Influence of the chlorophyll pigment structure on its transfer from an oily food matrix to intestinal epithelium cells.

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.

Digestive stability, micellarization, and uptake by Caco-2 human intestinal cell of chlorophyll derivatives from different preparations of pea (Pisum sativum L.).

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.

Physicochemical conditions modulating the pigment profile in fresh fruit (Olea europaea Var. Gordal) and favoring interaction between oxidized chlorophylls and endogenous Cu.

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.

Pigment-lipoprotein complexes in table olives (Cv. Gordal) with green staining 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.

Pectins as possible source of the copper involved in the green staining alteration of cv. Gordal table olives.

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.

Degradation of non-esterified and esterified xanthophylls by free radicals.

beta-Carotene and other xanthophylls present in pepper fruit as both free and esterified forms were oxidized using a free radical initiator (2,2'-azo-bis-isobutyronitrile). Capsorubin was degraded most slowly, followed by zeaxanthin, capsanthin, and beta-carotene. The presence of keto groups at the ends of the polyene chain could be a structural factor contributing to this difference in reactivity. It was also shown that whereas capsanthin and its esters and capsorubin and its esters were degraded at the same rate, zeaxanthin esters responded differently to the oxidation process, and were degraded more quickly than free zeaxanthin. The presence of unsaturated fatty acids (mainly linoleic) that esterify zeaxanthin help to accelerate the degradation of this xanthophyll and decreasing its antioxidant action. The antioxidant capacity of capsorubin and capsanthin (both in free and esterified form) exclusive to the genus capsicum should be taken into account.

Involvement of Copper and Zinc Ions in Green Staining of Table Olives of the Variety Gordal.

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.

Anomalous Transformation of Chloroplastic Pigments in Gordal Variety Olives during Processing for Table Olives.

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 e6 and Mg-phytyl-rhodin g7. 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 e6 and phytyl-rhodin g7. 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, ß-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.