Article pp.229-237 du Vol.25 n°3 (2005)

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ARTICLE ORIGINAL

ORIGINAL PAPER

Gamma irradiation delays the postharvest tomato fruit maturation and senescence

F. Chéour

¹

RÉSUMÉ

Ralentissement de la maturation et de la sénescence de la tomate par irradia- tion aux rayons gamma

Des tomates (Lycopersicum esculentum, cv. Sinkara) ont été irradiées aux rayons gamma pour vérifier les effets sur la dégradation des lipides membranaires au cours de l’entreposage et pour s’assurer que la méthode de détection de l’irradiation par la présence de l’o-tyrosine dans les protéines est applicable à ce fruit. Dans la première étude, les tomates ont été irradiées aux doses de 0, 1, 2, 3 ou 5 kGy et entreposées à l’obscurité à 8 °C pendant 21 jours. Pour vérifier si le ralentissement de la sénescence correspond à une protection des lipides membranaires, les contenus en chlorophylles et en caroténoïdes, ainsi que la composition lipidique des membranes ont été déter- minés dans les péricarpes au cours de la conservation des tomates. La teneur en chlorophylles diminue et celle des caroténoïdes augmente au cours de la maturation et de la sénescence de la tomate en relation avec la réduction des phospholipides. Le degré d’insaturation des phospholipides et de la fraction acides gras libres diminue.

Cependant, le rapport stérols sur phospholipides augmente. Les proportions relatives des différentes classes de phospholipides demeurent inchangées. Ainsi, le catabo- lisme des phospholipides est ralenti par les doses 1 et 2 kGy. Cependant, il est accé- léré par les doses 3 et 5 kGy. Dans la seconde étude, des tomates de la même variété ont été irradiées aux doses de 0 ou 20 kGy pour vérifier si la méthode de détection de l’irradiation par la présence de l’o-tyrosine dans les protéines est applicable à ce fruit.

Les analyses par GLC-SM des acides aminés libérés suite à l’hydrolyse des protéines n’ont pas révélé de pic(s) correspondant au temps de rétention de l’o-tyrosine chez le témoin et les tomates irradiées. Nous concluons que le ralentissement de la maturation et de la sénescence de la tomate par l’irradiation implique probablement une protection de ces lipides membranaires de la dégradation et que l’utilisation de la méthode d’o-tyrosine pour la détection de l’irradiation n’est pas applicable à ce fruit.

Mots clés

irradiation, rayon gamma, tomate, lipides, conservation, o-tyrosine.

SUMMARY

Tomato (Lycopersicum esculentum, cv. Sinkara) fruit was gamma irradiated at several doses to verify the effect on membrane lipid degradation during storage and if the

1. École Supérieure d’Horticulture et d’Élevage de Chott-Mariem – Sousse – Tunisie.

Correspondence: Foued Chéour, Ph. D – BP 49A – SFAX, 3099 – Tunisie – E-mail: [email protected] – Tél. : 216 98 289 744.

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detection of irradiation by o-tyrosine presence in proteins is applicable to tomato. The tomato fruit was irradiated at 0, 1, 2, 3 or 5 kGy and stored at 8°C for 21 days in the dark. To assess whether the delay of senescence by irradiation involved the protection of membrane lipids, chlorophyll and carotenoid contents, and the lipid composition of membrane were determined in the pericarp tissue during storage. Chlorophyll content decreased during storage, whereas that of the carotenoids increased in cor- relation with a reduction in the amount of phospholipids. The degree of unsaturation of phospholipids and free fatty acids decreased, whereas the ratio of sterol to phospholipid increased. The proportion of phospholipids classes did not change during senescence. The catabolism of phospholipids was delayed by 1 and 2 kGy, but accelerated by 3 and 5 kGy, as compared to the untreated sample. In a second study, tomato fruit, cv. Sinkara, was irradiated with 0 or 20 kGy to verify if the detection of irradiation by the o-tyrosine presence in proteins is applicable to tomato. Analysis by GLC-MS of amino acids liberated from proteins by hydrolysis did not show peaks of o-tyrosine in untreated and irradiated tomatoes. We conclude that the delay of senescence of tomato pericarp tissue by irradiation involved protection of membrane lipids from degradation and the use of o-tyrosine method for detection of irradiation was not applicable to tomato.

Keywords

gamma-irradiation, tomato, lipids, storage, o-tyrosine.

1 – INTRODUCTION

The effect of gamma irradiation on the preservation of fruit and vegetables has been previously investigated. Irradiation was used efficiently in the control of potato and onion germination, ripening of some tropical fruits such as mango, banana and papaya, the development of micro-organisms, and the proliferation of insects (KADER, 1986; SUMMER and PETERS, 1997; PRAKASH et al., 2000).

Although this technique was shown to delay senescence of some fruit and vegetables, opposite effects was observed for others (LARRIGAUDIÈRE et al., 1991). Indeed, the even gamma irradiation at a low dose of 1 kGy could accelerate maturation of broccoli (DOMIN- GUEZ-LOPEZ et al., 1988). A rise in respiration and metabolism was observed after gamma irradiation (KADER, 1986). Indeed, several authors have reported that the irradiation stimula- tes the production of ethylene continuation to injuries caused by irradiation (MAXIE et al., 1971). These observations could imply membrane deterioration. In fact, loss of cell membrane integrity is a characteristic of plants senescence. This is evident from progressive ultra-structural deterioration and from increased leakage of solutes (CHÉOUR et al., 1992).

Reduced membrane phospholipids content during senescence is an indication of membrane breakdown, as shown for senescing carnation petals (FOBEL et al., 1987). However, to the best of our knowledge, no study has shown clearly the effects of irradiation on the membrane preservation although studies of VOISINE et al. (1993) have demonstrated a modification of phospholipid catabolism in microsomal membranes of gamma irradiated cauliflower.

Although methods have been developed to establish if a fruit or a vegetable was irradiated or not and what dose was used, the results have only been reliable for spices (JEF- FRIES, 1983). Such methods are required for the application of national regulations on irradiation and for regulation of international fruit and vegetable trade. A press-release from the US National Bureau of Standards (Commerce New, June 16, 1986), confirmed by publication (KRAM and SIMIC, 1986), indicated that it was possible to detect whether meat had been irradiated and the dose used, by measuring the o-tyrosine content in the proteins by GLC-MS. Thus, WILLEMOT et al. (1989) tested this method with plant material and concluded that the method based on the presence of o-tyrosine in proteins was not applicable to strawberries.

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The objectives of this study were to evaluate the effect of several doses of gamma irradiation on tomato membrane lipid degradation during storage and to verify if the detection of irradiation by o-tyrosine presence in proteins is applicable to tomato fruit.

2 – MATERIELS AND METHODS

Two experiments were carried out to evaluate the influence of gamma irradiation on the tomato membrane lipid degradation as well as the presence of o-tyrosine in proteins.

In the first experiment, the effect of gamma irradiation on lipid membrane stabilisation during storage was verified. Tomato fruit was irradiated at doses 0, 1, 2, 3 or 5 kGy. The phospholipids (PL), the free fatty acids (FFA) and the free sterols (FS) were determined.

Greenhouse grown tomatoes (Lycopersicum esculentum, cv. Sinkara) were obtained from School of Horticulture and Breeding, Sousse, Tunisia. Tomatoes, pre-cooled and selected for uniformity of size and color (one-fourth to one-half red), and lack of wound, were stored in the dark in polyethylene 25-liter containers under a continuous air stream at 12°C and 85% HR for 0, 14 or 21 days. The levels of irradiation were chosen after preliminary tests showed delay of chlorophyll degradation below 2 kGy, and acceleration above 3 kGy.

Ripening and senescence of tomatoes were evaluated by determination of chlorophyll and carotenoid contents of pericarp tissue according to the methods described by BERGEVIN et al. (1993). The extraction was achieved using a chloroform/methanol mixture (2/1, v/v).

The absorbance at 480 and 664 nm was measured using a spectrophotometer (Hewlett- Packard, model 8451A Diode Array, Mississauga, ON). Pure solutions of lycopene and chlorophyll b pigments (Sigma, St-Louis, MO) were used to prepare standard curves. At the end of storage, pericarp tissues were fixed in a boiling water for 3 min to inactivate endogenous phospholipases.

Total lipids were extracted from the tissue using the procedure of BLIGHT and DYER (1959).

The lipids in the chloroform phase were separated using TLC on 250 µm silica gel G plates (Fisher Scientific Co., Ottawa, ON). Acetone/acetic acid/water (100/2/1, v/v) was used to separate the PL from galactolipids, hexan/diethyl ether/acetic acid (80/20/1; v/v/v) was used to separate the neutral lipids, and chloroform:methanol/acetic acid/water (80/15/15/3,5, v/v/v/

v) was used to separate PL. The lipids were visualized in iodine vapors and identified using authentic standards (Sigma, St-Louis, MO). The area corresponding to each class on the TLC plate was scraped into a test tube and transmethylated directly onto the silica gel with 14%

(w/v) BF3 in methanol (METCALFE and SCHMITZ, 1961). For quantitative determination of FA, a known amount of heptadecanoate (C17:0) was added as an internal standard. Methyl esters of FA were analyzed by GLC (Hewlett-Packard, model 5890A, Mississauga, ON) on a 30-m capillary DB 225 column (J & W Scientific, Rancho Cordova, CA) as described by MAKHLOUF et al. (1990). FS were silylated directly on the silica gel (COUTURE et al., 1989) and assayed by GLC using cholestane as a standard. Sterol trisilyl derivatives were separated by GLC on a 25-m capillary column (Hewlett-Packard, ULTRA 1, Mississauga, ON).

In the second experiment, where the presence of the o-tyrosine in proteins was verified after gamma irradiation, tomato fruit was irradiated at 0 or 20 kGy as described by WILLE- MOT et al. (1989) studies. Pure phenylalanine (Sigma) was irradiated at the same dose in a glass tube as a powder (20 mg) in 10 mL of 20% methanol solution (50 mg). For analysis of irradiated phenylalanine, an aliquot of 1 mg of the radiated sample was derivatized with N- methyl-N-(T-butyl-dimethylsilyl)-trifluoroacetamide (MTBSTFA, Regis Chemical, MORTON Grove, IL) and the derivatized tertio-butyldimethylsilyl (t-BDMS) was separated by GLC on 25-m capillary column (Hewlett-Packard, high performance cross-linked methyl silicone).

The temperature of the oven was maintained initially at 100°C over 10 min, increased to 300°C to 10°C/min and maintained during 2 min. Chromatograms were compared with those of untreated phenylalanine, o-tyrosine and p-tyrosine (Sigma). Analysis were carried out at different dates, and under varied conditions of GLC and retention times (Rt). Requi- red controls were included for each series of analysis. For the extraction and analysis of free amino acids of tomatoes, 60 g irradiated and control samples were extracted for

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20 min with ethanol (80%). The filtrate, concentrated under vacuum, was dissolved in 50 mL of 2M formic acid. Amino acids were purified by passage through an ion exchange resin column (10 cm ✕ 0,7 cm) (Ag 50W-X8 200/400, Bio-rad, Richmond, CA). Organic acids were eluted with 10 mL of 2M formic acid and amino acids with 20 mL of 4M NH₄OH.

Three aliquots were analyzed using GLC as described previously. For the extraction and analysis of amino acids, irradiated and control tomato samples were homogenized. Pro- teins were precipitated by saturation with Na₂SO₄ and dialyzed for 24 h. Amino acids liberated upon hydrolysis by 6M HCl at 100°C over 24 h were purified and analyzed similar to that for the free amino acids.

The identity of o-tyrosine peaks was confirmed by mass spectrometry (Hewlett-Packard model 5970, St-Louis, MO) coupled to a similar GLC as mentioned previously. The detector, a mass filter of hyperbolic quadrupole, was used in mode SIM (selected ion monitoring).

Spectres were compared with those of the bank of spectre NBS (National Bureau of Switch- boards), contained in the memory of the instrument, and that of the o-tyrosine reference.

Analysis of variance (ANOVA) of the results was carried out following a split-plot design (SNEDECOR and COCHRAN, 1957) by statistical analysis system (SAS) using general linear model (GLM) (SAS Institute Inc. 1989). Homogeneity of variance was verified by means of the standard Bartlett test (ANDERSON and MCLEAN, 1974). Each analysis was made in triplicate. Results are means of triplicate ± standard deviation. The experiments were repeated with similar results.

3 – RESULTS

3.1 Lipids membrane degradation

PL, FS and FFA of tomato pericarp tissues were measured during storage to verify if changes of chlorophyll and carotenoid contents were associated with an alteration in membrane lipid composition and if they are influenced by the irradiation.

Chlorophyll content of pericarp tissue decreased significantly during storage at 8°C for all treatments, however, that of the carotenoids increased significantly (P ≤ 0,001) (table 1).

Treatment with 1 and 2 kGy delayed the loss of chlorophyll and the synthesis of carotenoids, whereas, 3 and 5 kGy accelerated it.

Table 1

Change with time in pigments content and ratio of FS to PL of pericarp tissue during storage of gamma irradiated tomato fruit, cv. Sinkara, at 12°C for 14 days.

Values are expressed as means ± SD.

Irradiation

(kGy) Days Chlorophylls

(nm/g FW)

Carotenoids (nm/g FW)

FS/PL (µg /µg)

00 9,9 ± 0,9 6,7 ± 1,2 0,14 ± 0,01

0 14 0,1 ± 0,2 71,0 ± 32,1 0,26 ± 0,02

21 ≤ 0,1 147,2 ± 45,6 0,38 ± 0,04

1 14 7,8 ± 0,2 39,0 ± 32,1 0,17 ± 0,01

21 5,4 ± 0,3 99,2 ± 45,6 0,21 ± 0,03

2 14 8,1 ± 0,2 43,0 ± 32,1 0,18 ± 0,02

21 5,8 ± 0,2 97,2 ± 45,6 0,21 ± 0,01

3 14 2,1 ± 0,2 72,0 ± 32,1 0,23 ± 0,02

21 – 136,6 ± 45,6 0,39 ± 0,06

14 ≤ 0,1 77,3 ± 9,1 0,29 ± 0,03

5 21 – 162,7 ± 21,3 0,43 ± 0,04

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The PL content of tomato pericarps decreased significantly during storage at 8°C for all treatments (P ≤ 0,001) (figure 1). However, this decrease varied according to the dose of irradiation employed. Indeed, the rate of decline in PL level was less at 1 and 2 kGy, but greater at 3 and 5 kGy than in the control. This result was confirmed by the levels of degradation products of PL determined during storage (figure 2). Phosphatidic acid (PA), diacylglycerol (DAG) and free fatty acids (FFA) levels rose during storage subjected to the five treatments. PA and DAG production was affected by the irradiation dose. Doses 1 and 2 kGy reduced the production of PA and DAG during storage, whereas doses 3 and 5 kGy slightly enhanced their production. Levels of FFA increased in an essentially linear fashion throughout the storage period, but they were apparently not affected by the irradiation treatment (data not shown). The most important PL were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol (51.4, 34.6, 7.4 and 6.6%, respectively). The proportion of the PL classes did not change significantly (P ≥ 0,05) during storage for all treatments indicating that each class of PL declined at a similar rate.

The FS content showed no significant change under any of the treatments during storage (P ≥ 0,05). The loss of PL from the membrane was reflected with a shift in the ratio of FS to PL (table 1) (Pr ≤ 0,001). The ratio, which increased significantly for the control and even more to 3 and 5 kGy, hardly changed at 1 and 2 kGy and was closely correlated with the loss of chlorophylls and an increase of carotenoids (r = – 0,85 and 0,91, respectively).

Table 2 shows the FA composition of PL and FFA fractions. PL were rich in linoleic acid (18:2), linolenic acid (18:3), and their ratio of PUFA to saturated FA (mol%), 2.91, was greater than that of the FFA, 2.37. Loss of PUFA from both fractions during storage was reflected by a decrease in the ratio of PUFA to saturated FA (table 3) (P ≤ 0,001). The decrease was greater at 3 and 5 kGy and lower at 1 and 2 kGy for the both fractions than in the control (P ≤ 0,001).

60 80 100 120

0 1 2 3 5

Dose (kGy)

Phospholipids (%)

Day 0 Day 14 Day 21

Figure 1

Change with time in PL content of pericarp tissue during storage of gamma irradiated tomato fruit, cv. Sinkara, irradiated at 12°C for 14 days.

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Table 2

FA composition and ratio of PUFA to saturated FA (mol%) of the PL and FFA fractions of pericarp tissue during storage of gamma irradiated tomato fruit,

cv. Sinkara, at 12°C for 14 days. Values are expressed as means ± SD.

FA Fractions

PL FFA

16:0 25.6 ± 0.8 27,6 ± 0.6

18:0 – 2.1 ± 0.6

18:1 2.1 ± 0.6 1.9 ± 0.8

18:2 49.2 ± 1.6 55,7 ± 1.3

18:3 23,1 ± 0,9 12.7 ± 0.4

PUFA/S 2,91 ± 0,6 2,37 ± 0.4

0 10 20 30 40 50

0 1 2 3 5

PA ( g/g FW)

0 5 10 15 20 25 30

0 1 2 3 5

Dose (kGy)

DAG ( g/g FW)

Day 0 Day 14 Day 21

Figure 2

Change with time in PA and DAG contents of pericarp tissue during storage of gamma irradiated tomato fruit, cv. Sinkara, irradiated at 12°C for 14 days.

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Table 3

Change with time in the ratio of PUFA to saturated FA (mol%) to PL and fractions FFA in pericarp tissue during storage of gamma irradiated tomato fruit, cv. Sinkara,

at 12°C for 14 days. Values are expressed as means ± SD.

3.2 Detection of o-tyrosine in proteins

GLC of pure amino acids of reference (phenylalanine, p- and o-tyrosine) and the irradiated phenylalanine showed that the irradiation did not cause degradation of phenylalanine in p-tyrosine (R_t. 25.08) and/or in o-tyrosine (R_t. 24.02).

The analysis of free amino acids showed the presence of traces of o-tyrosine in control and irradiated tomatoes. The identity of these peaks was confirmed by mass spectrometry.

However, analysis of amino acids liberated from proteins by hydrolysis did not allow the detection of peaks corresponding to the control and irradiated tomatoes.

4 – DISCUSSION

The beneficial effect of irradiation on preservation of the quality of some fruit and vegetables, and the control of the development of mold has been demonstrated (KADER, 1986;

THOMAS, 1986; LALAGUNA, 1998). However, to generalize its application for other horticultu- ral products, it is important to know their physiological effect at various doses and to evaluate the risk of physical damage or metabolic deregulation since fruit and vegetables are characterised by their diversity.

A characteristic feature of senescence is membrane deterioration due to lipids degradation and ensuing destabilisation of the bilayer (CHÉOUR et al., 1992). Our results indicated that senescence of tomatoes, reflected by loss of chlorophyll and synthesis of lycopene, was delayed by irradiation as reported by CHÉOUR and MAHJOUB (2003) for strawberries.

The involvement of membrane lipid breakdown in tomato pericarp was indicated by several markers of lipids degradation during storage as reflected by a reduction in PL content, lar- ger ratio of FS to PL, increase in the level of PL degradation products PA, DAG and FFA, and losses of PUFA from PL and FFA. The levels of these markers of membrane lipid degradation changed in parallel with chlorophyll breakdown and lycopene synthesis, com- mon markers for tomato senescence. Protection of the membrane from lipid degradation by 1 and 2 kGy was indicated by a decrease in most of these changes. Irradiation effects on membrane preservation have been demonstrated by VOISINE et al. (1993). In fact, a modification of phospholipid catabolism in microsomal membranes of gamma irradiated cauliflower has been observed.

In contrast, senescence and the associated membrane lipid degradation were accelerated by 3 and 5 kGy. The contradictory results cited by CHÉOUR and MAHJOUB (2003) may be

Dose (kGy)

PL fraction Days after storage

FFA fraction Days after storage

0 14 21 0 14 21

PUFA/S

0 2,91 ± 0,27 2,57 ± 0,19 2,31 ± 0,14 2,37 ± 0,22 2,04 ± 0,09 1,99 ± 0,08

1 2,88 ± 0,11 2,83 ± 0,09 2,33 ± 0,07 2,27 ± 0,09

2 2,87 ± 0,13 2,84 ± 0,11 2,31 ± 0,09 2,26 ± 0,13

3 2,68 ± 0,23 2,23 ± 0,07 1,92 ± 0,08 1,85 ± 0,11

5 2,46 ± 0,20 2,19 ± 0,11 1,90 ± 0,13 1,87 ± 0,11

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due to high levels of irradiation. Acceleration of ripening and senescence of tomatoes can be the result of injuries caused to membranes by irradiation inducing their softening and predisposition to attack by pathogens (EL-ASSI et al., 1997). Tomato is rich in water and irradiation would provoke the formation of radiolysis products such that free radicals that accelerate the degradation of membrane lipids thus dissolution of walls and consequently its senescence (THOMAS, 1986). The cellular membrane implication in the process of senescence was well demonstrated by CHÉOUR et al. (1992). Free radicals could have induced degradation of PL and FA, thus liberated undergo the oxidation by the lipoxygenase during senescence of fruits (C^HÉOUR and M^AHJOUB, 2003).

In an effort to establish if irradiation produces o-tyrosine in proteins, tomatoes were irradiated by gamma rays at doses 0 or 20 kGy and analysed by GLC-MS. This method did not prove adequate for tomatoes. This result confirms that reported by WILLEMOT et al. (1989) who concluded that the method based on the presence of o-tyrosine in proteins was not applicable to strawberries. Traces of o-tyrosine could be found in the free state but not in proteins. The irradiation normally favors the formation of o-tyrosine from free phenylalanine or that in proteins. However, incorporation of o-tyrosine in proteins is not possible because there is no an enzyme that can accept it as a substrate (WILLEMOT et al., 1989).

In conclusion, results of this study show that irradiation at ≤ 2 kGy delay senescence and membrane lipid degradation of tomato pericarps. In contrast, doses ≥ 3 kGy accelerate senescence and lipid breakdown. The use of the o-tyrosine method for detection of irradiation does not seem applicable to tomatoes.

5 – ACKNOWLEDGMENT

The author is grateful to Mme Josée CHAMPAGNE (Laval University, Québec) for the help in this project and for the preparation of the manuscript.

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