Thermal degradation of antioxidant micronutrients in citrus juice: kinetics and newly formed compounds

The thermal degradation kinetics of vitamin C, two carotenoids (beta-carotene and beta-cryptoxanthin), and hesperidin, as a function of temperature, were determined for Citrus juice [Citrus sinensis (L.) Osbeck and Citrus clementina Hort. ex Tan]. The influence of dissolved oxygen on the rate of ascorbic acid degradation was also assessed. Analysis of kinetic data suggested a first-order reaction for the degradation of vitamin C and carotenoids. The kinetics parameters Dtheta, z, and Ea have been calculated. Following the Arrhenius relationship, the activation energy of ascorbic acid was 35.9 kJ mol-1 and agreed with the range of literature reported value. The results on vitamin C and carotenoids from citrus juice made it possible to validate the predicting model. Thermal degradation of carotenoids revealed differences in stability among the main provitamin A carotenoids and between these and other carotenoids belonging to the xanthophyll family. The activation energies for the two provitamin A carotenoids were 110 and 156 kJ mol-1 for beta-carotene and beta-cryptoxanthin, respectively. On the other hand, no degradation of hesperidin was observed during thermal treatment. Finally, the vitamin C in citrus juice was not as heat sensitive as expected and the main provitamin A carotenoids present in citrus juice displayed a relative heat stability. The high-performance liquid chromatography-diode array detection-mass spectrometry analysis of degradation products showed that the isomerization of the epoxide function in position 5,6 into a furanoxide function in position 5,8 was a common reaction for several xanthophylls. These findings will help determine optimal processing conditions for minimizing the degradation of important quality factors such as vitamin C and carotenoid in citrus juice.     

Carotenoid biosynthetic pathway in the citrus genus: number of copies and phylogenetic diversity of seven genes

The first objective of this paper was to analyze the potential role of allelic variability of carotenoid biosynthetic genes in the interspecific diversity in carotenoid composition of Citrus juices. The second objective was to determine the number of copies for each of these genes. Seven carotenoid biosynthetic genes were analyzed using restriction fragment length polymorphism (RFLP) and simple sequence repeats (SSR) markers. RFLP analyses were performed with the genomic DNA obtained from 25 Citrus genotypes using several restriction enzymes. cDNA fragments of Psy, Pds, Zds, Lcy-b, Lcy-e, Hy-b, and Zep genes labeled with [alpha-(32)P]dCTP were used as probes. For SSR analyses, two primer pairs amplifying two SSR sequences identified from expressed sequence tags (ESTs) of Lcy-b and Hy-b genes were designed. The number of copies of the seven genes ranged from one for Lcy-b to three for Zds. The genetic diversity revealed by RFLP and SSR profiles was in agreement with the genetic diversity obtained from neutral molecular markers. Genetic interpretation of RFLP and SSR profiles of four genes (Psy1, Pds1, Lcy-b, and Lcy-e1) enabled us to make inferences on the phylogenetic origin of alleles for the major commercial citrus species. Moreover, the results of our analyses suggest that the allelic diversity observed at the locus of both of lycopene cyclase genes, Lcy-b and Lcy-e1, is associated with interspecific diversity in carotenoid accumulation in Citrus. The interspecific differences in carotenoid contents previously reported to be associated with other key steps catalyzed by PSY, HY-b, and ZEP were not linked to specific alleles at the corresponding loci.     

Influence of mitochondria origin on fruit quality in a citrus cybrid

Sugar, organic acid, and carotenoid are the most important indicators of fruit taste and nutritional and organoleptic quality. These components were studied on fruit pulp of the cybrid between Willow leaf mandarin ( Citrus deliciosa Ten.) and Eureka lemon [ Citrus limon (L.) Burm.] and the two parents. The cybrid possessed nuclear and chloroplast genomes of Eureka lemon plus mitochondria from Willow leaf mandarin. The impact of new mitochondria on fruit quality was studied during the mature period. Levels of organic acids were slightly higher in the cybrid fruit pulp than in Eureka lemon. No significant difference in sugar and carotenoid content was observed between the cybrid and the lemon. Results confirm that the main genetic information for the biosynthesis of sugars, organic acids, and carotenoids is contained in the nucleus. In Citrus, cybridization can be used as a strategy to breed specific traits associated with mitochondrial genomes, such as male sterility, without affecting the main organoleptic and nutritional qualities.     

朱红橘果实成熟过程中类胡萝卜素的积累及相关基因的表达分析

为探究朱橘类果实成熟过程中类胡萝卜素的积累及相关基因的表达模式,本研究以朱红橘为材料,使用手持色差仪测定果实成熟过程中果皮和果肉的色差值,利用液相色谱串联质谱(LC-MS/MS)检测类胡萝卜素组分的含量变化,并利用实时荧光定量PCR(qRT-PCR)分析类胡萝卜素代谢相关基因的表达.结果表明,朱红橘成熟过程中,果皮和果...     

Quantification of carotenoids in citrus fruit by LC-MS and comparison of patterns of seasonal changes for carotenoids among citrus varieties

To quantify the 18 carotenoids on the basic routes of the carotenoid biosynthesis in plants simultaneously, a method for liquid chromatography-mass spectrometry (LC-MS) using atmospheric pressure chemical ionization was developed. With this method, the seasonal changes of carotenoids in the flavedo and juice sacs of 39 citrus varieties were analyzed. On the basis of the patterns of seasonal changes of carotenoids in both flavedo and juice sacs, 39 citrus varieties were classified. In flavedo, 39 varieties were classified into 5 clusters, in which the carotenoid profiles were carotenoid-poor, phytoene-abundant, violaxanthin-abundant, violaxanthin- and beta-cryptoxanthin-abundant, and phytoene-, violaxanthin-, and beta-cryptoxanthin-abundant, respectively. In juice sacs, they were classified into 4 clusters, in which the carotenoid profiles were carotenoid-poor, violaxanthin-abundant, violaxanthin- and phytoene-abundant, and violaxanthin-, phytoene-, and beta-cryptoxanthin-abundant, respectively. In flavedo, many citrus varieties, except for the carotenoid-poor and phytoene-abundant varieties, massively accumulated beta,epsilon-carotenoids (e.g., lutein), beta,beta-carotenoids (e.g., beta-cryptoxanthin and violaxanthin), and phytoene, in that order. In juice sacs, the accumulation order among beta,beta-carotenoids was observed. Violaxanthin accumulation preceded beta-cryptoxanthin accumulation in violaxanthin-, phytoene-, and beta-cryptoxanthin-abundant varieties. In each variety, the carotenoid profiles of the flavedo and juice sacs on the basis of the concentration in violaxanthin and beta-cryptoxanthin were similar, with the exception of a few varieties.     

Differences in the carotenoid content of ordinary citrus and lycopene-accumulating mutants

High-performance liquid chromatography, coupled with photodiode array detection, was used to analyze the carotenoid composition of peel and juice vesicle tissues of ordinary and lycopene-accumulating mutants (referred to as red mutants in this article) of orange, pummelo, and grapefruit. Thirty-six major carotenoids, including some cis-trans isomers, were separated on a C30 reversed phase column, and 23 of them were identified on the basis of retention times and spectral characteristics with authentic standards. Carotenoid profiles varied with tissue types, citrus species, and mutations. beta-Citraurin occurred in the peel of oranges but not in juice vesicles, whereas the reverse was found for violaxanthin, 9-cis-violaxanthin, and luteoxanthin. The diversity of carotenoids in peel and juice vesicle tissues and the fact that there was over 250 times higher content of total carotenoids in peels of Yuhuan pummelo than juice vesicles suggested that the biosynthesis of carotenoids in these two tissues was independent and exchange of carotenoids between the tissues was not likely. Lutein was observed in peels of pummelos and grapefruits and juice vesicles of ordinary pummelo but not in orange tissues. Accumulation of lycopene and beta-carotene was observed in red mutant citrus, except for the peel of Cara Cara red orange. Additionally, phytoene accumulated in all tissues except for the peel of Chuzhou Early Red pummelo. No obvious change in the total content of xanthophylls was observed in the Cara Cara red orange. Ordinary grapefruit (Marsh) tissues and pummelo (Yuhuan) juice vesicles were almost devoid of carotenoids, and in red mutants, the content of total carotenoids increased dramatically up to 790-fold. The different changes in carotenoid content and profiles in mutant(s) of different citrus species suggest that the underlying mechanisms for the mutations might be different.     

Effects of salinity stress on carotenoids, anthocyanins, and color of diverse tomato genotypes

One nonanthocyanin-accumulating (Ailsa Craig) and three anthocyanin-accumulating tomato genotypes (Anthocyanin fruit type, Atroviolaceum, and Sun Black) were analyzed to assess differences in their carotenoid and anthocyanin levels and color and to evaluate the effects of nutrient solutions with different salt concentrations on these parameters. The carotenoid content of control Atroviolaceum tomatoes was ca. 2-2.5-fold higher relative to the other two types, and the color of its puree could be visually distinguished from those of other genotypes. Salinity stress led in some cases to a 2-3-fold increase in the lycopene content. Saline treatment increased the accumulation of total anthocyanins in fruits of Sun Black (2-fold increase), while it reduced it in fruits of Anthocyanin (10-fold decrease). In general, the treatment increased the differences in color of different purees. These results indicate that salinity stress can lead to similar or higher increases in tomato carotenoids than those achieved by genetic engineering. In addition, these changes were accompanied by visually discernible color differences in tomato products. Our findings show the considerable potential of exploiting saline soils to obtain tomatoes with higher levels of secondary metabolites like carotenoids and anthocyanins.     

Determination of carotenoids, total phenolic content, and antioxidant activity of Arazá (Eugenia stipitata McVaugh), an Amazonian fruit

The fruit of Arazá (Eugenia stipitata McVaugh) native to the Colombian Amazon is considered a potentially economically valuable fruit for the Andean economy due to its novel and unique taste. The fruit has an intense yellow color, but its chemical composition and properties have not been well studied. Here we report the identification and quantitation of carotenoids in the ripe fruit using high performance liquid chromatography (HPLC) with photodiode array detector (PDA) and atmospheric pressure chemical ionization (APcI) mass spectrometry (MS/MS). The qualitative carotenoid profile of the fruit according to maturity stage was also observed. Furthermore, antioxidant activity of the peel and pulp were assessed using the ferric reducing ability of plasma (FRAP), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods, in addition to chemical indexes and total phenolic content. Multiple carotenoids were identified in the peel and pulp including four xanthophylls (free and esterified as their mono and diesters) and two carotenes. One of the xanthophylls was tentatively identified as zeinoxanthin, while the others were identified as lutein, zeaxanthin, and β-cryptoxanthin. Carotenes included α-carotene and β-carotene. The total carotenoid content was significantly higher in the peel (2484 ± 421 μg/100 g FW) than in the pulp (806 ± 348 μg/100 g FW) with lutein, β-cryptoxanthin, and zeinoxanthin as the major carotenoid components. The unique carotenoid composition of this fruit can differentiate it from other carotenoid-rich fruits and perhaps be useful in authentication procedures. Overall, results from this study suggest that Colombian Arazá may be a good edible source of carotenoids important in retinal health as well as carotenoids with provitamin A activity. Therefore, Arazá fruit can be used as a nutraceutical ingredient and in production of functional foods in the Colombian diet.     

Varietal and interspecific influence on micronutrient contents in citrus from the Mediterranean area

To specify the genotypic variation of Mediterranean Citrus juices, the contents of carotenoids, flavonoids, and vitamin C were determined by high-performance liquid chromatography. A selection of orange varieties and Mandarin species from the Mediterranean area (Citrus sinensis, Citrus deliciosa Ten, and Citrus clementina Hort. ex Tan) was evaluated using carotenoid profiles and flavanones contents. Among the eight varieties of orange (Salustiana, Hamlin, Shamouti, Pera, Valencia, Maltaise, Sanguinelli, and Cara-cara) and two Mandarin species, only three cultivars (Pera, Sanguinelli, and Shamouti) and the two Mandarin species displayed a high content of vitamin A (374, 381, and 272 ER L(-1) for the three orange cultivars and 1156 and 960 retinol equivalent (RE) L(-1) for the Mandarins) due to a high content of beta-cryptoxanthin. These same Citrus were also rich in hesperidin (502, 537, 552, 767, and 754 mg L(-1), respectively). Principal component analysis allowed the Mediterranean orange varieties and Mandarin species to be differentiated on the basis of nutritional criteria. Strong correlations were observed between beta-cryptoxanthin and hesperidin (r = 0.92) and between beta-cryptoxanthin and beta-carotene (r = 0.98). In contrast, vitamin C content was not correlated with carotenoids and flavanone glycosides. The Mandarin and orange group was quite distinct. The orange varieties could be divided in two groups. In addition, a diversity tree allowed a genetic approach to differentiating Citrus cultivars on the basis of Euclidian distances. This representation showed that the hybrid Clementine was nearer to its parent Mandarin than to its parent orange, suggesting that beta-cryptoxanthin was a dominant genetic factor. With regard to vitamin A, Mandarin and its hybrid Clementine appeared to be the best Citrus species.     

Orange, mandarin, and hybrid classification using multivariate statistics based on carotenoid profiles

A study was undertaken to differentiate citrus on the basis of a carotenoid profile obtained from the HPLC determination of 12 carotenoids found in saponified fresh juice. Mandarin, orange, and various hybrid varieties were analyzed to determine their carotenoid profiles. The resulting peak areas were analyzed using principal component analysis (PCA), canonical discriminate analysis (CDA), and Mahalanobis distances. These were used to develop models for classifying the juices into appropriate groups. Thirty-two samples were analyzed to determine classification techniques. Mandarin and orange juices were quite distinct, with the hybrids scattered throughout the mandarin and orange clusters using PCA. CDA resulted in three distinct groups with only a few of the hybrids in the orange grouping.     

Phenotypic diversity and delimitation between wild and cultivated forms of the genus <Emphasis Type="Italic">Pyrus</Emphasis> in North-eastern Spain based on morphometric analyses

We investigated morphological evidence that might allow wild Pyrus spp. to be distinguished from cultivated material (Pyrus communis L.) in the North-eastern Iberian Peninsula. 134 pear trees were identified in the wild and characterized by 13 quantitative and 13 qualitative leaf–shoot and fruit traits. The trees were visually classified into two preliminary groups of wild and cultivated material and discriminant functions, based on a reference collection for allocating individuals to one of the groups, were constructed. Both classifications were compared with a near-optimal numerical classification (the two-stage Ward-MLM strategy) using two criteria. The visual assignment of trees allocated 60% of trees to the wild group and 40% to the cultivated group. The overall discrepancy rate between the field classification and the discriminant analysis was low (17.4%). In general, wild individuals had smaller leaves, shorter petioles and more rounded and smaller fruits than their cultivated counterparts. They also had small-to-intermediate petiole widths, thorns on their shoots and straight or convex fruit profiles. However, the Ward-MLM strategy always formed better groups, in terms of the two criteria used, in all the continuous and categorical variables, for both leaf–shoot and fruit traits. Likewise, the agreement between classifications (discriminant analysis and Ward-MLM strategy) was only partial, with some Ward-MLM groups composed of both wild and cultivated material in similar proportions. This result suggests a limited success in identifying genuine wild individuals based on morphometric data, which can be ascribed either to poor phenotypic diversity and lack of distinguishing traits among species or to widespread crossability and subsequent development of hybrid/introgressant populations between wild and cultivated specimens.     

Identification of isolutein (lutein epoxide) as cis-antheraxanthin in orange juice

The carotenoid profile of orange juice is very complex, a common characteristic for citrus products in general. This fact, along with the inherent acidity of the product, which promotes the isomerization of some carotenoids, makes the correct identification of some of these pigments quite difficult. Thus, one of the carotenoids occurring in orange juice has been traditionally identified as isolutein, a term used to refer to lutein epoxide, although enough evidence to support that identification has not been given. In this study, the carotenoid previously identified as isolutein/lutein epoxide in orange juice has been isolated and identified as a 9 or 9'-cis isomer of antheraxanthin as a result of different tests. To support this identification, a mixture of geometrical isomers of lutein epoxide isolated from petals of dandelions was analyzed under the same conditions used for orange juice carotenoids to check that neither their retention times nor their spectroscopic features matched with those of the orange juice carotenoid now identified as a cis isomer of antheraxanthin.     

Flavonoids in tropical citrus species

HPLC with PDA and MS(2) detection was used to identify and quantify flavonoids in the tropical citrus species Citrus microcarpa , Citrus hystrix , Citrus medica var. 1 and 2, and Citrus suhuiensis . Most of these species contained high amounts of flavones, flavanones, and dihydrochalcone C- and/or O-glycosides, which were identified on the basis of HPLC retention times, cochromatography with available authentic standards, absorbance spectra, and mass spectral fragmentation patterns. Among the major compounds detected were apigenin-6,8-di-C-glucoside, apigenin-8-C-glucosyl-2″-O-rhamnoside, phloretin-3',5'-di-C-glucoside, diosmetin-7-O-rutinoside, hesperetin-7-O-neohesperidoside, and hesperetin-7-O-rutinoside. Most of the dihydrochalcone and flavone C-glycosides have not previously been detected in tropical citrus. C. microcarpa contained a high amount of phloretin-3',5'-di-C-glucoside. Most of the tropical citrus flavanones were neohesperidoside conjugates, which are responsible for imparting a bitter taste to the fruit. Only C. suhuiensis fruit contains rutinoside, a nonbitter conjugate.     

POTASSIUM FERTILIZATION AND FRUIT PRODUCTION OF PAGE CITRUS ON A PUNSIRUS ROOTSTOCK: QUANTITATIVE AND QUALITATIVE TRAITS

Determination of optimum rates of potassium (K) for high citrus production with great qualitative traits is of both agricultural and economical significance, particularly when performing long–term experiments. A five–year field experiment was conducted in the Citrus Research Center of Tonekabon, Iran in a silty clay loam. The objectives were to: 1) to apply different rates of K fertilizer and determine the optimum rates for citrus high production, and 2) to evaluate the effects of K fertilization on the fruit quantitative and qualitative traits for the production of tasty and great quality fruits for fresh or long consumption. Fifteen–year citrus trees were fertilized with five rates of potassium at control, 750, 1500, 2250 and 3000 g tree^?1 on the basis of a completely randomized block design in five replicates. Fruit parameters were determined. Potassium significantly increased fruit yield and qualitative traits at the optimum amount of 1500 g tree^?1.     

Change in carotenoids and antioxidant vitamins in tomato as a function of varietal and technological factors

The change in the carotenoid and bioantioxidant content of tomato as a function of varietal and technological factors was investigated in the present work. No great differences were found between cultivars for fresh consumption (salad tomatoes) and those for processing in ascorbic acid content. The concentration of ascorbic acid ranged between 14.6 and 21.7 mg/100 g fresh weight of ripe tomato fruit. Processing cultivars contained higher amounts of tocopherols, particularly alpha-tocopherol than tomatoes for fresh consumption. Significant differences could be obtained between the examined varieties with regard to carotenoid concentration. The different tomatoes varied not only in the total carotenoid content but also in the qualitative distribution of some pigments such as lycopene, beta-carotene and lutein. During heat-based processing, ascorbic acid, tocopherols, and carotenoids showed different role and response. Ascorbic acid, alpha-tocopherol quinone, and beta-carotene were the most susceptible components toward thermal degradation.     

Dual role of the pigmentation gene B in affecting carotenoid and vitamin E content in squash (Cucurbita pepo) mesocarp

The yellow and orange colorations of the mesocarp of pumpkins and squash of Cucurbita pepo are due to the presence of carotenoid pigments that also greatly contribute to their nutritional value. Carotenoids, as well as tocopherols (vitamin E), are isoprenoid compounds formed by branches of a common biosynthetic pathway. Photodiode array HPLC analysis was used to simultaneously determine the content and composition of fruit-flesh carotenoids and tocopherols in five pairs of near-isogenic lines differing in the allelic state of genes previously identified as having profound effects on fruit color. The dominant B allele promoted carotenoid accumulation up to 5-fold and prevented tocopherol accumulation in all genetic backgrounds. The dominant L-2 allele doubled carotenoid content and, in combination with the dominant B allele, increased carotenoid content by 10-15-fold as compared to the recessive l-2 allele. The genes D and L-1 had no significant effects on mesocarp tocopherols or carotenoids content. These results indicate that the B gene, which affects both carotenoids and tocopherols, may play a regulatory role in the flux of the isoprenoid pathway products.