Expression of genes involved in metabolism of phenolic compounds by Lactobacillus pentosus and its relevance for table-olive fermentations.

Genes with the potential to code for enzymes involved in phenolic compound metabolism were detected in the genome of Lactobacillus pentosus IG1, isolated from a green olive fermentation. Based on homology, these genes could code for a 6-P-ß Glucosidase, two different Tannases, a Gallate decarboxylase and a p-Coumaric decarboxylase. Expression of up to seven of these genes was studied in L. pentosus IG1 (olive fermentation) and CECT4023T (corn silage), including responses upon exposure to relevant phenolic compounds and different olive extracts. Genes potentially coding Tannase, Gallate decarboxylase and p-Coumaric acid decarboxylase significatively increased their expression upon exposure to such compounds and extracts, although it was strain dependent. In general, both the genetic organization and the characteristics of gene expression resembled very much those described for Lactobacillus plantarum. In accordance to the observed induced gene expression, metabolism of specific phenolic compounds was achieved by L. pentosus. Thus, methyl gallate, gallic acid and the hydroxycinamic acids p-coumaric, caffeic and ferulic were metabolized. In addition, the amount of phenolics such as tyrosol, oleuropein, rutin and verbascoside included in a minimal culture medium was noticeably reduced, again dependent on the strain considered.

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.

Quantification of bioactive compounds in Picual and Arbequina olive leaves and fruit.

BACKGROUND: Olive leaves and fruit possess bioactive substances such as phenolic compounds and triterpenic acids that can be obtained from olive by-products generated during olive oil extraction. The aim of the present study was the characterization and quantification of these compounds in Picual and Arbequina cultivars from different locations and throughout two seasons in both olive leaves and fruit. RESULTS: The major phenolic compound identified in the leaves was oleuropein, and the total content of phenolic compounds in this material reached 70 g kg-1 fresh weight. The leaves were also rich in triterpenic acids (20 g kg-1 fresh weight), with oleanolic acid being the most concentrated among them. With regard to olives, oleuropein and demethyloleuropein were the main phenolic compounds in the pulp of Picual and Arbequina cultivars, and the total concentration of these phenolic compounds reached 3.5% fresh weight. Olives can also be an important source of triterpenic acids, although this is mainly the skin part, where the maslinic and oleanolic acids are concentrated. CONCLUSION: Olive leaves can contain up to 70 g kg-1 phenolic compounds and 20 g kg-1 triterpenic acids, and olive fruit can contain up to 35 g kg-1 of the former and 3 g kg-1 of the latter. It must also be noted that this level was constant both between seasons and orchard locations. © 2016 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 .

Assays to control the development of the green staining alteration in Spanish-style green olives of the Gordal variety.

BACKGROUND: Olives of the Gordal variety processed according to the Spanish style sometimes develop an alteration in color known as green staining (GS), due to the formation of harmless copper-chlorophyll complexes, which makes the product less valuable. The aim of this study was to investigate methods to minimize the impact that this alteration has for the table olive industry. RESULTS: Calcium chloride, sorbic, benzoic and ascorbic acids and SO2 did not inhibit the development of the alteration in olives packed under their own fermentation brine or new fresh brine. It was also discovered that the incubation of olive samples at 45 °C for 20 days accelerates the formation of GS and can be a very useful tool to predict the incidence of the alteration in advance. By applying this test to numerous industrial tanks for four consecutive seasons, it was found that GS was mainly present in olives harvested at the beginning of the season. CONCLUSION: The formation of GS in olives of the Gordal variety is time- and temperature-dependent, and none of the additives tested avoided or retarded the development of the alteration. However, an accelerated test to predict the development of GS formation has been proposed that could contribute to minimize the effects of the alteration. © 2015 Society of Chemical Industry.

Production of 4-ethylphenol in alperujo by Lactobacillus pentosus.

BACKGROUND: Alperujo is the paste generated from the two-phase extraction system of olive oil. This wet pomace must be stored for several months and, during this period, the formation of 4-ethyphenol provokes a strong off-odour. The aim of this work was to identify the microorganisms able to produce this volatile phenol. RESULTS: Yeast and bacterial strains were isolated from stored alperujo and tested for their ability to metabolize p-coumaric acid and form 4-ethylphenol. Among them, Lactobacillus pentosus was the microorganism that both in synthetic medium and alperujo gave rise to 4-ethylphenol formation. This microorganism did not grow in alperujo acidified to pH 2, thereby confirming that acidification as the best method to control odour emissions during alperujo storage. CONCLUSION: Lactic acid bacteria, particularly Lactobacillus pentosus, can be responsible for the formation of the off-odour caused by 4-ethylphenol during the storage of alperujo. This odour can be prevented by acidifying the alperujo.

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.

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.

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.

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.

Reuse of KOH Solutions during Black Ripe Olive Processing, Effect on the Quality of the Final Product and Valorization of Wastewaters as Possible Fertilizer Product.

A high volume of water is needed to produce black ripe olives, which also entails a significant volume of wastewater with a high organic and inorganic contaminant charge. To reduce this problem, the reuse of KOH solutions (lyes) in a new process was studied. Once the lyes were removed from the tanks, KOH was then added for a new darkening process. Reusing the lyes up to four times gave rise to a product with similar physico-chemical and organoleptic characteristics as obtained with fresh solutions. The application of this process reduced coadjutant consumption by 32% and water by 20%, while global wastewater presented a high K content whose concentration could be valorized as a fertilizer by replacing commercial potassium nitrate.

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.

Packing black ripe olives in acid conditions.

The type of container (airtight and pouches with different O2 permeability) and packing conditions (cover brine, air or N2 atmosphere) has been studied to preserve black ripe olives in acid medium for a year. Unlike the traditional sterilized product, these acidified olives only needed pasteurization to assure its microbial safety, the absence of acrylamide being an additional advantage. Surprisingly, an increase in the oxygen diffusion through the films (i) faded the black color of the olives, (ii) softened the fruit that lost around 33% of its initial firmness in only 6 months, and (iii) produced the lipid́s oxidation forming volatile compounds that transmitted an abnormal flavor which tasters identified as rancid. Therefore, ripe olives in acid medium must be packed in airtight containers such as glass jars, cans o metallic pouches with cover brine or N2 atmosphere. The addition of calcium is recommended to avoid olive softening.

Effect of fertilisation with black table olive wastewater solutions on production and quality of tomatoes cultivated under open field conditions.

This study was aimed at making progress on the valorisation of table olive wastewater that currently represent a big environmental problem for factories. Concentrates from vacuum evaporation of the wastewater generated during processing of black ripe olives treated with KOH were tested as fertilisers of tomato plants in open field assays for three consecutive crops. Fertilisation was performed by drip irrigation every 15 days; the first treatment being 15 days after transplanting, and a total of five fertilisation treatments were carried out. A phytotoxic effect was not observed on plants or fruit in any case but higher yield (fruit/plant and g/plant) was obtained in comparison to irrigation with only tap water. Moreover, the combined use of the olive concentrate with inorganic nitrogen supply (NH4NO3), in order to comply with the nutrient needs of plants, was also tested. The results demonstrated that the olive concentrates could be a good substitute for inorganic potassium (as KNO3) during the cultivation of tomato plants without any negative effect on the tomato quality (pH, °Brix, sugars, organic acids) or content of bioactive substances (phenolic compounds and carotenoids).

Supplementing the Diet of Dairy Goats with Dried Orange Pulp throughout Lactation: I. Effect on Milk Performance, Nutrient Utilisation, Blood Parameters and Production Economics.

Dried orange pulp (DOP) can be incorporated into ruminant diets, but no reports have considered this strategy during the entire lactation period in goats. Two experiments were performed using lactating Payoya goats. In experiment 1, to study the effect, over 180 days, of DOP on milk yield and composition, blood metabolites and economic values, 44 primiparous goats were allocated into three groups: control diet (concentrate plus lucerne) and DOP40 and DOP80 diets, in which DOP replaced 40% and 80%, respectively, of the cereals. Nutrient digestibility and rumen fermentation were also studied (experiment 2). The DOP diets did not affect milk yield and composition. DOP triggered lower intake and digestibility of ether extract and crude protein. Ruminal fermentation was unaffected by DOP, except for a decrease in butyrate for DOP80. The energy balance was unaltered by diet while the balance and retention of nitrogen decreased. Regarding plasma biochemistry, DOP supplementation caused changes that could indicate an improvement in hepatic function and reduced muscular damage and oxidative muscular stress. Moreover, DOP80 provided a profit increase of EUR 3.27/goat. In conclusion, the partial replacement of cereals by DOP is a profitable and healthy nutritional strategy in dairy goats and is suitable for the entire lactation period without compromising productivity.

Chemical hydrolysis of oleuropein affected by the type of organic acid.

The debittering of natural table olives is a very slow process. The effect of acetic, lactic and citric acids on the hydrolysis rate of oleuropein was studied in vitro and at pilot plant scale. The acid hydrolysis of oleuropein was faster with lactic and citric acids than acetic acid, running the experiments at the same pH of 3.8-4.0 units. The temperature exerted a high effect of the hydrolysis of oleuropein in a range of 10-30 °C and the concentration of the organic acid did not show a significant trend. Moreover, the in vitro results were confirmed with three lots of olives that presented a higher content of oleuropein after 3-7 months of preservation when they were processed with acetic acid rather than lactic acid and the opposite for hydroxytyrosol. These results open the possibility of accelerating the debittering of natural olives by preserving them with lactic acid instead of acetic acid.

Progress on green table olive processing with KOH and wastewaters reuse for agricultural purposes.

Table olive wastewaters are seriously polluting and a difficult to treat effluent worldwide, mainly due to their high content in sodium. An alternative approach could be the treatment of the olives with KOH instead of NaOH, in order to reuse the olive streams as biofertilizers. In this study, the debittering of olives with KOH was investigated at pilot plant scale in two olive seasons. The results indicated that a concentration between 1.7 and 2.0% of KOH (similar to that employed with NaOH) led to a fermented product with the same physicochemical and organoleptic characteristics than the traditional one. The spent lyes and washing waters from the KOH treatments were gathered and vacuum evaporated, giving rise to a concentrated solution rich in potassium (52 g/L) that was tested as biofertilizer in open tomato fields. Furthermore, the drip irrigation of the tomato plants with a combined olive solution and mineral fertilizer (NH4NO3) produced similar tomato yield and quality than the irrigation with only mineral fertilizer (NH4NO3 + KNO3). Overall, it has been demonstrated that Spanish-style green olives can be processed with KOH and the effluents valorized to be used as biofertilizer.

Bactericidal activity of glutaraldehyde-like compounds from olive products.

The bactericidal effects of several olive compounds (nonenal, oleuropein, tyrosol, the dialdehydic form of decarboxymethyl elenolic acid either free [EDA] or linked to tyrosol [TyEDA] or to hydroxytyrosol [HyEDA]), other food phenolic compounds (catechin, epicatechin, eugenol, thymol, carvacrol, and carnosic acid), and commercial disinfectants (glutaraldehyde [GTA] and ortho-phthalaldehyde [OPA]), were tested against strains of Pseudomonas fluorescens, Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli. It was found that the bactericidal activities of olive GTA-like compounds (EDA, HyEDA, and TyEDA) were greater than those exerted by several food phenolic substances. Surprisingly, these olive antimicrobials were as active as the synthetic biocides GTA and OPA against the four bacteria studied. Thus, it has been proposed that the bactericidal activity of the main olive antimicrobials is primarily due to their dialdehydic structure, which is similar to that of the commercial biocides GTA and OPA. Our results clearly reveal that olive GTA-like compounds possess a strong bactericidal activity even greater than that of other food phenolic compounds or synthetic biocides.

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.