aureus, and the same water extracts against S. aureus and Escherichia coli. To the best of our knowledge, this study represents the first report of the characterisation of pomegranate genetic resources from Istria at different levels, including the molecular, chemical, antimicrobial and nutritional properties.

Influence of phenols mass fraction in olive (Olea europaea L.) paste on volatile compounds in Buza cultivar virgin olive oil.

The influence of the phenolic content in olive paste of cv. Buza increased by the addition of an aqueous solution of phenolic extract of freeze-dried olive pulp (cv. Istarska bjelica) on the final products of the lipoxygenase pathway in oil was studied. Increases by 12, 38, and 56% for ripe fruits (maturity index = 4.0) and by 38% for unripe fruits (maturity index = 1.2) were examined. Phenols in the olive paste were determined according to the HPLC method, whereas volatiles in oil were determined according to SPME-GC-MS. A significant negative effect on Z-3-hexenal and E-2-hexen-1-ol (Tukey's test, p < 0.05) was found for ripe fruits (average decreases of 55 and 60%, respectively), but not for the unripe sample. Positive effects in both ripening levels were found for Z-3-hexenyl acetate (average increase of 68% for ripe and a double increase for unripe fruits) and total C5 compounds (average increase of 32% for ripe and an increase of 30% for unripe fruits).

Monitoring of quorum-sensing molecules during minifermentation studies in wine yeast.

At high cell density or under low nutrient conditions, yeasts collectively adapt their metabolism by secreting aromatic alcohols in what is known as quorum sensing. However, the mechanisms and role of quorum sensing in yeast are poorly understood, and the methodology behind this process is not well established. This paper describes an effective approach to study quorum sensing in yeast fermentations. The separation, detection, and quantification of the putative quorum-sensing molecules 2-phenylethanol, tryptophol, and tyrosol have been optimized on a simple HPLC-based system. With the use of a phenyl HPLC column and a fluorescence detector, the sensitivity of the system was significantly increased. This allowed extraction and concentration procedures to be eliminated and the process to be scaled down to 2 mL minifermentations. Additionally, an innovative method for rapid viable-cell counting is presented. This study forms the basis for detailed studies in kinetics and regulation of quorum sensing in yeast fermentation.

New vitamin E isomers (gamma-tocomonoenol and alpha-tocomonoenol) in seeds, roasted seeds and roasted seed oil from the Slovenian pumpkin variety 'Slovenska golica'.

The Stajerska region in north-eastern Slovenia and the Styria region in southern Austria have a long tradition of growing pumpkins (Cucurbita pepo L.) as an oil crop. GC-MS determination of the free and esterified minor compounds in oil of roasted pumpkin seeds from the Slovenian C. pepo L. variety 'Slovenska golica' revealed the presence of two previously unreported compounds: alpha-tocomonoenol and gamma-tocomonoenol. Using the GC-MS data, reference samples (Crude Palm Oil) and tocopherol and tocotrienol standards it was possible to assign and quantify alpha-tocomonoenol (17.6±0.6µg/g) and gamma-tocomonoenol (118.7±1.0µg/g) compounds in roasted 'S. golica' seed oil using HPLC. The concentrations of alpha-tocopherol and gamma-tocopherol were 77.9±1.9µg/g and 586.0±4.6µg/g, respectively. Surprisingly the gamma-tocotrienol concentration found was only 6.9±0.2µg/g. Analysis of the seeds from which the oil was pressed showed the initial gamma-tocotrienol amount was even lower (1.6±0.1 and 2.2±0.1µg/g in the ground and roasted seeds, respectively) than in the roasted seed oil.

Stereospecific analysis of triacylglycerols as a useful means to evaluate genuineness of pumpkin seed oils: lesson from virgin olive oil analyses.

In Slovenia two superb vegetable oils with high added nutritional value are produced: "Ekstra devisko oljcno olje Slovenske Istre (extra virgin olive oil from Slovene Istra)" and "Stajersko prekmursko bucno olje (pumpkin seed oil from Slovenia)". Their quality and genuineness must be monitored as adulteration can easily be undertaken. Olive oil genuineness determination experiences can show how analyses following an experience data-driven decision tree gathering several chemical determinations (fatty acids, (E)-isomers of fatty acids, sterol and tocopherol determinations) may be helpful in assessing the pumpkin seed oil from Slovenia genuineness. In the present work a set of HPLC triacylglycerol determinations was performed, based on the nine main triacylglycerols (LLLn, LLL, PLL, LOO, PLO, OOO, POO, SPL, and SLS) on a limited number of different pumpkin seed oils from northeastern Slovenia. The performed determinations showed that stereospecific analyses of triacylglycerols together with other chemical determinations can be useful in building a protocol for the evaluation of the genuineness of pumpkin seed oil from Slovenia.

Chemical changes in extra virgin olive oils from Slovenian Istra after thermal treatment.

Changes in the physico-chemical parameters of extra virgin olive oils after heating for 142h at 100°C with an air flow 10L/h were investigated. The experimental study was carried out on the two predominant olive cultivars in Slovenian Istra - cv. Istrska belica and cv. Leccino. The data obtained showed that oils from Istrska belica were more stable than those from Leccino. Peroxide values and spectrophotometric data showed higher amounts of oxidation products in oils from Leccino than in those from Istrska belica. After thermal treatment fatty acid composition was changed more in Leccino oils; particularly the amounts of polyunsaturated fatty acids dropped significantly, while α-tocopherol was completely depleted in all samples. The content of total biophenols decreased from 598mg/kg to 241mg/kg in Istrska belica oils and from 391mg/kg to 176mg/kg in Leccino oils. HPLC data showed that transformation of secoiridoid biophenols to the simple biophenols, tyrosol and hydroxytyrosol took place.