María Isabel Mínguez-Mosquera

Lipoxygenase activity during pepper ripening and processing of paprika

Abstract The crude enzymatic extract obtained directly by trituration of the fruits of Capsicum annuum in phosphate buffer followed by filtration was shown to be suitable for the measurement of lipoxygenase activity. The use of Triton X-100 did not improve extraction and the use of DTT to protect the enzyme against oxidizers was not necessary. The optimum pH was 7 for the extraction and 6.5 for measurement of activity. The number of previous harvests from the plant had a marked effect on the levels of lipoxygenase activity during ripening of the fruits from two varieties of pepper. During drying, there was a progressive loss of activity in both varieties.

An evaluation of the basis and consequences of a stay-green mutation in the navel negra citrus mutant using transcriptomic and proteomic profiling and metabolite analysis.

A Citrus sinensis spontaneous mutant, navel negra (nan), produces fruit with an abnormal brown-colored flavedo during ripening. Analysis of pigment composition in the wild-type and nan flavedo suggested that typical ripening-related chlorophyll (Chl) degradation, but not carotenoid biosynthesis, was impaired in the mutant, identifying nan as a type C stay-green mutant. nan exhibited normal expression of Chl biosynthetic and catabolic genes and chlorophyllase activity but no accumulation of dephytylated Chl compounds during ripening, suggesting that the mutation is not related to a lesion in any of the principal enzymatic steps in Chl catabolism. Transcript profiling using a citrus microarray indicated that a citrus ortholog of a number of SGR (for STAY-GREEN) genes was expressed at substantially lower levels in nan, both prior to and during ripening. However, the pattern of catabolite accumulation and SGR sequence analysis suggested that the nan mutation is distinct from those in previously described stay-green mutants and is associated with an upstream regulatory step, rather than directly influencing a specific component of Chl catabolism. Transcriptomic and comparative proteomic profiling further indicated that the nan mutation resulted in the suppressed expression of numerous photosynthesis-related genes and in the induction of genes that are associated with oxidative stress. These data, along with metabolite analyses, suggest that nan fruit employ a number of molecular mechanisms to compensate for the elevated Chl levels and associated photooxidative stress.

Metabolites Involved in Oleuropein Accumulation and Degradation in Fruits of Olea europaea L.: Hojiblanca and Arbequina Varieties

The biosynthetic pathway of oleuropein (from 7-ketologanin, oleoside-11-methyl ester, 7-β-1-d-glucopyranosyl-11-methyl oleoside, and ligstroside to oleuropein) was investigated in two fruit species of Oleaceae, namely, Arbequina and Hojiblanca. Main oleuropein precursors and their metabolites, produced by the enzymatic hydrolysis mediated by β-glucosidase, were identified and quantified to establish the oleuropein transformation pathway. Changes in the concentration of these compounds were measured by direct control of in vivo fruit tissue during their ripening. High contents of aglycones at the initial stage of the process were caused by the high activity of β-glucosidase, which supports that oleuropein biosynthesis is coupled with enzymatic hydrolysis, producing its aglycone form. The low oleuropein content at this initial stage was caused by the imbalance between catabolic and anabolic pathways, favoring the former ones. Once the main polyphenol synthesis phase was completed, the biosynthetic capacity diminished and the content of all compounds decreased. Mass balance revealed that precursors of oleuropein, which are rapidly transformed by β-glucosidase and esterases, scarcely contributed to the accumulation of oleuropein. The biosynthetic pathway proposed by Damtoft applies for both varieties, but our study reveals that the β-glucosidase enzyme is involved in oleuropein synthesis. This enzyme shows high substrate specificity to oleuropein, which consequently is degraded to its aglycone form, with diminished efficacy of oleuropein biosynthesis. Different enzymatic activities of varieties will result in oleuropein accumulation and metabolic transformation of phenols.

Control of olive oil adulteration with copper-chlorophyll derivatives

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 and carotenoid degradation mediated by thylakoid-associated peroxidative activity in olives (Olea europaea) cv. hojiblanca.

A peroxidative activity was found in solubilized thylakoid membranes of olives (Olea europaea) cv. hojiblanca that catalyses degradation of chloroplast pigments in the presence of H2O2 and 2,4-dichlorophenol (DCP). The intermediate products of this degradation were analyzed using HPLC with diode array detection and the results indicated that 13(2)-OH-chlorophyll a and 13(2)-OH-chlorophyll b were the primary catabolites. The peroxidative activity assosiated with the thylakoid membranes affected, not only chlorophyll a and chlorophyll b, but also other accessory pigments in the photosynthetic process, such as the carotenoids. Quantitatively, the progressive decrease of the ratios Chl a/b and total Chls a+b/carotenoids indicated a more rapid disappearance of Chl a than of Chl b and a faster degradation of Chls a+b than of carotenoids.

Thermal degradation kinetics of chlorophyll pigments in virgin olive oils. 1. Compounds of series a.

Virgin olive oils (VOO) collected at three maturation stages were thermodegraded to determine the degradation kinetics of series a chlorophyll pigments. The proposed degradation mechanism involves reactions that alter the structure of the isocyclic ring of pheophytin, originating intermediary products such as pyropheophytin, 13(2)-OH-pheophytin, and 15(1)-OH-lactone-pheophytin, and reactions that affect the porphyrin ring, producing colorless compounds. The marked effect of temperature has been pointed out in these competitive processes with the formation of pyropheophytin and the significantly higher value of its kinetic constant. No significant effect of the oily medium on the reaction mechanisms of pyropheophytin and 15(1)-OH-lactone-pheophytin has been found, comparing kinetic and thermodynamic parameters determined in the three VOO matrices of different pigment contents (high, medium, and low). The reaction mechanism of 13(2)-OH-pheophytin, by contrast, was affected by the medium; the reaction rate was the same for all of the matrices only at the isokinetic temperature (51 degrees C).

β-Glucosidase involvement in the formation and transformation of oleuropein during the growth and development of olive fruits (Olea europaea L. cv. Arbequina) grown under different farming practices.

The present study investigates oleuropein metabolism, as well as the involvement of β-glucosidase during the growth, development, and ripening of olive fruit. The results show that in olive fruit the in vivo formation and transformation of oleuropein takes place in three different stages. The first one is characterized by a net accumulation of oleuropein and occurs in the immature fruit. In the second stage, associated with the green and light-green fruits, oleuropein content is maintained practically constant, and finally, a third stage that begins in the green-yellow fruit is characterized by a progressive decline of the oleuropein concentration. Our findings confirm that in the absence of β-glucosidase the Damtoft-proposed pathway is active and that net synthesis of oleuropein is unquestionable. β-Glucosidase activity plays a key role in the oleuropein metabolism catalyzing its in vivo hydrolysis.

Carotenoid levels during the period of growth and ripening in fruits of different olive varieties (Hojiblanca, Picual and Arbequina)

During fruit growth and development, carotenoid accumulation follows the same qualitative pattern in three olive varieties (Olea europaea L.). In the stage of ripening, the Arbequina variety is differentiated from Hojiblanca and Picual by its possession of esterified xanthophylls. The Chl a/b ratio is higher in Arbequina than in Hojiblanca and Picual throughout the life cycle of the fruit, while the percentage of lutein is always lower, and that of beta-carotene higher. Independent of the high (Hojiblanca and Picual) or low (Arbequina) pigment content, the chlorophyll/carotenoid ratio (a + b)/(x + c) is similar for the three varieties. There is evident carotenoid breakdown at the onset of ripening in the fruits of the Hojiblanca and Picual varieties, while in Arbequina there is a new period of carotenoid accumulation. As ripening proceeds in Arbequina fruits, a slow carotenoid-breakdown process is initiated.

Stability of Paprika without supplementary antioxidants during storage under industrial controlled conditions.

Different quality parameters of paprika samples stored under controlled conditions (temperature 4 degrees C and relative humidity 70%) and without reconstitution of the antioxidant levels were analyzed. These included carotenoid composition, ASTA values (as specified by the American Spice Trade Association), fatty acid composition, and peroxide index, in order to determine the progress of autoxidative reactions and directly correlate the loss of carotenoid fraction with the development of prooxidative processes. Evolution of the carotenoid content indicated that autoxidative reactions minimally took place and that coloring capacity was maintained. Peroxide values were very low (1 mequiv/kg) and reached values of 3 mequiv/kg at the end of the storage period. Control of microbial flora during storage also showed how the storage conditions preserved quality of the paprika, as the flora was kept at levels similar to those of the beginning. Therefore, controlled storage conditions were enough to preserve and keep the overall quality of paprika without reconstitution or addition of antioxidants to the product.

Differences in the Activity of Superoxide Dismutase, Polyphenol Oxidase and Cu-Zn Content in the Fruits of Gordal and Manzanilla Olive Varieties

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.