Effect of roasting on tocopherols of gourd seeds (Cucurbita pepo)

Se ha estudiado el efecto del tostado a alta temperatura sobre la vitamina E en semillas de calabaza Cucurbita pepo. El tostado a 140°C durante 5 min indujo una hinchazón de la semilla con un aumento en el volumen del 43 %, y una pérdida de peso del 6,5 %. La semilla adquirió la apariencia deseada después del tostado. La actividad del agua en la semilla cruda fue de 0,544 y disminuyó durante el tostado a 0,105. Las semillas de Cucurbita pepo contenían un 51,0 % de grasa. El contenido de tocoferol de las semillas tostadas fue de 68mg/100g y el de las semillas no tostadas de 107mg/100g de aceite. El γ-tocoferol representó el 96% de los tocoferoles totales. La pérdida de tocoferoles totales durante el tostado fue del 36% siendo la má alta la del β-tocoferol con un 50%; la del α-tocoferol fue del 41% y la del γ-tocoferol del 36%.


INTRODUCTION
The Cucurbitaceae family consists of 90 genera and approximately 700 species.The Cucurbitaceae are characterised by long flexible stems, a crawling or climbing growth habit and fruits that differ widely in
The majority of the Cucurbitaceae used as food are found in five genera: Citrullus (water melons and wild colocynths), Cucumis (cucumbers, gherkins and melons), Lagenaria (gourds), Sechium (chayotte) and Cucurbita.The last one is the most important genus economically and is represented by four species: the musky pumpkin Cucurbita maxima, the gourd Cucurbita moschata, the gourd of Siam Cucurbita ficifolia and the gourd Cucurbita pepo.
The Cucurbitacae have long been cultivated not only for food but also for their medicinal properties.Particular medical properties have been attributed to each part of the fruit and the plant.In the south of France, a slice of gourd pulp (Lagenaria) mixed in one liter of water was used as laxative to treat intestinal diseases and to satisfy thirst.The seeds are noted as an effective remedy against Taenia, a parasitic worm that is paralyzed by the action of cucurbitine (3amino-3-carboxypyrrolidine) contained in the skin of the seed (Armougom, 1998).
Increasingly, consumers want foods that not only taste good but are also good for their health.This is reflected in the demand for food products that have optimal bioavaliblity of essential fatty acids, vitamins, and antioxidants.
Vitamin E is an essential liposoluble vitamin which was discovered by Evans and Bishop in 1922.The term vitamin E is a general descriptor of tocopherols and all their derivatives which present the biological activity of the RRR α-tocopherol (Machlin, 1984;Tomassi and Silano, 1986).Recommended dietary allowances for vitamin E are around 10 to 30mg for an adult in good health (Veris Inc., 1993).
Tocopherols are antioxidant molecules and their primary task is to prevent the damage caused by free radicals on tissues.This is achieved by donating a hydrogen atom to a peroxide radical which results from the degradation of unsaturated lipids and as a consequence tocopherols are minor but omnipresent components in membranes (Kamal-Eldin and Appelquist, 1996).
Tocopherols are particularly sensitive to heating at high temperatures (Barrera-Arellano et al., 2002).As a result, most tocopherols are lost or destroyed during the refining of vegetable oils.Most commercial vitamin E is thus prepared by chemical synthesis.In order to increase the shelf-life of food, the addition of antioxidants during food processing is common.However because of the possible toxic effects of synthetic antioxidants, industry is increasingly turning to natural antioxidants.Many authors consider that natural vitamin E is more active than the synthetic analogue because the chemical synthesis produces many isomers with less biological activity (Horwitt, 1986).A mixture containing 60% in weight of natural a, g and d isomers is largely used as an additive (Shimada et al., 2000).
α-tocopherol is the most easily absorbed by the intestinal wall.A high plasma content of α-tocopherol decreases the natural absorption of γ and δtocopherol (Huang and Appel, 2003).α-tocopherol has the highest antioxidant activity (Chunhieng, 2003).This activity decreases from α to δ-tocopherol (Dziezak, 1986).The tocotrienols are more effective antioxidants because they are unsaturated.
In blood and animal cells, α-tocopherol is the most abundant and represents 87% of the tocopherols in the plasma (Gonzales, 1990).ß and γ-tocopherol represent 2 and 10 % of plasmatic vitamin E respectively (De Leenheer et al., 1988).
The α isoform has many functions similar to the γ isoform, such as, for example, increasing the activity of superoxide dismutase in arterial plasma and cells.γ-tocopherol has some specific biological properties which are different from those of αtocopherol.People who have high plasmatic concentrations of γ-tocopherol tend to get less cancer of the prostate, while those who have low levels tend to have more cancer of the higher aerodigestive tract (Jiang et al., 2001).
Along with selenium, present in Cucurbita pepo seed (0.3 ppm) (Kreft et al., 2002), vitamin E can act on the ageing of cells.This can be proven from the fact that the product resulting from the metabolism of g tocopherol plays an important role in the prevention of cardiac diseases and in the regulation of blood pressure by controlling the drainage of water and of different metabolites in the body.
The liver selectively exports α-tocopherol, using lipoproteins as carriers, to other tissues of the body.The liver eliminates other forms of vitamin E that the chromane cycle has not totally methylated (Schmidt and Nikoleit, 1993).One hypothesis is that the antioxidant activity of tocopherols in the body is linked to their chiral structure and to the mechanism of membrane transfer (Meydani et al., 1986).The RRR α-tocopherol is more readily accepted by the lipoproteins of the membranes than other forms such as SRR α-tocopherol (Ingold et al., 1987).
However, γ-tocopherol may be a more potent cancer chemopreventive than α-tocopherol (Campbell et al., 2003).Thus, it is considered that the structure of γ-tocopherol confers on it a better stability against oxidation at high temperatures and consequently its loss is less and its effectiveness against oxidation is greater than α-tocopherol (Cheng et al., 1987).
This study is related to the effect of roasting at high temperature (140°C) on vitamin E present in the oil of hulled gourd seed Cucurbita pepo Lady Godiva variety.

Samples
The hulled seed of Cucurbita pepo Lady Godiva variety is derived from a mutant closely related to the variety Styriaca, which is widely grown in North-East Europe, particularly in Austria.In France, our samples were produced by the company "Les fleurs de Jaussely" (81470 Aguts, France).
The hulled character of the gourd seed lends itself to efficient analysis as losses associated with process manipulation are reduced.
For each experiment, 75 g of seeds were roasted at 140°C for 5min, 24h before the extraction of oils.To protect tocopherols, the roasted seeds were hermetically sealed in plastic packaging.

Moisture content and water activity
Analysis of the moisture content was carried out by drying 4 lots of 10 g of seeds at 105 ± 2°C for 24h.The water activity was determined with an Aqualab A w meter.The plastic cap of the Aqualab was filled with ground seeds and the analysis was done in two replicates at 24 +/-1°C.The water activity (A w ) is a good indicator of the preservation potential of the product and its microbiological stability with time.Below a threshold of A w = 0.62, there is usually no notable fungal development (Guiraud, 1998).

Extraction of oil
The gourd seeds were crushed using a domestic crusher and dried in an oven at 105°C for 24 h.In order to obtain a sufficient quantity of oil, we used 20 g of dried seeds.The extraction was done by Soxhlet with petroleum ether for 6 hours.

Analysis and quantification of tocopherols by HPLC
Tocopherol quantification (Figure 1) was carried out using Thermo Separation Products (TSP) with a P1000XR pump, using 4 external tocopherol standards (Tocopherol set, Calbiotech TM ).The standard solutions were prepared every day and G. FRANÇOIS, B. NATHALIE, V. JEAN-PIERRE, P. DANIEL AND M. DIDIER were preserved in HPLC grade hexane in flasks without actinic activity.These tests were repeated 4 times on each oil before and after roasting to obtain the most representative possible equations calculated by linear regression (Table 1).Standard samples were made at concentrations of 1, 2, 2.5, and 5μg/mL, which is the maximum limit of the detection threshold of the fluorometer (TSP FL3000).2mg of oil were dissolved in 25mL hexane for HPLC, then analyzed directly by HPLC.
A hypersil silica column (250mm x 4,6 x 5μm) coupled with a 100μL injection loop was used for separation.The mobile phase was a mixture of hexane and dioxane 97/3 v/v (flow: 1mL/min).The detection of tocopherols was done using a fluorometer, which made it possible to inject and proportion the oil directly in hexane.Excitation was set at 290nm, thus inducing fluorescence emission by tocopherols at 330nm.

Specific volume
During heating, the pumpkins seeds swell.To measure this swelling, a graduated test-tube was used.The volume occupied by a measured quantity of 75g of seeds was measured in 3 replicates before and after treatment.

Seed roasting
Roasting was carried out in a coffee roaster (Probat TM Type BRZ 2) at 140°C for 5min.Seventy five grams of raw seeds were placed in a rotary metal drum (Diameter 11 cm, length 18 cm, 1.7 Kw) which was heated by electrical resistance.The seeds were simultaneously roasted by contact with the metal plate, and also by the hot air in the drum.Under these conditions, the seeds obtained are generally appreciated by a panel of tasters.

Characterisation of roasted seeds
The water activity A w of raw seed was 0.544 +/-0.002.A w decreased considerably during roasting at 140°C for 5 min to only 0.105 +/-0.002.Thus, in both cases, the seeds were stable with respect to potential growth of micro-organisms (Guiraud, 1998).The roasting of seeds will therefore increase sanitary quality by increasing microbiological stability with time.
Specific volume as well as the weight of the seeds were measured before and after roasting.We calculated the swelling and the loss in weight due to the drying of seeds at 140°C for 5 min.Roasting at 140°C for 5 min caused an increase in volume of almost 43.2 +/-4.4%, and a loss of weight of 6.4 +/-0.24% which can be explained by the drying of seeds.The loss in density was -34.6+/-1.8%.The seed thus acquired the desired puffed-up appearance.

• Calibration
The results were obtained from four repetitions.An average curve of peak areas relating to the four selected concentrations was then plotted using a linear regression calculation.The coefficients of correlation R 2 were close to 1 (Table 1).

• Analysis of Cucurbita pepo seeds
Cucurbita pepo seeds contained 51.0 +/-1% of fat, which is comparable to the data given by Ucciani (1995).
Figure 1 shows the analysis of tocopherol standards and Figure 2 the analysis of the tocopherols from roasted seeds at 140°C for 5min.The seeds had a tocopherol content of 107.4 +/-2.9 mg/100g oil, similar to other oleaginous seeds such as soybean (98mg/100g oil) and maize (81.6mg/100g oil).This content was higher than those of olive (11mg/100g oil), palm (38mg/100g oil) and sunflower (70mg/100g oil) (Kamal-Eldin and Andersson, 1997).Murkovic and Pfannhauser (2000) analysed 15 samples of pumpkin seed oil and found a vitamin E content of between 100 and 600 mg/100g oil.
γ-tocopherol, which represented 89.7% of the total amount of tocopherols detected in the EFFECT OF ROASTING ON TOCOPHEROLS OF GOURD SEEDS (CUCURBITA PEPO)

Figure 1 HPLC analysis of standard tocopherols
Cucurbita pepo seed, was the most significant, just as it is in many other vegetable oils (Table 2 and Figure 2).
Roasting caused a loss in all tocopherols (Table 3).But, the loss was not the same for all the tocopherol molecules.The loss of β-tocopherol was the highest at 50%, for α-tocopherol it was 41%, while for γtocopherol it was 36%.For δ-tocopherol, the loss was less at 25%.
Antioxidant capacity decreases from α-tocopherol to γ-tocopherol (Dziezak, 1986).γ-tocopherol is thus more resistant to oxidation due to ambient air.In the roasted oil, its percentage increased and stayed very high at 90% (Tab.3), whereas α-tocopherol decreased most notably from 7.1 to 6.7%.

CONCLUSIONS
Tocopherols have already been the subject of many studies describing their content in plant materials and some papers describe the effect of physical treatments like frying at 180°C (Barrera-Arellano et al., 2002) and subjecting them to microwave heating (Yoshida et al., 1991;1999), but none on the effect of roasting.
Our results showed that roasting at 140°C for 5min affected the content of tocopherols by preferentially reducing (36%) those with the greatest antioxidant capacity (α-tocopherol: loss of 41%) thus concentrating δ (loss of 25%) and γtocopherols (loss of 36%), which have the least antioxidant capacity (Gottstein and Grosch, 1990).γ-tocopherol which is less active, is more stable at high temperatures than α-tocopherol.compounds during the frying of potato chips (Neff et al., 2003).Barrera-Arellano et al. (2002) showed the same tendency with a quicker loss of αtocopherol than the other tocopherols and a better preservation of δ-tocopherol at frying temperatures.After treatment at 140°C for 5min, the amount of tocopherols in roasted seeds remained significant with 68mg per 100g of seeds (Table 3).Yoshida et al. (1992) showed that the stability of these compounds was connected to the influence fatty acids present in the lipid fraction.When vegetable oils were heated, the levels of free fatty acids increased through hydrolysis of the triglycerides.They also showed that the accumulation of short chain fatty acids which are saturated was linked to the reduction of tocopherols in coconut and palm oils.The lipid fraction of Cucurbita pepo seed contains essentially long polyunsaturated fatty acids (62% of C18:2) (Younis et al., 2000) which protect the tocopherol fraction against oxidation by scavenging free oxygen.
In conclusion, the most sensitive tocopherols are those that are more susceptible to oxidation.However, the differences observed in the loss of tocopherols would be in favour of their oxidative role over their degradation at roasting temperature (140°C).Barrera-Arellano et al. (2002) showed the opposite tendency for the loss of tocopherols during frying at high temperatures and they estimated that the loss is due to the chemical degradation of tocopherol molecules.It could be explain by the difference in the experiment duration.In fact, Barrera-Arellano et al. (2002) studied frying for 2 to 10 hours, which could explain the degradation.In our case, roasting was done only for 5 min and the oxidation, more than the chemical degradation, is certainly the reason for the loss in tocopherols.
It would be interesting to conduct a further study on the combined effect of oxidation on fatty acids and tocopherols and in particular the pro-oxidative or co-oxidative effect of tocopherols when they are subject to roasting temperature as suggested by Kamal-Eldin and Andersson (1997) who proposed the hypothesis that α-tocopherol could be involved in pro-oxidation in oils rich in 18:3 fatty acid.Another instructing technology could be the roasting by microwave tested by Yoshida et al. (1999) who showed a good stability of all tocopherols for 6 to 8 min and a preservation of 80% of tocopherols after 20 min of roasting.
Figure 2 HPLC analysis of tocopherols from roasted Curcubita pepo seeds at 140°C for 5min