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19 Páginas.-- 1 Tabla.-- 5 Figuras ; 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 ; This work was supported by the Spanish Government Projects AGL2013‐42739‐R and AGL2016‐76820‐R (AEI/FEDER, UE). ; Peer reviewed

17 Páginas.-- 3 Tablas.-- 3 Figuras ; The aim of this work was to assess the contents of phenolic and triterpenic substances in commercial olives produced in the USA and Spain, which currently use different olive cultivars and elaboration methods The phenolic compounds identified in black ripe olives were hydroxytyrosol, hydroxytyrosol-4-glucoside, hydroxytyrosol acetylated, salidroside, tyrosol, luteolin-7-glucoside, p-coumaric acid and comselogoside.The concentration of these substances in American black ripe olives was lower than 50 mg/kg whereas it was higher than 150 mg/kg in Spanish olives regardless of whether the American processors used the Manzanilla cultivar, which contains higher contents of these substances in the harvested fruit than the Hojiblanca cultivar. Moreover, the concentration of triterpenic acids (oleanolic and maslinic) in American black ripe olives was lower than 200 mg/kg whereas it was higher than 400 mg/kg in the Spanish product. These results indicate that the method used by American processors to elaborate black ripe olives gives rise to lower contents in bioactive substances than the Spanish method, and it must be related to the number of alkali/washing cycles used during processing. Hence, olive processors worldwide must keep in mind the increasing consumer demand for healthy products rich in bioactive substances when developing their elaboration methods. ; This work was partially supported by the Project AGL-2013-42739-R and the European Union FEDER funds. ; Peer reviewed

27 Páginas, 3 Tablas, 5 Figuras ; The development of a method for darkening black ripe olives during the washing step with a single NaOH treatment and preservation liquid was studied. Olives of the Hojiblanca cultivar were darkened at pilot plant scale, packed, sterilized, and analyzed after 2 mo of storage at ambient temperature. It was found that the use of a mixture of preservation liquid:water at a ratio of 1:1 during the first washing gave rise to darker olives with slightly better firmness and no effect on sensory quality. However, care must be taken with the concentration of acetic acid in the preservation solution, as a content of this organic acid higher than 25 g/L can cause adverse effects on olive color due to the low pH that can be reached in the flesh of the fruit. Additionally, the re‐use of the preservation solution in the first washing resulted in enrichment in antioxidant compounds of the packed product. Black ripe olives processed with preservation liquid had a total phenolic content of 629 mg/kg, whereas those with only tap water had 376 mg/kg, in particular hydroxytyrosol and hydroxytyrosol‐4‐glucoside. These findings mean that it is possible to get darker olives with higher contents in bioactive substances by reusing the preservation liquid during the darkening step of black ripe olives. ; This work was supported by the Spanish Government Project AGL‐2013‐42739‐R and the European Union FEDER funds. The authors thank Alejandra Expósito de la Cerda for her assistance with the experimental work. ; Peer reviewed

Olives can reduce their bitterness by oleuropein oxidation via an enzymatic reaction. In this study, olives of the Manzanilla and Hojiblanca varieties preserved in acidified brine were submitted to oxidation tests under overpressure of oxygen (0.3 bars) for 3 days. It was found that the oxidation of fruit preserved for more than 3–4 months showed a reduction in the concentration of oleuropein, but with great variability (28–100%). By contrast, when olives were preserved for only one month, the oxidation treatment was able to decrease more than 90% of the content of oleuropein in the fruit. These oxidised olives were stored again in the acidified medium for 6 months and no spoilage or organoleptic problems were detected. Oxidation gave rise to darker olives (L* parameter diminished from 40 to 34). Moreover, the oxidised fruit was enriched with free phenols such as hydroxytyrosol and tyrosol as a consequence of the acid hydrolysis of phenolic glucosides during the preservation step. ; This work was supported by the Project AGL-2013-42739-R and the European Union FEDER funds. ; Peer Reviewed

This study investigates the application of electro-coagulation and ozonation technologies for the partial depuration of ferrous solutions deriving from the color fixation stage of ripe olive processing. Different operational conditions were investigated along with the possibilities of combining the two techniques sequentially. In electro-coagulation the best depuration conditions were obtained using a current density of 25 mA cm-2 for 40 min; COD elimination reached 30%, the phenols and color of the solutions were also considerably reduced and the dissolved Fe was completely removed. On the contrary, the microbial population mainly composed of lactic acid bacteria and yeasts, hardly decreased. The application of ozonation also reduced the total phenols, color and COD of solutions, but did not decrease the iron concentration. However, it considerably affected the microbial population in a higher proportion than electro-coagulation. In light of the results obtained, the best working conditions consist of applying 40 min electro-coagulation with aluminum in the anode and iron in the cathode at 25 mA cm-2 current density, followed by the storage of a partially purified solution bubbling ozone to reduce the microorganism population and even eliminate it completely when 6 g O3 L-1 were added. This would allow for a possible reuse of these solutions for preparing new fixing color solutions or as cover brine in packaging. © 2014 Elsevier B.V. All rights reserved. ; This work was supported by the Spanish Government (project AGL2010-15494 partially financed from European regional development funds, ERDF) and Junta de Andalucía (through financial support to group AGR-125). ; Peer Reviewed

Benthic diatoms are well known bioindicators of water quality, used in many aquatic ecosystems. Since diatom-based monitoring of water quality is required by European legislation, the search for methods that facilitate this task has become more relevant. The aim of this study was to test the reliability of DNA metabarcoding combined with high-throughput sequencing (HTS) techniques in the bioassessment of 22 Mediterranean shallow ponds in Spain. For each pond, Trophic Diatom Index (TDI) was calculated from inventories obtained by using light microscopy, and then molecular (HTS) methods. Ponds were subsequently classified into five water quality classes. Our results showed a good correspondence between both methods, especially after applying a correction factor depending on the biovolume of the cells. This correspondence led to the assignment to the same quality class in 59% of the ponds. The determination and quantification of valves or DNA sequences was one of the main pitfalls, mainly those related to the variability in the relative abundances of some species. Accordingly, ponds with similar relative abundances for the dominant species were assigned to the same quality class. Moreover, other difficulties leading the discrepancies were the misidentification of some species due to the presence of semi-cryptic taxa, the incompleteness of the reference database and the bioinformatic protocol. Therefore, the validation of DNA-based methods for the identification of freshwater diatoms represents an important goal, as an alternative to traditional ones in Mediterranean shallow ponds.

The aim of this work was to obtain ripe olives enriched in bioactive compounds of phenolic nature. For this, two partially purified solutions rich in phenols from the table olive processes were used as cover in the packaging: washing waters of Spanish-style green olives or previous preservation acid solutions of ripe olives. Both solutions were nano-filtered through a 4000 Da membrane and vacuum evaporated at pilot plant scale. Regenerated liquids were employed at 50% and 100% in the packing brine. Results demonstrated that it is possible to achieve a >3-fold higher concentration of phenolic compounds in olives than with the traditional method when using the regenerated washing water at 100% to enrich the packing brine. Moreover, a <2-fold higher concentration was found in olives enriched with the regenerated preservation solution at 100%. It must also be noted that the color, texture and flavor of the olives were not affected by the use of regenerated washing water as packing solution. Industrial relevance: Among the different types of commercial table olives, black ripe have the lowest content in phenolic compounds. The methodology developed in this work will allow the enrichment of black olives in these bioactive substances, in particular hydroxytyrosol, which has been attributed to many beneficial properties for human health. The methodology is simple and easy to implement by processors. Hence, these results will contribute to the nutritional valorization of black ripe olives. ; This work was supported by the Spanish Government Projects AGL2013-42739-R and AGL2016-76820-R (AEI/FEDER, UE), and the Junta de Andalucía through financial support to group AGR-125. The authors thank Alejandra Expósito for her assistance with the experimental work. ; Peer Reviewed

In the present study, a dose-response model was used to investigate the susceptibility (NIC) and resistance (MIC) of the lactic acid bacteria and yeast populations with respect to five chemical preservatives (fumaric and pyruvic acids, cinnamaldehyde, sodium metabisulphite and natamycin) with potential application in table olives. Results were compared with respect to potassium sorbate, a well-known preservative habitually used in olive packaging. Sodium metabisulphite was the most efficient preservative to control lactic acid bacteria growth (MIC, 50 ppm), followed by cinnamaldehyde (1060 ppm) while pyruvic acid required higher concentrations (3211 ppm). Natamycin (25 ppm) was highly efficient against yeasts, followed by cinnamaldehyde (125 ppm), potassium sorbate (553 ppm), sodium metabisulphite (772 ppm) and pyruvic acid (3038 ppm). Fumaric acid, in the range assayed (0–2000 ppm), did not show any inhibitory effect against these two microbial groups. This survey presents for the first time a comparative study of the efficiency of potential preservatives to control the growth of table olive related microorganisms. Further studies should be performed to validate their effects and interactions in the food matrix. ; The research leading to these results has received funding from INTERACEITUNA (Organización Interprofesional de la Aceituna de Mesa, Spain) and the Junta de Andalucía. FNAL thanks to the Spanish Government and CSIC for his Ramón y Cajal postdoctoral research contract, while VRG thanks the AgriFood Campus of International Excellence (ceiA3), Bank of Santander, Spanish Government and 'Aloreña de Málaga' Olive Manufacturing Association for her pre-doctoral fellowship (training and formation program of Ph.D. in companies). ; Peer Reviewed

Olives can debitter naturally without the use of NaOH but it is a very slow process. The purpose of this work was to evaluate the influence of both temperature and chemical characteristics of brine on the oleuropein hydrolysis rate in natural table olives. Two different phases were established for natural debittering. During the first 1–2 months of brining, a low concentration of NaCl (60 g/L) and acetic acid (2 g/L) together with a low storage temperature (10 °C) were the processing conditions that promoted a rapid hydrolysis of the bitter phenol because these mild conditions facilitated the action of endogenous enzymes (β-glucosidase and esterase). Thereafter, higher concentrations and temperature of storage (140 g/L NaCl, 16 g/L acetic acid and 40 °C) favored the chemical hydrolysis of oleuropein during long term (a few months) storage. These results will contribute to the knowledge of the natural debittering of table olives and they will help processors to accelerate their elaboration methods. ; This work was supported by projects AGL 2009-07512 and AGL 2013-42739-R from the Spanish Government and European Union (European Regional Development Funds). Eva Ramírez thanks MINECO for her FPI fellowship. Thanks also to Alejandra Expósito for technical assistance ; Peer Reviewed

The bitter taste of olives is mainly caused by the phenolic compound named oleuropein and the mechanism of its hydrolysis during the processing of natural green olives was studied. First, a rapid chemical hydrolysis of oleuropein takes place at a high temperature of 40 °C and at a low pH value of 2.8, but the chemical hydrolysis of the bitter compound is slow at the common range of pH for these olives (3.8-4.2). However, decarboxymethyl elenolic acid linked to hydroxytyrosol and hydroxytyrosol have been found in a high concentration during the elaboration of natural green olives. When olives were heated at 90 °C for 10 min before brining, these compounds are not formed. Hence, the debittering process in natural green olives is due to the activity of β-glucosidase and esterase during the first months of storage and then a slow chemical hydrolysis of oleuropein happens throughout storage time. ; This work was supported by projects AGL 2009-07512 and AGL-2013-42739-R from the Spanish Government and European Union (European Regional Development Funds). Eva Ramírez wishes to thank MINECO for her FPI fellowship. Thanks also to Alejandra Expósito for technical assistance. ; Peer Reviewed

A ripe olive model system was used to evaluate the potential inhibiting effects on acrylamide formation from a set of amino acids and water-soluble vitamins. The system was based on ripe olive juice heated at 121 C for 30 min in a stainless steel tubular reactor. The most potent acrylamide inhibitors were proline and sarcosine, both with inhibition rates of ∼75% at a 100 mM level. In addition, glycine, ornithine, taurine, and γ-amino butyric acid were effective (50-65% inhibition) while the rest of the compounds demonstrated weak or non-significant effects. Acrylamide contents in the model system were found to be highly correlated with the corresponding contents in the real product. The kinetic pattern for the formation of acrylamide in the absence and presence of two selected amino acids, added separately or together, was well fitted using a simple logistic function. © 2013 Elsevier Ltd. All rights reserved. ; This study was supported in part by the European Union (FEDER funds) and the Spanish government through Project AGL 2010-19178. Financial support from the Junta de Andalucía (Group AGR-208) is also acknowledged. ; Peer Reviewed

The present study focused on investigating a peculiar spoilage of traditional Aloreña de Málaga table olives characterized by the formation of whitish and soft regions on the olive surface. To determine its causes, the main microbiological and physicochemical changes in 50 commercial packages were monitored until the alteration in appearance (63 days). Colour and firmness of fruits deteriorated progressively in the packaged olives during storage. Sugar in brines (7.5 g/L) remained stable during the first month and then decreased gradually (5.0 g/L) while, in parallel, the lactic acid bacteria population and titratable acidity increased. After two months of storage, evidence of spoilage was noticed, coinciding with the maximal lactic acid bacteria populations in fruit (>6.5 log10 CFU/g). The spoilage affected ¿25% of the fruits within packages. The microbial species detected in the product after packaging were Enterobacter gergoviae and Lactobacillus plantarum among bacteria, and Candida tropicalis, Candida parapsilosis, and Lodderomyces elongisporus among the yeasts while Lactobacillus pentosus was the dominant species in the spoilt packages. A specific biotype of L. pentosus was only detected in the damaged fruits. Further studies will be made to confirm the association between the spoilage and the presence of this L. pentosus biotype. ; The research leading to these results has received funding from Junta de Andalucía Government through the PrediAlo project (AGR-7755: www.predialo.science.com.es). FNAL wishes to express thanks to the Spanish government for his RyC postdoctoral research contract while VRG would like to thank ceiA3, Spanish Government, Bank of Santander, IG-CSIC and 'Aloreña de Málaga' Olive Manufacturing Association for her pre-doctoral fellowship. ; Peer Reviewed

This work focuses on the persistence of the putative probiotic bacteria Lactobacillus pentosus TOMC-LAB2 on green Spanish-style Manzanilla olives according to different packaging conditions and storage temperatures. The lactic acid bacteria population decreased with time but the highest survival counts (and lowest yeasts) at the end of storage (8 months) were observed in plastic pouches under nitrogen atmosphere and glass jars with brine stored at 20°C. Molecular techniques showed a 100% presence of the putative probiotic bacteria in biofilms adhered to olive epidermis, while it was absent in PPB (plastic pouches with brine) and in olives stored at 7°C. No changes in NaCl, pH or combined acidity were observed during the storage except for a slight increase in titratable acidity at 20°C. The color of the fruits was stable but degraded at 20°C for olives in plastic pouches with brine. ; The research leading to these results has received funding from the EU's Seventh Framework Program (FP7/2007-2013) under grant agreement n°243471 (PROBIOLIVES) and the Junta de Andalucía (through financial support to group AGR-125). Thanks to JOLCA for supplying the fruits and the packages and to ASEMESA for its support. FNAL thanks the Spanish Government and CSIC for his Ramón y Cajal postdoctoral research contract, while VRG wishes to thank the AgriFood Campus of International Excellence (ceiA3), Bank of Santander, Spanish Government, and 'Aloreña de Málaga' Olive Manufacturing Association for her predoctoral fellowship (training program of PhD in companies). ; EUR 1,172 APC fee funded by the EC FP7 Post-Grant Open Access Pilot ; Peer reviewed

© 2014 Rodríguez-Gómez, Romero-Gil, García-García, Garrido-Fernández and Arroyo-López. Dairy products are currently the main carriers of probiotic microorganisms to the human body. However, the development of new matrices for probiotic delivery is convenient for intolerant to milk (or its derivatives) and those requiring low-cholesterol diet consumers. The present work focused on the fortification of previously fermented green Spanish style olives with the autochthonous putative probiotic bacteria Lactobacillus pentosus TOMC-LAB2. The fortification was carried out by inoculating the bacteria into the packing brines using Manzanilla fruits from three different processes: (i) spontaneously fermented (F1), (ii) fermented using L. pentosus TOMC-LAB2 as starter (F2), and (iii) spontaneously fermented and then thermally treated (F3). Data showed that all inoculated treatments had higher population levels (5.49, 4.41, and 6.77 log10 cfu/cm2) than their respective controls (1.66, 4.33, and 0.0 log10 cfu/cm2, for F1, F2, and F3 treatments, respectively). The presence of L. pentosus TOMC-LAB2 on olive surface was confirmed by rep-PCR, with a recovery frequency at the end of the shelf life (200 days) of 52.6, 57.9, and 100.0% for F1, F2, and F3 treatments, respectively. Thus, results obtained in this work show the ability of this microorganism to survive under packing conditions for long period of times as well as to colonize the olive surface which is the food finally ingested by consumers. This opens the possibility for the development of a new and simply probiotic fortified olive product. ; he research leading to these results has received funding from the EU's Seventh Framework Programme [FP7/2007-2013] under grant agreement no 243471 (PROBIOLIVES). We also thank the Spanish Government for financial support (projects AGL2009-07436/ALI, and AGL2010-15529/ALI partially financed by European regional development funds, ERDF), and the Junta de Andalucía (through financial support to group AGR-125). Thanks to JOLCA and ASEMESA for supplying the fruits and their own expertise for the development of this work. Francisco N. Arroyo-López wishes to express thanks for his Ramón y Cajal postdoctoral research contract (Spanish government). ; Peer Reviewed

37 Páginas.-- 2 Tablas.-- 3 Figuras.-- 5 Tablas suplementarias ; Knowledge of the microbial biodiversity through the different stages of black ripe olive processing is minimal. This study applied a metagenomic approach to evaluate the bacterial and fungal communities present in samples of two olive cultivars collected from an industrial producer. High-throughput sequencing and analysis of the V2-V3 region of the bacterial 16S rRNA genes showed that genus Acetobacter and the lactic acid bacteria (LAB) Lactobacillus and Oenococcus were the most abundant during most of the process. Other LAB genera (Enterococcus, Lactococcus, Weisella, Leuconostoc and Streptococcus), as well as Enterobacteriaceae and Vibrio were also detected, increasing their abundance in the last step before packaging. Some genera, such as Pseudoalteromonas, Alteromonas, Marinomonas and Oenococcus, had never been previously detected in table olives. The fungal community was characterized by the presence of Pichia membranifaciens, Kregervanrija fluxuum and members of the family Dispodascaceae. Although black ripe olives are sterilized before trading, the consequences that these findings may have on the quality of this product are discussed. ; This research was supported by the Spanish Government (Projects AGL2013-42739-R and AGL2016-76820-R), and partially financed by the European Union (European Regional Development Funds, ERDF). The authors gratefully thank Mrs. Alejandra Expósito for her skilful experimental work in the laboratory. ; Peer reviewed