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The use of solid phase extraction columns of octadecyl (C18) to get a fat free pigment extract in combination with reversed phase ion pair HPLC permits the separation of chlorophylls and its derivates, carotenoids, xantophylis and metalochlorophyllic complexes in a maximum period of one hour.

Alkaline treatment is a key stage in the production of green table olives and its main aim is rapid debittering of the fruit. Its action is complex, with structural changes in both the skin and the pulp, and loss of bioactive components in addition to the bitter glycoside oleuropein. One of the components seriously affected are chlorophylls, which are located mainly in the skin of the fresh fruit. Chlorophyll pigments are responsible for the highly-valued green color typical of table olive specialties not preserved by fermentation. Subsequently, the effect on chlorophylls of nine processes, differentiated by NaOH concentration and/or treatment time, after one year of fruit preservation under refrigeration conditions, was investigated. A direct relationship was found between the intensity of the alkali treatment and the degree of chlorophyll degradation, with losses of more than 60% being recorded when NaOH concentration of 4% or greater were used. Oxidation with opening of the isocyclic ring was the main structural change, followed by pheophytinization and degradation to colorless products. To a lesser extent, decarbomethoxylation and dephytylation reactions were detected. An increase in NaOH from 2% to 5% reduced the treatment time from 7 to 4 h, but fostered greater formation of allomerized derivatives, and caused a significant decrease in the chlorophyll content of the olives. However, NaOH concentrations between 6% and 10% did not lead to further time reductions, which remained at 3 h, nor to a significant increase in oxidized compounds, though the proportion of isochlorin e 4 -type derivatives was modified. Chlorophyll compounds of series b were more prone to oxidation and degradation reactions to colorless products than those of series a . However, the latter showed a higher degree of pheophytinization, and, exclusively, decarbomethoxylation and dephytylation reactions.

Pistachio roasting before oil extraction increases consumer preference but may cause changes in the oil composition. In this work, the effect of different roasting conditions on the physical parameters, oxidative stability, and pigment composition of pistachio oil extracted by pressure was studied. Density value of pistachio oil was reduced with severe roasting conditions (125 °C), while viscosity increased slightly. This adverse effect was compensated by a significant increase in both oxidative stability and, especially, in the content of chlorophyll and carotenoid pigments. Pistachio roasting temperature had a clear impact on the color of the pistachio oils, changing from yellow in oils from raw or minimally roasted pistachios (50-75 °C) to brilliant green in oils from pistachios subjected to higher temperature treatments (100-125 °C). An increase in temperature favored the pigment transfer to the oil. The green oils had a total pigment content between 2.3 and 4 times higher than the yellow oils., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The carotenoid composition of strawberry tree (Arbutus unedo) fruits has been characterised in detail and quantified for the first time. According to the total carotenoid content (over 340 μg/g dw), mature strawberry tree berries can be classified as fruits with very high carotenoid content (>20 μg/g dw). (all-E)-Violaxanthin and 9Z-violaxanthin were found to be the major carotenoid pigments, accounting for more than 60%, responsible for the bright colour of the flesh of ripe fruits. In addition other 5,6-epoxide carotenoids, such as (all-E)-neoxanthin, (9'Z)-neoxanthin (all-E)-antheraxanthin and lutein 5,6-epoxide, together with (all-E)-lutein, (all-E)-zeaxanthin and (all-E)-β-carotene were found at high levels (>5-20 μg/g dw). The LC-MS (APCI+) analysis of the xanthophyll fraction in their native state (direct extract) revealed that most of them (>90%) were totally esterified with saturated fatty acids (capric, lauric, myristic, palmitic and stearic). Monoesters, homodiesters and heterodiesters of (all-E)-violaxanthin and 9Z-violaxanthin were the major pigments., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

High irradiances may lead to photooxidative stress in plants, and non-photochemical quenching (NPQ) contributes to protection against excess excitation. One of the NPQ mechanisms, qE, involves thermal dissipation of the light energy captured. Importantly, plants need to tune down qE under light-limiting conditions for efficient utilization of the available quanta. Considering the possible redox control of responses to excess light implying enzymes, such as thioredoxins, we have studied the role of the NADPH thioredoxin reductase C (NTRC). Whereas Arabidopsis thaliana plants lacking NTRC tolerate high light intensities, these plants display drastically elevated qE, have larger trans-thylakoid ΔpH and have 10-fold higher zeaxanthin levels under low and medium light intensities, leading to extremely low linear electron transport rates. To test the impact of the high qE on plant growth, we generated an ntrc-psbs double-knockout mutant, which is devoid of qE. This double mutant grows faster than the ntrc mutant and has a higher chlorophyll content. The photosystem II activity is partially restored in the ntrc-psbs mutant, and linear electron transport rates under low and medium light intensities are twice as high as compared with plants lacking ntrc alone. These data uncover a new role for NTRC in the control of photosynthetic yield., (© 2015 John Wiley & Sons Ltd.)

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The carotenoid profile of sixty potato cultivars (commercial, bred, old and native cultivars) has been characterised in order to provide information to be used in selective breeding programs directed to improve the nutritional value of this important staple food. Cultivars were segregated into three groups according to the major pigment in the carotenoid profile: violaxanthin (37 cultivars; especially those with higher carotenoid content), lutein (16 cultivars), and neoxanthin (7 cultivars). Other minor carotenoids were antheraxanthin, β-cryptoxanthin and β-carotene, while zeaxanthin was absent in all sample. The total carotenoid content ranged from 50.0 to 1552.0 μg/100 g dry wt, with an average value of about 435.3 μg/100 g dry wt. Sipancachi, Poluya and Chaucha native cultivars showed the highest carotenoid content (1020.0, 1478.2 and 1551.2 μg/100 g dry wt, respectively). Xanthophyll esters were present in most cultivars, mainly as diesterified forms, being observed a direct correlation between the carotenoid content and the esterified fraction, suggesting that the esterification process facilitates the accumulation of these lipophilic compounds within the plastids. Therefore, the presence of xanthophyll esters should be a phenotypic character to be included in the breeding studies, and more efforts should be dedicated to the understanding of the biochemical process leading to this structural modification of carotenoids in plants., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The changes in chloroplastic pigments, mineral nutrients, and characteristics related to freshness were studied during storage and packing of cracked seasoned olives. Cracking produced an initial loss in green pigments and color degradation. Later, storage caused a progressive degradation of chlorophylls and carotenoids, with a slower rate in refrigerated fruits (which preserved the greenish tones better), but after packing (and storage at room temperature), the differential effect disappeared and, at the end of the study, all olives showed similar pigment transformations, which were correlated with CIE a* and hue. Processing led to a Na content increase in olive flesh (and Ca and Zn, when added) but marked losses in the other mineral nutrients. Sodium metabisulfite and ZnCl₂ promoted LAB growth while inhibiting yeast, thus enhancing product stability, and erythorbic acid caused yeast growth and firmness deterioration.

The present work proposes an analytical method able to detect in an adulterated olive oil sample the addition of the copper complexes of chlorophylls (E 141i). The method consists of a pigment extraction in liquid phase and subsequent analysis by HPLC-DAD. The profile of chlorophyll pigments of an olive oil is determined essentially by its content in pheophytins (a and b), but in no case any copper derivative. Different samples of colorant E 141i have been analyzed, the natural coloring additives used to adulterate vegetable oils. The 99.59+/-0.52% of the chlorophyll pigments present in the different samples of E 141i colorant are not those of an olive oil (more than 75% are cupro-derivatives). Thus, the simple detection of one of the compounds in an olive oil indicates adulteration. The major chlorophyll derivative in all the E 141i colorants samples is Cu-pyropheophytin a and its limit of detection (LOD) defined at a signal-to-noise ratio of about 3 was 6.58 ng/g.

Chlorophyll a, chlorophyll b, and the Mg-free chlorophyll derivatives pheophytin a, pheophytin b, pyropheophytin a, pheophorbide a, and pyropheophorbide a, dissolved in an oily matrix, were subjected to a simulated in vitro digestion procedure coupled with uptake by human intestinal Caco-2 cells. The native chlorophylls showed greater instability to the digestive process than the Mg-free chlorophyll derivatives. In addition to pheophytinization reactions, allomerization and oxidation to uncolored compounds were found to greater extents for the former. After digestion, the pigment dispersion degree in the colloid system (aqueous-"micellar" phase) showed significant differences (p < 0.05) among series a and series b derivatives. However, when a mixture of pheophytin a and pheophytin b was digested, there was a positive effect for pheophytin b. Both the dispersion degree and the accumulation rate by the Caco-2 intestinal epithelial cells were significantly higher (p < 0.05) for dephytylated chlorophyll derivatives. Differences in the transport route were also found. Whereas phytylated chlorophyll derivatives showed passive absorption by simple diffusion, the dephytylated ones showed passive absorption by facilitated diffusion in the lower range of concentrations tested. These results showed that the structural modifications of the chlorophyll pigments, mainly the de-esterification of phytol, significantly increased--by an estimated 65-fold--its transfer from the food matrix to the intestinal epithelial cells during digestion, making it more bioaccessible. The possible relationship between the phototoxicity associated with pheophorbide and the high bioaccessibility demonstrated in this work is discussed.

The digestive stability, efficiency of micellarization, and cellular accumulation of the chlorophyll pigments of different preparations of pea were investigated, using an in vitro digestion procedure coupled with human intestinal Caco-2 cells. Fresh pea (FP), cooked fresh pea (CFP), frozen pea (FZP), cooked frozen pea (CFZP), and canned pea (CP) were subjected to simulated digestion. Although after digestion the pigment profile was modified for all samples, except CP, allomerization reactions and greater destruction of chlorophylls were observed in only FP, which should be due to enzymes in FP that were denaturalized in the rest of the test foods. A pigment extract of CFZP was also subjected to in vitro digestion, showing a positive effect of the food matrix on the pigment digestive stability. The transfer of the chlorophyll pigments from the digesta to the micellar fraction was significantly more efficient in CFZP (57%, p < 0.0001), not significantly ( p > 0.05) different between CFP, FZP, and CP (28-35%), and lowest in FP (20%). Pheophorbide a stood out as the most-micellarized chlorophyll derivative in all of the samples, reaching levels of up to 98%. Incubation of Caco-2 cells with micellar fractions at the same concentration prepared from each test food showed that pigment absorption was considerably lower ( p < 0.006) in cells incubated with FP, whereas there were no differences among the rest of the preparations. Therefore, factors associated with the food matrix could inhibit or mediate the chlorophyll pigment absorption. These results demonstrated that the industrial preservation processes of peafreezing and canningas well as the cooking have a positive effect on the bioaccessibility and bioavailability of the chlorophyll pigments with respect to the FP sample, emphasizing CFZP with greater bioaccesibilty degree.

The changes in allomerized chlorophyll during the growth and development of the olive fruit as well as during the main operations of its processing as green table olive (alkaline treatment and lactic fermentation) were investigated to study their influence in the color alteration known as green staining (GS). Chlorophyll synthesis coincided in time with the maximum content in allomerized intermediates, weeks before the fruits were harvested for processing. The alkaline treatment caused a subsequent chlorophyll oxidation independent of the high or low initial content of allomerized chlorophylls. However, this oxidation was directly related with the oxidizing capacity of the alkaline solution and the cell deterioration caused. The later maintenance of the fruits in osmotic solutions at different pHs that reproduce the pH reduction caused by the lactic fermentation showed that at pH below 4.5 the insertion of Cu into the chlorophyll molecule was produced in certain fruits; the extent of this reaction was greatest when the prior formation of oxidized chlorophylls exceeded 23%. This apparent relationship between chlorophyll oxidation and copper chlorophyll complexes was investigated in table olives with GS alteration.

In table olives showing the green staining alteration, extracts of pigment-lipoprotein complexes were obtained from the zone altered and the rest of the fruit. In the altered zone of the olive, the surrounding components of pigments were affected, with the degeneration of proteins and phospholipids forming the pigment-lipoprotein complexes. There was also less interaction between the pigments and the membrane lipids. These results suggested a greater loss of cell integrity in the green-stained zone of the fruit, allowing the migration and local accumulation of Cu-metallochlorophyll complexes, macroscopically visible as the form of green staining alteration.

The pectic and pigment compositions and Ca and Cu contents of the alcohol-insoluble solid (AIS) residues were determined in cv. Gordal olives treated with NaOH solution and kept at different constant pH values (3.5-6.5). The same controls were made in table olives presenting green staining alteration. The ratio between the various pectin fractions of the more acid pH experiment samples remained similar in fruits not showing green staining. In altered fruits, the protopectin fraction was lower, and the calcium pectate or EDTA soluble pectins were higher. Regarding the presence of Ca and Cu in the AIS, it was observed that, whereas Ca levels fell at the most acid pH values, those of Cu increased. The concentration of Ca was higher in the AIS of altered olives than in nonaltered ones. The same trend was seen for the zone with or without green staining of an altered fruit. In the case of Cu, the relationship was the opposite: a decrease in the levels of AIS Cu in fruits and zones of fruits with green staining. This result was correlated with the highest concentration of Cu-chlorophyll complexes found in such samples and suggested that pectins might act as a reservoir of Cu involved in the alteration.

Activity of the enzymes superoxide dismutase (SOD) and polyphenol oxidase (PPO) as well as Cu-Zn content have been monitored during the thirteen weeks growth of both Gordal and Manzanilla olive variety fruits. These metalloenzymes, with Cu and Zn in the prostetic group, are involved in controlling the redox balance in the chloroplast environment. The results indicated that, under similar phenological and environmental conditions, there are periodic peaks of SOD activity in both varieties, followed by fluctuations in the copper content of the fruit. This was interpreted as a common and simultaneous response to situations of oxidative stress, and this response was more intense in the variety Gordal. The enzyme PPO showed an activity peak at start of growth and then practically disappeared. Thus, its activity cannot be correlated with situations of stress or with changes of Cu and Zn in the fruit.

Metallochlorophyllic complexes of copper are present in green table olives, showing the alteration known as green staining. They were formed stepwise in such a way that new metallochlorophyll derivatives were detected as the fruits became more altered. Cu-15-glyoxylic acid pheophytin a was the first compound formed, followed in order by Cu-pheophytin a, Cu-15-glyoxylic acid pheophytin b, Cu-15-formyl-pheophytin a, and Cu-pyropheophytin a. Pigment analysis in fruits classified according to the surface area affected by green staining showed a progressive increase in the concentration of all copper complexes with the course of the alteration. The metallochlorophyll derivatives of copper were present both in the part of the fruit affected by the alteration and in the remainder part, although the amount was significantly greater in the former. On the other hand, not all the copper present in the fruits was accessible for the formation of such compounds, since the concentration of copper complexes found in olives with the maximum degree of alteration observed did not exceed 20% of the total copper of the fruit.

In Spanish green table olives showing the alteration known as green staining, new chlorophyll derivatives have been identified: the copper complexes of 3(1),3(2)-didehydrorhodochlorin-15-glyoxylic acid-17(3)-phytyl ester a and b (Cu-15-glyoxylic acid pheophytins a and b), and 3(1),3(2)-didehydro-15(1)-hydroxy-15(1)-hydroxyrhodochlorin- 15-acetic acid delta-lactone-15(2)-methyl-17(3)-phytyl ester a and b (Cu-15(1)-OH-lactone-pheophytins a and b). These compounds were isolated by normal phase thin-layer chromatography and identified from their UV-visible and mass spectra and by co-chromatography with authentic standards. The chromatographic and spectroscopic characteristics and the molecular mass for a new allomerized chlorophyll derivative and its copper complex are reported. The corresponding molecular structure according to the molecular mass has been proposed. The characterization of the latter compounds enables their possible detection in processed fruits and vegetables. The present work gathers new advances in the study of the color alteration in table olives and this is the first time that copper complexes of oxidized chlorophylls are detected in foodstuffs.

The results of a qualitative and quantitative study of pigments, carried out during the processing of Gordal variety olives for table use, has provided valuable information on the type, extent, and mechanism of degradation of the chlorophylls and carotenoids present in the fresh fruit. Unexpected results were obtained, since the initial treatment of the fruits with NaOH did not provoke chlorophyllase activity. However, the alkaline pH brought about the oxidation of chlorophylls, giving rise to Mg-phytyl-chlorin e 6 and Mg-phytyl-rhodin g 7 . Subsequently, as a consequence of the acid pH generated in the fermentation medium, these compounds were transformed into the corresponding Mg-free derivatives, phytyl-ch1orin e 6 and phytyl-rhodin g 7 . At the same time, those chlorophylls which initially escaped transformation were converted into their corresponding pheophytins. In addition, small amounts of pyropheophytin a , pheophorbides a and b , and pyropheophorbide a were detected, As far as the carotenoid fraction is concerned, β-carotene and lutein remained unaltered throughout processing, and only those components with molecular structures sensitive to the acid medium were affected. The total balance of pigment material indicated that there was a slow but progressive decrease in the concentration of the chlorophyllic and carotenoid fractions, which indicates that a certain amount of these pigments is degraded into colorless products.

The presence of metalochlorophyllic complexes of copper has been detected in table olives showing the alteration known as green staining. These compounds are absent in the healthy fruit. The possible implication of fungicidal treatment of olive trees in this alteration has been studied. No alteration was produced in table olives prepared with fruit from trees with and without fungicidal treatment and the differences found between copper levels in the fruit were not significant. The possibility that the copper involved in this alteration is of extraneous origin was, therefore, discarded. On the other hand, there were no significant differences in the levels of copper in random samples of fruits with and without green staining. Therefore, although the green-staining alteration is the result of the formation of complexes of copper with chlorophyll derivatives, it seems clear that the simple presence in the fruits of copper, by itself, does not lead to the appearance of green-staining.

Chlorophyllase activity in olive fruit (Olea europaea) was measured using the enzyme solubilized from the protein precipitate. The reaction was stopped by freezing the mixture at -20 degrees C, to avoid dilution of the sample and consequent reduction of the substrate levels to below the detection limits of the analytical system. Separation of the substrates and products of the enzymatic reaction was performed by reverse-phase HPLC using a gradient solvent system of water and ion suppressor/methanol/acetone. These conditions allowed direct resolution of the reaction mixture prior to centrifugation, without the need for the transfer of any of the components to other solvents. Olive chlorophyllase in the crude enzymatic extract showed maximum activity at 50 degrees C and the optimum pH was 8.5 in acetate-phosphate-borate buffer for all substrates used, chlorophylls (a and b) and pheophytins (a and b). The Km and Vmax values obtained for hydrolysis of these substrates showed that chlorophyllase had a greater affinity for chlorophyll b, while the highest maximum rate of reaction occurred with pheophytin a. Substrate inhibition was observed with pheophytin b.