New and Rapid Analytical Method Using HPLC-MS Detection for Acrylamide Determination in Black Ripe Olives.

The presence of acrylamide, a known human carcinogen, in various heated foods raises significant concerns among consumers. Therefore, the development of a good analytical method is of paramount interest to the scientific community. Keeping this in view, a rapid, simple, reliable, and low-cost analytical method was developed and validated for acrylamide quantification in black ripe olives. The method consisted of the water extraction of the compounds from crushed olives with the addition of (13C3)acrylamide as an internal standard. The quantification was performed using high-pressure liquid chromatography and mass detection with positive electrospray ionization. The limits of detection and quantification were determined to be 4 and 11 µg/kg, respectively. The developed method exhibited excellent results in terms of accuracy (98.4-104.8%) and intra- and inter-day precision limits, both less than 20%. This new method was carried out by analyzing 15 samples of Spanish commercial black ripe olives, revealing a wide range of values, from 79 to 1068 µg/kg of fruit. The new protocol reduces the analysis time to just one hour per sample versus the minimum 24 h required by gas chromatography and mass detection, meaning that it could be a good option for the routine analysis of acrylamide in black ripe olives, and may be extendable to the analysis of this compound in other foods.

Olive Leaf Processing for Infusion Purposes.

Olive leaf is a by-product rich in bioactive compounds, such as polyphenols and triterpenic acids, with numerous biological activities for human health. Nowadays, the existence of dry olive leaves marketed for infusion elaboration is lacking. During the elaboration process, the drying and grinding stages are critical for the conservation of bioactive compounds, and, precisely, the existing research on olive leaf production procedures is quite scarce. This work aimed to study and model the dehydration process using a forced-air oven and infrared with air convection systems. In addition, different grinding grades were studied. The kinetic constant and activation energy during dehydration were obtained. Drying temperatures above 50 °C produced a decrease in the phenolic concentration of olive leaves; however, it has been observed that prior storage of 24 h at room temperature considerably reduced the loss of phenols. Likewise, it was observed that the higher the degree of grinding, the greater the diffusion of both bioactive compounds and colored compounds. Therefore, the drying and grinding stages were closely related to the content of beneficial compounds and the appearance of the infusions, and their optimization was of crucial importance to produce dried olive leaves rich in biocompounds for use as healthy infusions.

Chemical and Enzymatic Characterization of Leaves from Spanish Table Olive Cultivars.

Olive leaves are generated as by-products in the olive industry and contain substances with biological properties that provide health benefits. Although these compounds have been characterized in many leaves from olive cultivars devoted to olive oil extraction, few data are available on leaves from the processing of table olives. In this study, the concentration of polyphenols, triterpenic acids, sugars and enzymatic activities (polyphenol oxidase, peroxidase, ß-glucosidase and esterase) were determined in the leaves of the olive tree (Olea europaea L.) of cvs. Aloreña, Cacereña, Empeltre, Hojiblanca, Manzanilla, Verdial, Gordal and Morona. The mean total phenolic content in olive leaves reached 75.58 g/kg fresh weight, and oleuropein was the main polyphenol identified (89.7-96.5%). The main triterpenic acid identified was oleanolic acid, and the main sugar was mannitol, with mean values of 15.83 and 22.31 g/kg, respectively. However, the content of these biocompounds was influenced by the type of cultivar and the orchards of origin. The highest oleuropein content was found in the Manzanilla variety, while the Gordal had the highest triterpene and mannitol content. In particular, the phenolic content could also be affected by endogenous enzymatic activities. High polyphenol oxidase, peroxidase and ß-glucosidase activity and low esterase activity were detected, compared to the fruit. Similar to the phenolic compounds, enzymatic activities varied with the harvesting season. The lowest phenolic content corresponded to the highest polyphenol oxidase activity detected during spring. The rest of the enzymatic activities also varied throughout the year, but no common trend was observed.

Industrial Processing of Algerian Table Olive Cultivars Elaborated as Spanish Style.

Olives from the Sigoise, Verdale, and Sevillana cultivars were elaborated as Spanish-style table olives by four Algerian factories, and the quality and food safety of the industrial table olives have been studied by the analysis of physicochemical and microbiological parameters. Differences were observed between the treatments carried out by the different factories throughout the manufacturing process, especially during the washing stage, but no significant differences were found between the analyzed samples for the concentration of sugars and polyphenols. The final pH values reached at the end of fermentation ranged between 5.04 and 4.27, and the titratable acidity was above 0.4% for all samples. Lactic and acetic acids were produced in mean concentrations of 0.68% and 0.21% as a result of lactic acid bacteria (LAB) and yeast metabolism, respectively. However, the presence of butyric, isobutyric, and propionic acids was also detected, and was related to the growth of undesirable spoilage microorganisms, responsible for secondary fermentations. The high-throughput sequencing of bacterial DNA suggested the dominance of LAB species belonging to genera Lactiplantibacillus, Leuconostoc, Pediococcus, Oenococcus, or Enterococcus. The Enterobacteriaceae family was detected during the first days of brining and in only one sample after 120 days of fermentation. Other spoilage microorganisms were found, such as Lentilactobacillus buchneri or the Pectinatus and Acetobacter genera, capable of consuming lactic acid and these played an essential role in the onset of spoilage. The Clostridium and Enterobacter genera, producers of butyric and propionic acids, were responsible for the malodorous fermentation present in the industrial samples that were analyzed. The study concluded that the safety of the table olives analyzed was compromised by the presence of undesirable microorganisms and microbial stability was not guaranteed. The elaboration process can be improved by reducing the washing steps and the time should be reduced to avoid the loss of fermentable matter, with the goal of reaching a pH < 4.0 after the fermentation and preventing the possibility of the growth of spoilage microorganisms and foodborne pathogens.

Characterization of bioactive compounds in commercial olive leaf extracts, and olive leaves and their infusions.

A large spectrum of beneficial health properties has been attributed to olive leaves. This study was undertaken to characterize the bioactive compounds of commercial olive leaf extracts and olive leaves and their infusions. High variability of bioactive compounds was found among commercial samples. Polyphenol was detected in a range of 44-108 g kg-1 and 7.5-250 g kg-1 for olive leaves and olive leaf extracts, respectively. The main phenol was oleuropein, representing 74-94% of total phenols. However, only 17-26% of polyphenols were diffused to the aqueous phases when olive leaf infusions were prepared. Triterpenic acids were found in a range of 26-37 g kg-1 in olive leaves, but not detected in the infusions. Hence, the absence of the latter substances and the low oleuropein diffusion in olive leaf infusions make new studies necessary to maximize the presence of these bioactive compounds in the final product.

Influence of iron redox state on black ripe olive processing.

BACKGROUND: The oxidation and polymerization of the phenolic compounds present in black ripe olives give rise to their black shiny colour, which is fixed by the addition of ferrous salts such as ferrous gluconate. Among the many variables that can affect the success of this fixation step, the influence of pH on this process was assessed on fruits of the Hojiblanca cultivar processed at pilot plant scale. Hence the aim of this work was to gain knowledge on the development of this fixation step, in particular on olives that were oxidized with a single alkaline treatment. RESULTS: It was found that the penetration of the ferric cation into the olive flesh was an extremely slow process and it led to less-dark olives. Besides, at a pH higher than 6 the oxidation of the ferrous to the ferric cation was a very rapid reaction, and olives that were processed with initial pH adjusted to 4 units had a darker colour than those without pH correction. Ultrafiltration/nanofiltration of oxidized olive solutions spiked with ferric ions or even pure oxidized ferric solutions through membranes of 10, 3 and 1 kDa revealed that ferric ions formed high-molecular structures that inhibited the penetration of the ion into the olive flesh and therefore the fixation of the black colour. CONCLUSION: The results presented in this study suggest that the formation of ferric ions during the colour fixation step of black ripe olives must be avoided to obtain darker olives. Consequently, the initial pH of the ferrous gluconate solution must be corrected to avoid oxidation of the ferrous to ferric cations during the fixation step. This study will contribute to the improvement of the colour fixation step of black ripe olive processing. © 2018 Society of Chemical Industry.

New by-products rich in bioactive substances from the olive oil mill processing.

BACKGROUND: Olive oil extraction generates a large amount of residue consisting mainly of the pomace and leaves when using a two-phase centrifugation system. The aim of this study was to assess the content of phenolic and triterpene compounds in the by-products produced in Spanish olive oil mills. RESULTS: Olive pomace had concentrations of phenolic and triterpene substances lower than 2 and 3 g kg-1 , respectively. The leaves contained a high concentration of these substances, although those collected from ground-picked olives had lost most of their phenolic compounds. Moreover, the sediment from the bottom of the olive oil storage tanks did not have a significant amount of these substances. By contrast, a new by-product called olive pomace skin has been revealed as a very rich source of triterpenic acids, the content of which can reach up to 120 g kg-1 in this waste product, maslinic acid comprising around 70% of total triterpenics. CONCLUSION: Among the by-products generated during extraction of olive oil, olive pomace skin has been discovered to be a very rich source of triterpenic acids, which can reach up to 120 g kg-1 of the waste. These results will contribute to the valorization of olive oil by-products. © 2017 Society of Chemical Industry.

The NUTRAOLEOUM Study, a randomized controlled trial, for achieving nutritional added value for olive oils.

BACKGROUND: Virgin olive oil, a recognized healthy food, cannot be consumed in great quantities. We aim to assess in humans whether an optimized virgin olive oil with high phenolic content (OVOO, 429 mg/Kg) and a functional one (FOO), both rich in phenolic compounds (429 mg/Kg) and triterpenic acids (389 mg/kg), could provide health benefits additional to those supplied a by a standard virgin olive oil (VOO). METHODS/DESIGN: A randomized, double-blind, crossover, controlled study will be conducted. Healthy volunteers (aged 20 to 50) will be randomized into one of three groups of daily raw olive oil consumption: VOO, OVOO, and FOO (30 mL/d). Olive oils will be administered over 3-week periods preceded by 2-week washout ones. The main outcomes will be markers of lipid and DNA oxidation, inflammation, and vascular damage. A bioavailability and dose-response study will be nested within this sustained- consumption one. It will be made up of 18 volunteers and be performed at two stages after a single dose of each olive oil. Endothelial function and nitric oxide will be assessed at baseline and at 4 h and 6 h after olive oil single dose ingestion. DISCUSSION: For the first time the NUTRAOLEUM Study will provide first level evidence on the health benefits in vivo in humans of olive oil triterpenes (oleanolic and maslinic acid) in addition to their bioavailability and disposition. TRIAL REGISTRATION: The Trial has been registered in ClinicalTrials.gov ID: NCT02520739 .

Composition of pigments and colour changes in green table olives related to processing type.

Brownish colourations in Natural green table olives (non-treated with alkali) make this product less attractive to consumers than Spanish-style green table olives (treated with alkali), which develop a more appreciated bright golden-yellow colour. These colour differences were studied in relation to changes in the composition of chlorophyll and carotenoid pigments, as well as polyphenolic compounds and polyphenol oxidase enzyme (PPO) activity. Natural green olives showed a different chlorophyll profile than Spanish-style. However, all the chlorophyll pigments formed in both processing types were Mg-free derivatives (mostly pheophytins) with similar colourations, ranging from grey to green brownish. In the carotenoid fraction no appreciable differences were found between both processing types. The fruit's brownish colour was mainly due to polymeric substances with a size of >1000 daltons and polyphenolic nature, resulting from an enzymatic oxidation by PPO of the o-diphenolic compounds present in the fresh fruits.

Endogenous enzymes involved in the transformation of oleuropein in Spanish table olive varieties.

The main Spanish table olive varieties supplied by different olive cooperatives were investigated for their polyphenol compositions and the endogenous enzymes involved in their transformations during two growing seasons. Olives of the Manzanilla variety had the highest concentration in total polyphenols, followed by the Hojiblanca and Gordal varieties. The Gordal and Manzanilla cultivars showed the highest polyphenol oxidase activities. The Gordal cultivar presented a greater ß-glucosidase and esterase activity than the others. An important influence of pH and temperature on the optimal activity of these enzymes was also observed. The polyphenol oxidase activity increased with temperature, and peroxidase activity was optimal at 35 °C. The ß-glucosidase and esterase activities were at their maximum at 30 and 55 °C, respectively. The oxidase and ß-glucosidase activities were at their maximum at the pH of the raw fruit. These results will contribute to the knowledge of the enzyme transformation of oleuropein in natural table olives.

Assessment of Helicobacter pylori eradication by virgin olive oil.

BACKGROUND: A recent study conducted by Medina et al. disclosed that virgin olive oil has a bactericidal effect in vitro against Helicobacter pylori because of its contents of certain phenolic compounds with dialdehydic structures. We carried out two clinical trials to evaluate the effect of virgin olive oil on H. pylori-infected individuals. MATERIALS AND METHODS: Two different pilot studies were performed with 60 H. pylori-infected adults. In the first study, thirty subjects who tested positive for H. pylori received 30 g of washed virgin olive oil for 14 days, and after 1 month, the patients took 30 g of unwashed virgin olive oil for another 14 days. In a second study, a group of 30 subjects received 30 g of a different virgin olive oil for 14 days. Helicobacter pylori-infection status was checked by the urea breath test. RESULTS: Helicobacter pylori was eradicated in 8 of 30 individuals when microorganism status was checked after 4-6 weeks from the first clinical intervention although 12 of 30 individuals did not show H. pylori infection at 24-72 hour of the last oil dose. Eradication rates were 27 and 40% by intention to treat and per protocol, respectively. Moreover, only 3 of 30 individuals were H. pylori negative after 4-6 weeks from the second clinical intervention but 5 of 30 were negative at 24-72 hour of the last oil dose. Eradication rates were 10 and 11% by intention to treat and per protocol, respectively. It must also be noted that 13 subjects withdrew from the studies because of taste and nausea drawbacks. CONCLUSIONS: The administration of virgin olive oil showed moderate effectiveness in eradicating H. pylori. Further studies are needed to confirm these findings, especially with longer periods, different administration conditions, and several types of olive oils.

Debittering of olives by polyphenol oxidation.

In this study, green olives preserved in acidified brine were debittered by subjecting them to an overpressure of oxygen or air for 1-3 days. It was demonstrated that fruits lost their bitter taste due to the enzymatic oxidation of the phenolic compounds, in particular, the glucoside oleuropein. Hence, the concentrations of both o-diphenols and, to a lesser extent, monophenols decreased in the olives with oxidation. This process also gave rise to a darkening effect on the superficial and interior color of the olives, which turned from yellow-brown to brown. Likewise, the effect of several variables on the oxidation rate of the olives, such as type of gas (oxygen, air), temperature, overpressure level, and size of the olives, was also studied. Results indicate that a new debittering method which could be a promising alternative to the treatment of fruits with NaOH is available to the industry. In addition, a new product with different color and texture from the traditional table olives is presented.

Antimicrobial activity of olive oil, vinegar, and various beverages against foodborne pathogens.

The survival of foodborne pathogens in aqueous extracts of olive oil, virgin olive oil, vinegar, and several beverages was evaluated. Vinegar and aqueous extracts of virgin olive oil showed the strongest bactericidal activity against all strains tested. Red and white wines also killed most strains after 5 min of contact, black and green tea extracts showed weak antimicrobial activity under these conditions, and no effect was observed for the remaining beverages (fruit juices, Coca-Cola, dairy products, coffee, and beer). The phenolic compound content of the aqueous olive oil and virgin olive oil extracts could explain their antibacterial activity, which was also confirmed in mayonnaises and salads used as food models. Virgin olive oil in mayonnaises and salads reduced the counts of inoculated Salmonella Enteritidis and Listeria monocytogenes by approximately 3 log CFU/g. Therefore, olive oil could be a hurdle component in certain processed foods and exert a protective effect against foodborne pathogens when contaminated foods are ingested.

Comparison of analytical methodologies based on 1h and 31p NMR spectroscopy with conventional methods of analysis for the determination of some olive oil constituents.

The present study was designed to assess the agreement between analytical methodologies based on 1H and 31P NMR spectroscopy and conventional analytical methods (titration, gas chromatography, and high performance liquid chromatography) for measuring certain minor and major constituents (free acidity, fatty acids, iodine value, and phenolic compounds) of olive oil. The standard deviations of the NMR method were comparable to those of the conventional methods, except perhaps those of the total hydroxytyrosol and total tyrosol. Linear regression analyses showed strong correlations between NMR and conventional methods for free acidity, total hydroxytyrosol, total tyrosol, total diacylglycerols, (+)-pinoresinol, (+)-1-acetoxypinoresinol, and apigenin; good correlations for linoleic acid, free hydroxytyrosol, and free tyrosol; and weak correlations for oleic acid, linolenic acid, saturated fatty acids, and luteolin. Furthermore, a method comparison study was conducted and the agreement between NMR and conventional methods was evaluated by using the Bland and Altman statistical analysis. The distribution of the data points in the bias plot showed that 96.4% and 100% of the measurements of free acidity and iodine value, respectively, were within the limits of agreement of the two methods. For the remaining constituents of olive oil, the percentage of measurements, located within the limits of agreement, ranged from 94% to 98.5%.

In vitro activity of olive oil polyphenols against Helicobacter pylori.

Helicobacter pylori is linked to a majority of peptic ulcers and to some types of gastric cancer, and resistance of the microorganism to antibiotic treatment is now found worldwide. Virgin olive oil is an unrefined vegetable oil that contains a significant amount of phenolic compounds. Under simulated conditions, we have demonstrated that these substances can diffuse from the oil into the gastric juice and be stable for hours in this acidic environment. In vitro, they exerted a strong bactericidal activity against eight strains of H. pylori, three of them resistant to some antibiotics. Among the phenolic compounds, the dialdehydic form of decarboxymethyl ligstroside aglycon showed the strongest bactericidal effect at a concentration as low as 1.3 microg/mL. Although the experimental conditions are different from other reported works, this bactericidal concentration is much lower than those found for phenolic compounds from tea, wine, and plant extracts. These results open the possibility of considering virgin olive oil a chemopreventive agent for peptic ulcer or gastric cancer, but this bioactivity should be confirmed in vivo in the future.

Polyphenol changes during fermentation of naturally black olives.

The individual evolution of phenolic compounds has been studied during the natural fermentation of black olives for the first time. Cyanidin 3-rutinoside and cyanidin 3-glucoside were the main anthocyanins identified in fresh olives, and they were not detected after 1 month of storage either in brine or in olive. The fruit colors were different when aerobic or anaerobic conditions were used and as a consequence of the different anthocyanin polymerizations that took place. At time zero, the polyphenols observed in the olive juice were hydroxytyrosol-4-beta-glucoside, oleuropein, hydroxytyrosol, tyrosol, salidroside, and verbascoside and, after 12 months, the main phenol was hydroxytyrosol. The polyphenol content in the oil phase of olives was also analyzed. The dialdehydic form of elenolic acid linked to hydroxytyrosol and tyrosol, oleuropein aglycon, and ligstroside aglycon were the main compounds found at the beginning of fermentation but were not detected after 3 months. In contrast, hydroxytyrosol, hydroxytyrosol acetate, tyrosol, and tyrosol acetate were the main polyphenols detected in the oil phase of the final product. The acid hydrolysis of the initial glucosides (in olive juice) and the aglycons (in oil phase) was, therefore, the main reaction that took place during fermentation.

Effect of cultivar and processing method on the contents of polyphenols in table olives.

Polyphenols were determined by HPLC in the juice and oil of packed table olives. The phenolic compositions of the two phases were very different, hydroxytyrosol and tyrosol being the main polyphenols in olive juice and tyrosol acetate, hydroxtyrosol acetate, hydroxytyrosol, tyrosol, and lignans (1-acetoxypinoresinol and pinoresinol) in oil. The type of processing had a marked influence on the concentration of polyphenols in olive juice and little on the content in oil. The analyses carried out on 48 samples showed that turning color olives in brine had the highest concentration in polyphenols ( approximately 1200 mg/kg), whereas oxidized olives had the lowest ( approximately 200 mg/kg). Among olive cultivars, Manzanilla had a higher concentration than Hojiblanca and Gordal. The type of olive presentation also influenced the concentration of polyphenols in olives, decreasing in the order plain > pitted > stuffed. The results obtained in this work indicate that table olives can be considered a good source of phenolic antioxidants, although their concentration depends on olive cultivar and processing method.

Influence of thermal treatments simulating cooking processes on the polyphenol content in virgin olive oil.

Virgin olive oils were subjected to simulated common domestic processing, including frying, microwave heating, and boiling with water in a pressure cooker. The impact of these processes on polyphenol content and physicochemical characteristics of oils was assessed. Thermal oxidation of oils at 180 degrees C caused a significant decrease in hydroxytyrosol- and tyrosol-like substances. In contrast, oils heated for 25 h still retained a high proportion of the lignans 1-acetoxypinoresinol and pinoresinol. Thermal oxidation also resulted in a rapid degradation of alpha-tocopherol and the glyceridic fraction of oils. Microwave heating of oils for 10 min caused only minor losses in polyphenols, and the oil degradation was lower than that in thermoxidation assays. Again, lignans were the least affected polyphenols and did not change during microwave heating. Boiling a mixture of virgin olive oil and water in a pressure cooker for 30 min provoked the hydrolysis of the secoiridoid aglycons and the diffusion of the free phenolics hydroxytyrosol and tyrosol from the oil to the water phase. Losses of polyphenols were detected only at pH lower than 6. Moreover, alpha-tocopherol and the glyceridic fraction of oils were not modified during this process. It is worth noting that all the heating methods assayed resulted in more severe polyphenols losses and oil degradation for Arbequina than for Picual oil, which could be related to the lower content in polyunsaturated fatty acids of the latter olive cultivar. These findings may be relevant to the choice of cooking method and olive oil cultivar to increase the intake of olive polyphenols.

Validation of a method for the analysis of iron and manganese in table olives by flame atomic absorption spectrometry.

There is no generally accepted method for determination of the amounts of iron and manganese in table olives. Application of flame atomic absorption spectrometry to the analysis of both elements has been examined to validate a method that may be used by the industry's quality control laboratory as well as by the laboratories of regulatory agencies. The method has detection limits of 0.106 and 0.022 mg/L and quantification limits of 0.271 and 0.057 mg/L, for Fe and Mn, respectively, referred to the solution to be measured. There was no significant effect due to the matrix, but a slight bias due to the presence of Ca has been detected. Recoveries were excellent, and the method was robust. Influence of operator, HCl and Mg salt compounds, calcination equipment, or dates on results was not found. Relative errors were, in general, below 4% for both cations, and repeatability was below 3.43 and 0.38 mg/kg of olive paste for Fe and Mn, respectively. The method is proposed for the analysis of Fe and Mn in ripe olives and table olives in general.