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 β-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 β-glucosidase and esterase activities were at their maximum at 30 and 55 °C, respectively. The oxidase and β-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.

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 10min 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.

20Brownish colourations in Natural green table olives (non-treated with alkali) make this 21 product less attractive to consumers than Spanish-style green table olives (treated with 22 alkali), which develop a more appreciated bright golden-yellow colour. These colour 23 differences were studied in relation to changes in the composition of chlorophyll and 24 carotenoid pigments, as well as polyphenolic compounds and polyphenol oxidase 25 enzyme (PPO) activity. Natural green olives showed a different chlorophyll profile than 26 Spanish-style. However, all the chlorophyll pigments formed in both processing types 27were Mg-free derivatives (mostly pheophytins) with similar colourations, ranging from 28 grey to green brownish. In the carotenoid fraction no appreciable differences were 29 found between both processing types. The fruit's brownish colour was mainly due to 30 polymeric substances with a size of >1,000 daltons and polyphenolic nature, resulting 31 from an enzymatic oxidation by PPO of the o-diphenolic compounds present in the 32 fresh fruits. 33 34

The objective of this work was to investigate the debittering process of dry-salted olives. Fruits of the Manzanilla cultivar were put into layers of coarse salt and their physicochemical and microbiological parameters were assessed during the dehydration step. A correlation between the content in salt of the olive juice and the ratio salt/olives was found: the lower the ratio, the higher the concentration of salt in the dry-salted olives. The population of lactic acid bacteria on the olive surface was not significant while yeasts, molds and Enterobacteriaceae were the predominant microbiota. In addition, the analyses of phenolic compounds in the olive flesh revealed that most of them disappeared during the dehydration process, in particular the bitter glucoside oleuropein. Likewise, freshly harvested olives were pasteurized and submitted for dehydration but in this case the degradation of oleuropein and the rest of polyphenols did not occur. All these findings suggest that olive debitter during the dry-salting process due to the enzymatic oxidation of oleuropein in the olive flesh resulting in no leaching of the phenolic glucoside in the generated brine.Practical applications: This is the first report focusing on the debittering process of dry-salted olives. An enzymatic pathway for oleuropein degradation has been found, and it opens up the possibility to optimize this reaction taking into consideration the variables involved such as phenolic compounds, oxidative enzyme, and oxygen. The ratio salt/olives must be as low as 0.4 to reach a high concentration of salt in the juice of dehydrated olives needed for the chemical and microbial stability of the product.

Fermented or acidified vegetable foods are considered microbiologically safe although the survival of certain pathogens has occasionally been reported in these products. The aim of this research was to investigate the fate of Escherichia coli, Salmonella enterica, Listeria monocytogenes and Staphylococcus aureus when they were added to different industrial olive brines, as well as to correlate their survival with the presence of phenolic and oleosidic substances. Brines of different cultivars prepared following the Spanish-style method or preserved in acidified brine were inoculated with a cocktail of four strains of each species. The evolution of their populations was analyzed by cultural methods when the brines were kept at 4 ºC or room temperature and in aerobiosis or anaerobiosis. All the pathogens investigated died off but their death rate was variable depending on the composition of the brines in phenolic compounds, temperature and oxygen availability. The time needed to reduce the inoculated pathogen populations by 5 log oscillated between less than 5 minutes and up to 17 days in the least deleterious conditions.

16Olives can debitter naturally without the use of NaOH but it is a very slow process. The 17 purpose of this work was to evaluate the influence of both temperature and chemical 18 characteristics of brine on the oleuropein hydrolysis rate in natural table olives. Two 19 different phases were established for natural debittering. During the first 1-2 months of 20 brining, a low concentration of NaCl (60 g/L) and acetic acid (2 g/L) together with a 21 low storage temperature (10 ºC) were the processing conditions that promoted a rapid 22 hydrolysis of the bitter phenol because these mild conditions facilitated the action of 23 endogenous enzymes (β-glucosidase and esterase). Thereafter, higher concentrations 24 and temperature of storage (140 g/L NaCl, 16 g/L acetic acid and 40 ºC) favored the 25 chemical hydrolysis of oleuropein during long term (a few months) storage. These 26 results will contribute to the knowledge of the natural debittering of table olives and 27 they will help processors to accelerate their elaboration methods. 28 29

The autoxidation process of vitamin C in orange juice is the most important cause of quality loss during its storage. We evaluated the enrichment with different concentrations of a phenolic extract in a commercial orange juice for some qualitative parameters such as the content of vitamin C, phenolic compounds, and antioxidant activity. The lowest concentration of phenols produced the most stable enriched juice. In particular, we observed an inverse correlation between phenolic concentration and vitamin C retention in enriched juices. DPPH assay results confirmed this trend, correlated more to the vitamin antioxidant effect. TEAC results instead, were similar for the different juice samples, probably influenced by the phenolic content. The application of this study is the production for the industry of new functional drinks such as juices enriched with phenolic ingredients that show increased stability concerning those without addiction.

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