Biochemical characterization of blood orange,sweet orange,lemon, bergamot and bitter orange

This paper reports on the composition of aroma compounds and fatty acids and some physico-chemical parameters (juice percentage, acidity and total sugars) in five varieties of citrus: blood orange, sweet orange, lemon, bergamot and bitter orange. Volatile compounds and methyl esters have been analyzed by gas chromatography. Limonene is the most abundant compound of monoterpene hydrocarbons for all of the examined juices. Eighteen fatty acids have been identified in the studied citrus juices, their quantification points out that unsaturated acids predominate over the saturated ones. Mean concentration of fatty acids varies from 311.8 mg/l in blood orange juice to 678 mg/l in bitter orange juice.     

Effects of thermal treatments on pectinesterase activity determined in blood oranges juices

The citrus processing industry aims to maintain turbidity and attractive colour of the juice obtained from Sicilian blood oranges. Nevertheless, the presence of pectinesterase (PE, E.C. 3.1.1.11) causes the loss of these peculiar characteristics, due to precipitation of colloids and very fine pieces of pulp in suspension, with negative effects on colour and clarification of the juices. This study compares total PE activity of Sicilian blood oranges (Sanguinello, Moro, Tarocco) with the blonde cultivar Navel, checking enzyme stability with various pasteurisation times and temperatures conditions. Decimal reduction time and temperature (D and z) as well as the kinetic constant (k) were established to optimise and increase the shelf-life of the pasteurised juice. Finally, a heat treatment (85 °C × 3 min) of both microbiological and enzymatic efficacy has been developed that does not compromise anthocyanin stability; this treatment could be used by the citrus fruit processing industry as a valid alternative in the production of blood orange juices.     

Pattern recognition and genetic algorithms for discrimination of orange juices and reduction of significant components from headspace solid‐phase microextraction

Introduction – Orange (Citrus sinensis L.) juice comprises a complex mixture of volatile components that are difficult to identify and quantify. Classification and discrimination of the varieties on the basis of the volatile composition could help to guarantee the quality of a juice and to detect possible adulteration of the product. Objective – To provide information on the amounts of volatile constituents in fresh‐squeezed juices from four orange cultivars and to establish suitable discrimination rules to differentiate orange juices using new chemometric approaches. Methodology – Fresh juices of four orange cultivars were analysed by headspace solid‐phase microextraction (HS‐SPME) coupled with GC‐MS. Principal component analysis, linear discriminant analysis and heuristic methods, such as neural networks, allowed clustering of the data from HS‐SPME analysis while genetic algorithms addressed the problem of data reduction. To check the quality of the results the chemometric techniques were also evaluated on a sample. Results – Thirty volatile compounds were identified by HS‐SPME and GC‐MS analyses and their relative amounts calculated. Differences in composition of orange juice volatile components were observed. The chosen orange cultivars could be discriminated using neural networks, genetic relocation algorithms and linear discriminant analysis. Genetic algorithms applied to the data were also able to detect the most significant compounds. Conclusions – SPME is a useful technique to investigate orange juice volatile composition and a flexible chemometric approach is able to correctly separate the juices. Copyright © 2009 John Wiley & Sons, Ltd.     

固定化果胶酶澄清果汁的条件及效果

利用固定化果胶酶对四种不同果汁澄清条件及效果进行研究,结果表明,固定化果胶酶澄清四种不同果汁的效果明显,其中澄清桔汁的果胶酶重复使用20次以上,酶活力及透光率仍可维持在80%以上,其最适反应条件是:果汁浓度50%;pH 3.0~3.5;温度45~50℃;反应时间2小时;酶量每毫升果汁0.05 g固定化果胶酶;澄清时间20小时。     

Functional identification of organic cation transporter 1 as an atenolol transporter sensitive to flavonoids

Atenolol, a β1-adrenergic receptor blocker, is administered orally and its intestinal absorption has recently been indicated to be mediated by carrier protein and reduced markedly by ingestion of some fruit juices, such as apple and orange juices. This could be postulated to be a problem arising from the interaction of some components of fruit juices with atenolol at a transporter involved in its intestinal uptake, but the responsible transporter and its interacting components have not been identified yet. In an attempt to examine that possibility, we could successfully find that human organic cation transporter 1 (OCT1/SLC22A1), which is suggested to be expressed at the brush border membrane of enterocytes, is highly capable of transporting atenolol. In this attempt, OCT1 was stably expressed in Madin-Darby canine kidney II cells and the specific uptake of atenolol by the transporter was found to be saturable, conforming to the Michaelis-Menten kinetics with the maximum transport rate (V_max) of 4.00 nmol/min/mg protein and the Michaelis constant (K_m) of 3.08 mM. Furthermore, the OCT1-specific uptake was found to be inhibited by various flavonoids, including those contained in fruit juices that have been suggested to interfere with intestinal atenolol absorption. Particularly, phloretin and quercetin, which are major components of apple juice, were potent in inhibiting OCT1-mediated atenolol transport with the inhibition constants of 38.0 and 48.0 µM, respectively. It is also notable that the inhibition by these flavonoids was of the noncompetitive type. These results indicate that OCT1 is an atenolol transporter that may be involved in intestinal atenolol uptake and sensitive to fruit juices, although its physiological and clinical relevance remains to be further examined.     

Inactivation of Escherichia coli and Shigella in acidic fruit and vegetable juices by peroxidase systems

AIMS: To study the bactericidal properties of the lactoperoxidase (LPER)-thiocyanate and soybean peroxidase (SBP)-thiocyanate systems at low pH, their efficiency for inactivation of Escherichia coli and Shigella in acidic fruit and vegetable juices, their effect on colour stability of the juices and interaction with ascorbic acid. METHODS AND RESULTS: Three-strain cocktails of E. coli and Shigella spp. in selected juices were supplemented with the LPER or SBP system. Within 24 h at 20 degrees C, the LPER system inactivated both cocktails by > or = 5 log10 units in apple, 2-5 log10 units in orange and < or = 1 log10 unit in tomato juices. In the presence of SBP, browning was significant in apple juice and white grape juice, slight in pink grape juice and absent in orange or tomato juice. Ascorbic acid protected E. coli and Shigella against inactivation by the LPER system, and peroxidase systems significantly reduced the ascorbic acid content of juices. CONCLUSIONS: Our results suggest a different specificity of LPER and SBP for SCN-, phenolic substrates of browning and ascorbic acid in acidic juices. The LPER system appeared a more appropriate candidate than the SBP system for biopreservation of juices. SIGNIFICANCE AND IMPACT OF THE STUDY: This work may open perspectives towards the development of LPER or other peroxidases as biopreservatives in acidic foods.     

A label-free differential quantitative mass spectrometry method for the characterization and identification of protein changes during citrus fruit development

Background

Citrus is one of the most important and widely grown commodity fruit crops. In this study a label-free LC-MS/MS based shot-gun proteomics approach was taken to explore three main stages of citrus fruit development. These approaches were used to identify and evaluate changes occurring in juice sac cells in various metabolic pathways affecting citrus fruit development and quality.

Results

Protein changes in citrus juice sac cells were identified and quantified using label-free shotgun methodologies. Two alternative methods, differential mass-spectrometry (dMS) and spectral counting (SC) were used to analyze protein changes occurring during earlier and late stages of fruit development. Both methods were compared in order to develop a proteomics workflow that could be used in a non-model plant lacking a sequenced genome. In order to resolve the bioinformatics limitations of EST databases from species that lack a full sequenced genome, we established iCitrus. iCitrus is a comprehensive sequence database created by merging three major sources of sequences (HarvEST:citrus, NCBI/citrus/unigenes, NCBI/citrus/proteins) and improving the annotation of existing unigenes. iCitrus provided a useful bioinformatics tool for the high-throughput identification of citrus proteins. We have identified approximately 1500 citrus proteins expressed in fruit juice sac cells and quantified the changes of their expression during fruit development. Our results showed that both dMS and SC provided significant information on protein changes, with dMS providing a higher accuracy.

Conclusion

Our data supports the notion of the complementary use of dMS and SC for label-free comparative proteomics, broadening the identification spectrum and strengthening the identification of trends in protein expression changes during the particular processes being compared.     

Visible Light Image-Based Method for Sugar Content Classification of Citrus

Visible light imaging of citrus fruit from Mie Prefecture of Japan was performed to determine whether an algorithm could be developed to predict the sugar content. This nondestructive classification showed that the accurate segmentation of different images can be realized by a correlation analysis based on the threshold value of the coefficient of determination. There is an obvious correlation between the sugar content of citrus fruit and certain parameters of the color images. The selected image parameters were connected by addition algorithm. The sugar content of citrus fruit can be predicted by the dummy variable method. The results showed that the small but orange citrus fruits often have a high sugar content. The study shows that it is possible to predict the sugar content of citrus fruit and to perform a classification of the sugar content using light in the visible spectrum and without the need for an additional light source.     

A reversed-phase HPLC method for measurement of 5-hydroxymethyl furfuraldehyde and furfuraldehyde in processed juices.

An HPLC method using a reversed-phase macroreticular PLRP-S column and phosphate buffer as eluent is described for the analysis of L-ascorbic acid degradation products, 5-hydroxymethyl furfuraldehyde and furfuraldehyde, in processed fruit juices. Measurement of the levels of 5-HMF and furfuraldehyde in citrus juices against time showed the presence of 5-HMF (0.45 mg l-1) even at zero time. An assessment on the effect of the additives on the formation of 5-HMF of reconstituted single-strength orange juice showed virtually the same results for all the samples stored at 4 degrees C and 20 degrees C, irrespective of the additive. For citrus juice samples which had been subjected to accelerated degradation, those that showed the highest decomposition of L-ascorbic acid, produced the highest level of 5-HMF. The presence of furfuraldehyde in any of the samples was not detected, probably due to the fact that furfuraldehyde was formed in such small amounts which are below the minimum detectability limit of the method (0.050 mg l-1).     

Citrus abscission and Arabidopsis plant decline in response to 5-chloro-3-methyl-4-nitro-1H-pyrazole are mediated by lipid signalling

The compound 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP) is a pyrazole-derivative that induces abscission selectively in mature citrus (Citrus sinensis) fruit when applied to the canopy and has herbicidal activity on plants when applied to roots. Despite the favourable efficacy of this compound, the mode of action remains unknown. To gain information about the mode of action of CMNP, the effect of application to mature citrus fruit and Arabidopsis thaliana roots was explored. Peel contact was essential for mature fruit abscission in citrus, whereas root drenching was essential for symptom development and plant decline in Arabidopsis. CMNP was identified as an uncoupler in isolated soybean (Glycine max) mitochondria and pea (Pisum sativum) chloroplasts and an inhibitor of alcohol dehydrogenase in citrus peel, but not an inhibitor of protoporphyrinogen IX oxidase. CMNP treatment reduced ATP content in citrus peel and Arabidopsis leaves. Phospholipase A₂ (PLA₂) and lipoxygenase (LOX) activities, and lipid hydroperoxide (LPO) levels increased in flavedo of citrus fruit peel and leaves of Arabidopsis plants treated with CMNP. An inhibitor of PLA₂ activity, aristolochic acid (AT), reduced CMNP-induced increases in PLA₂ and LOX activities and LPO levels in citrus flavedo and Arabidopsis leaves and greatly reduced abscission in citrus and delayed symptoms of plant decline in Arabidopsis. However, AT treatment failed to halt the reduction in ATP content. Reduction in ATP content preceded the increase in PLA₂ and LOX activities, LPO content and the biological response. The results indicate a link between lipid signalling, abscission in citrus and herbicidal damage in Arabidopsis.     

Novel expression patterns of carotenoid pathway-related genes in citrus leaves and maturing fruits

Carotenoids are abundant in citrus fruits and vary among cultivars and species. In the present study, high performance liquid chromatography (HPLC) and real-time polymerase chain reaction (PCR) were used to investigate the expression patterns of 23 carotenoid biosynthesis gene family members and their possible relation with carotenoid accumulation in fresh flavedo, juice sacs and leaves of Valencia orange during fruit maturation. Violaxanthin and lutein mainly accumulated in fruit (flavedo and juice sacs) and leaves (young and mature), respectively, accounting for nearly 79 %, 57 %, 53 % and 70 % of corresponding total carotenoids in February. Violaxanthin content quickly began to increase in flavedo in December, but the increase in juice sacs began later in January. In mature leaves, lutein content was three times that in young leaves; α-carotene and β-carotene were also much higher in mature leaves than in flavedo or juice sacs. Generally most of the carotenoid biosynthesis gene members were expressed at higher levels in flavedo than in juice sacs, and the expression of some continued to increase in flavedo during fruit maturation. All CHYB members expressed at high levels and had similar patterns in juice sacs. Interestingly, the capsanthin capsorubin synthase (CCS) members had similar expression levels and patterns in flavedo and juice sacs. Differences in gene expression between leaf and fruit tissues were noted, pointing to some tissue specificity for certain members of the gene families associated with carotenogenesis. The expression patterns of these 23 citrus carotenoid biosynthesis gene members were also compared with their expression patterns in other plants. Taken together, these first-hand expression data will be useful to define the tissue-specific roles of each gene member in accumulation of different carotenoids in citrus leaves and maturing fruits.     

Role of Alicyclobacillus acidoterrestris in the development of a disinfectant taint in shelf-stable fruit juice

AIMS: This study was undertaken to identify the bacterium and metabolic products contributing to a disinfectant taint in shelf-stable fruit juice and to determine some of the growth conditions for the organism. METHODS AND RESULTS: Microbiological examination of tainted and untainted fruit juice drinks detected low numbers of acid-dependent, thermotolerant, spore-forming bacteria in the tainted juices only. The presence of omega-cyclohexyl fatty acids was confirmed in two of the isolates by cell membrane fatty acid analysis. The isolates were subsequently identified as Alicyclobacillus acidoterrestris by partial 16S rDNA sequencing. Studies on the isolates showed growth at pH 2.5-6.0 and 19.5-58 degrees C. Gas chromatography/mass spectrometry (GC/MS) was used to identify and quantify 2,6-dibromophenol (2,6-DBP) and 2,6-dichlorophenol (2,6-DCP) in the tainted juice. Challenge studies in a mixed fruit drink inoculated with the two isolates and the type strain of A. acidoterrestris, incubated at 44-46 degrees C for 4 d, showed the production of both metabolites, which were confirmed and quantified by GC/MS. CONCLUSIONS: The results show that A. acidoterrestris can produce 2,6-DBP and 2,6-DCP in shelf-stable juices. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report detailing experimental methodology showing that A. acidoterrestris can produce 2,6-DCP in foods. Control of storage temperatures (to < 20 degrees C) immediately after processing may provide an effective control measure for the fruit juice industry to prevent spoilage by A. acidoterrestris.     

Decreased hydrogen sulphide content in valencia oranges after freeze damage

The hydrogen sulphide content of headspace gases above freshly extracted Valencia orange juice from freeze-damaged and control fruit was determined. The average value for control fruit was about three times higher than that for fruit suffering moderate to heavy freeze damage, indicating a potential use as an indicator of freeze damage to fresh citrus fruit.     

Essential oils of citrus fruit stimulate oviposition in the Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae)

Female Mediterranean fruit flies (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) perceive both qualitative and quantitative aspects of citrus fruit chemistry. However, the behavioural and biological adjustments of this response remain largely unknown. In the present study, the ovipositional responses of gravid female medflies to essential oils (i.e. the most critical resistance factor to medfly infestation) of various citrus fruits are investigated. In dual‐choice (treatment versus distilled water control) experiments, females lay significantly more eggs into pre‐punctured hollow oviposition hemispheres (domes) provided with 1 µL of citrus peel oil from sweet orange, satsuma mandarin, bitter orange, grapefruit and lemon compared with odourless domes. No‐choice tests show a weak effect of lemon essential oils in stimulating oviposition. The female ovipositional response to sweet orange oil (the most active in eliciting oviposition) is dose‐dependent. Additionally, limonene, the most abundant chemical in all citrus oils, stimulates oviposition, whereas linalool, a representative compound of immature citrus fruit associated with high toxicity against immature stages of fruit flies, has a significant deterrent effect. In further no‐choice tests, females lay approximately 23% fewer eggs in limonene (93%) (amount found in orange oil) and 60% fewer eggs in limonene 93% plus linalool 3% (approximately 10‐fold the amount found in orange oil) mixtures, relative to sweet orange oil. The results suggest that the limonene content accounts largely (but not completely) for the ovipositional responses observed in sweet orange oil, whereas high linalool proportions are capable of significantly masking and/or disrupting its stimulatory effects in citrus oils. The importance and practical implications of these findings with respect to understanding how citrus fruit chemistry influences the ovipositional responses of medfly is discussed.     

Immobilization of tomato (Lycopersicon esculentum) pectinmethylesterase in calcium alginate beads and its application in fruit juice clarification

Clarity of fruit juices is desirable to maintain an aesthetically pleasing quality and international standards. The most commonly used enzymes in juice industries are pectinases. A partially-purified pectinmethylesterase from tomato was entrapped in calcium alginate beads and used for juice clarification. The activity yield was maximum at 1 % (w/v) CaCl₂ and 2.5 % (w/v) alginate. The immobilized enzyme retained ~55 % of its initial activity (5.7 × 10^?2 units) after more than ten successive batch reactions. The K_m, pH and temperature optima were increased after immobilization. The most effective clarification of fruit juice (%T₆₂₀ ~60 %) by the immobilized enzyme was at 4 °C with a holding time of 20 min. The viscosity dropped by 56 % and the filterability increased by 260 %. The juice remains clear after 2 months of storage at 4 °C.     

Induction of a Citrus gene highly homologous to plant and yeast thi genes involved in thiamine biosynthesis during natural and ethylene-induced fruit maturation

Maturing citrus fruit undergo pigment changes which can be enhanced by exogenous ethylene. In order to identify genes induced by ethylene in citrus fruit peel, we cloned the gene c-thi1. mRNA corresponding to c-thi1 increased gradually in the peel during natural fruit maturation and in response to ethylene. GA₃ pretreatment reduced the inductive effect of ethylene. Levels of c-thi1 increased also in juice sacs but the effect of ethylene was much less prominent. c-thi1 is homologous to yeast and plant genes encoding for an enzyme belonging to the pathway of thiamine biosynthesis. The data suggest that thiamine is involved in citrus fruit maturation.     

Recent Advances in the Regulation of Citric Acid Metabolism in Citrus Fruit

The regulation of citric acid metabolism during fruit ripening has a major impact on the production of high-quality fruit. The impact of citric acid on organoleptic fruit quality attributes, fruit storage performance and the synthesis of several secondary metabolites has led to an exponential increase in research efforts during the last two decades. Recent research has focused on the relationship among citric acid biosynthesis, transportation, storage, and utilization. Among citrate metabolic processes, activities of a proton pump, especially the plasma membrane H⁺-ATPase on tonoplast and citrate catabolism in cytosol play important roles in the regulation of citrate accumulation in cell vacuoles. Moreover, we highlight recent advances and provide an overview of citrate metabolism, postharvest physiology of citrate metabolism, and the influence of agro-climatic factors on citrus fruits. It is the first review that provides a comprehensive model for citrate metabolism in citrus fruit juice sacs. We anticipate that this model for the regulation of citrate metabolism will facilitate the study of fruit acidity in citrus and other nonclimacteric fruits.     

Sugar utilization by citrus juice cells as determined by [14C]-sucrose and [14C]-fructose feeding analyses

Sugar utilization by mature citrus juice cells was investigated in light of previous reports suggesting the inability of these cells to phosphorylate hexoses. Grapefruit juice sac cells were incubated in solutions of [¹⁴C]-sucrose or [¹⁴C]-fructose for 16 h during which ¹⁴CO₂ evolution was measured by trapping into soluene. After the incubation period, tissue was extracted in 5 % trichloroacetic acid or 80 % ethanol and extracts separated and identified by thin layer chromatography. Fructose was taken up and metabolized more rapidly than sucrose. In both cases, significant amounts of ¹⁴CO₂, [¹⁴C]-pyruvic and [¹⁴C]-citric acid were recovered after incubation. In separate experiments, hexokinase activity in tissue extracts was found to co-sediment with mitochondrial fractions but was not detected in the soluble fractions as previously reported. The data indicated that, contrary to earlier observations, mature citrus fruit juice cells contain the enzymatic machinery to metabolize soluble sugars. This is consistent with the glycolytic utilization of sugars in cells undergoing anaerobic respiration.     

Longevity of multiple species of tephritid (Diptera) fruit fly parasitoids (Hymenoptera: Braconidae: Opiinae) provided exotic and sympatric-fruit based diets

While adult parasitic Hymenoptera in general feed on floral and extrafloral nectars, hemipteran-honeydews and fluids from punctured hosts, Diachasmimorpha longicaudata (Ashmead), an Old World opiine braconid introduced to tropical/subtropical America for the biological control of Anastrepha spp. (Tephritidae), can survive on fruit juices as they seep from injured fruit. An ability to exploit fruit juice would allow such a parasitoid to efficiently forage for hosts and food sources simultaneously. Two New World opiines, Doryctobracon areolatus (Szepligeti) and Utetes anastrephae (Viereck), are also prominent Anastrepha parasitoids and are roughly sympatric. All three species were provided with: (1) pulp and juice diets derived from a highly domesticated Old World fruit (orange, Citrus sinensis L.) that is only recently sympatric with the Mexican flies and parasitoids and so offered little opportunity for the evolution of feeding-adaptations and (2) a less-domesticated New World fruit (guava, Psidium guajava L.), sympatric over evolutionary time with D. areolatus and U. anastrephae. Both sexes of D. longicaudata died when provided guava pulp or juice at a rate similar to a water-only control. D. areolatus and U. anastrephae, presumably adapted to the nutrient/chemical constituents of guava, also died at a similar rate. Survival of all three species on orange pulp and juice was greater than on water, and often equaled that obtained on a honey and water solution. In confirmatory experiments in Mexico, D. areolatus and U. anastrephae, as well as other tephritid parasitoids Doryctobracon crawfordi (Viereck) and Opius hirtus (Fisher), all died at a significantly higher rates when provided guava in comparison to a honey and water diet. Such a result is likely due to guavas being repellent, innutritious or toxic. D. longicaudata clearly consumed guava juice tagged with a colored dye. Dilutions of orange and guava juice resulted in shorter lifespans than dilutions of orange juice and water demonstrating that there while diluted orange juice provided nutrition the addition of guava created toxicity. Given the differences in fruit-food quality, adult opiine food sources would not be obtainable at all oviposition sites and in the case of guava, more additional sites and foraging for food than previously postulated may be required.