Quality characteristics and microbiological safety evaluation of oils extracted from gamma irradiated almond ( Prunus dulcis Mill . ) seeds

Se ha evaluado las caracteristicas fisico-químicas y la descontaminación microbiana de aceites extraídos de semillas de almendras (variedades Misión y Price) gamma-irradiadas a dosis absorbidas de 2-10 kGy. La radiación gamma no ejerce ningún efecto considerable en la composición próximal de las semillas. Las características fisico-químicas tales como la densidad y el índice de refracción de los aceites, extraídos a partir de semillas gamma-irradiadas, permanecieron casi sin afectar; el índice de yodo disminuye mientras que el valor de saponificación, la materia insaponificable y los ácidos grasos libres aumentan. El estado oxidativo y el contenido de tocoferoles de los aceites de almendra se vieron afectados negativamente, mientras que el perfil de ácidos grasos se modifica ligeramente debido al estrés de la radiación. Curiosamente, los efectos sobre los atributos de calidad del aceite fueron más pronunciados a dosis de radiación más altas (> 6 kGy). Además, la contaminación microbiana se eliminó por completo en los aceites irradiados a una dosis absorbida de 6,0 kGy. Se puede concluir a partir de los presentes hallazgos que la radiación tiene un efecto positivo o negativo considerable en algunos atributos de la aceite de almendras. Por lo tanto, se debe aplicar una magnitud apropiada de radiación gamma para el tratamiento de semillas de almendra con el fin de retener los máximos beneficios nutritivos.


INTRODUCTION
It is well known that vegetable oils undergo oxidative deterioration during processing and storage resulting in the formation of hydroperoxides, aldehydes, ketones, and carboxylic acids which decrease the nutritive and organoleptic value of the products (Richardsa et al., 2005;Bhatti et al., 2010).Oxidation not only causes rancidity in oils and lowers their nutritional value; but the the oxidation products formed exhibit harmful effects on the health of consumers (Muik et al., 2005;Azim et al., 2009;Barros et al., 2011;Rohman et al., 2011).
Irradiation treatment can protect food commodities from oxidation, insects infestation and microbial contamination during storage and processing (Yusof et al., 2007;Alighourchi et al., 2008;Thomas et al., 2008;Braghini et al., 2009).Development of this preservation technique is based on the consideration that high energy irradiation might affect minutely the nutritive value of stored food.However, a study of the relationship between radiation absorbed doses and possible changes in the composition of food stuffs must be carried out in order to comprehensively assess the acceptability of irradiated processed foods (Azim et al., 2009).
Exposing foods to ionizing radiation is similar to a heat treatment, either thermal or microwave, that generates minute and mostly undetectable changes in the chemical composition of food due to its selectivity and high efficiency with which it is QUALITY CHARACTERISTICS AnD MICROBIOLOGICAL SAFETY EvALUATIOn OF OILS ExTRACTED FROM GAMMA… present research was undertaken to investigate the physico-chemical and biological characteristics of oils extracted from two locally available varieties of gamma irradiated almond seeds.

Sample collection and gamma irradiation
Two varieties of almond, namely Mission and Price, were obtained from the local dry fruit market and further authenticated by the Department of Botany, University of Agriculture, Faisalabad, Pakistan.The samples were packed in polyethylene bags and then exposed to the gamma-radiation for the doses of 2, 4, 6, 8 and 10 KGy using Cs-137 gamma radiation source at the nuclear Institute of Agriculture and Biology (nIAB), Faisalabad, Pakistan.A non-irradiated sample, kept under the same storage conditions, was used as a control.

Oil extraction
The irradiated and non-irradiated almond seeds (var.Misson and Price) were crushed using an electric grinder and the oils were extracted with n-hexane using a Soxhlet extractor for 7-8 hours in a water bath.After extraction, the solvent was evaporated under vacuum in a rotary evaporator (n-n Series) coupled with an aspirator and a digital water bath SB-651 (Eyela, Rikakikai Co. Ltd., Tokyo, Japan) at 45 °C and the extracted oils were stored at 4 °C until further analyses (Wijeratne et al., 2006).

Proximate seed parameters
After oil extraction from the control and irradiated almond seeds, the residue was subjected to proximate analyses following the standard methods.Protein contents were determined using the Kjeldahl apparatus according to AOAC method (AOAC, 1990).The fiber content was estimated by taking 2 g of defatted ground residual sample.The samples were boiled with 250 mL of 0.25M H 2 SO 4 , followed by the filtration and washing of insoluble residues.The residues were then boiled with 250 mL of 0.313 M naOH, followed by separation, washing, and drying.The dried residues were weighed and ashed at 600 °C using a muffle furnace (Eyela, TMF-2100, Tokyo, Japan); the loss in mass was determined gravimetrically transferred to the orbiting electrons in the atoms constituting food molecules and consequently produce free radicals.The free electrons are rapidly trapped by surrounding atom-forming anions.Most of the absorbed radiant energy is used to generate free radicals and to induce chemical reactions between radicals or between radicals and other molecules.Only a fraction of the absorbed energy is converted into heat, therefore, it is similar to a cold pasteurization technique (Siddhuraju et al., 2002).According to Graham et al., (2002) water is the predominant molecule in all living systems and is the principal primary reactant during radiorization.The free radicals formed in water radiolysis have the ability to recombine with the newly formed radical cations and react with other components in the food matrix to form secondary changes and thus stabilize organic compounds.
Almond (Prunus dulcis Mill.), a member of the Rosaceae family, is cultivated globally, with about 28% of its worldwide production derived from the Mediterranean region (Chen et al., 2005;Wijeratne et al., 2006).Due to its high nutritional value, the almond is incorporated as an important ingredient in various confectionery products.Regular intake of almonds has been known to reduce cholesterol levels and lipoprotein profiles significantly which might be linked to its high-oleic lipids (Martins et al., 2003;Moure et al., 2007;Cordeiro and Monteiro, 2001).Apart from its nutritive value, the almond is reported to contain a wide variety of phenolics and flavonoids which posses interesting biological effects such as sedative, anti-inflammatory, anti-hyperlipidemic, anti-tumour and antioxidant activities (Chen et al., 2005;Milbury et al., 2006;Esfahlan et al., 2010).
Until now there have been no previous studies reported on irradiated almond seeds.As gamma irradiation is investigated to affect the physicochemical characteristics and nutritive quality of several foods; this prompts the need to evaluate the effect of such a treatment on the quality characteristics of almond oil.Besides, the oil extracted from the almond kernel, due to its considerably higher moisture content, is more receptive to microbial contamination and rancidity during storage, therefore, it would be worthwhile to improve its shelflife by using an irradiation process.In this regard, the I.A. BHATTI, M. IQBAL, F. AnWAR, S.A.SHAHID AnD M. SHAHID those of pure standards.The FA composition was reported as a relative percentage of the total peak area.nonadecanoic acid was used as internal standard.All of the quantitative measurements were monitored using a Chromatography Station for Windows (CSW32) data handling software (Data APEx, Pague 5, The Czech Republic).

Tocopherol contents of oils
The tocopherol composition of almond oils (irradiated and non-irradiated) was studied using a high performance liquid chromatograph, model LC-10A series coupled with a liquid pump LS 10AS, a system controller SCL-10A, a Supelco C18 column (250 × 4.6 mm; 5 µm), a fluorescence detector RF-530 and an injector loop of 20-µL (Rheodyne, USA).The column was operated at 30 °C.A mobile phase consisting of a mixture of acetonitrile and methanol (1:1 v/v) at a flow rate of 1.3 mL min -1 was used.A sample for the chromatographic analysis was prepared by dissolving 1 g of almond oil into 2 mL of freshly distilled 2-propanal in a 5 mL sample vial.Tocopherol isomers in the eluent were detected using a fluorescence detector set at an emission wavelength of 325 nm and an excitation of 295 nm.Qualitative and quantitative measurements for the individual tocopherols were performed by comparing the retention time and area of the unknown with those of pure standards of α-, γ-, and δ-tocopherols.

Microbiological analysis of oils
The oil, extracted from non-irradiated and irradiated almond seeds, to the absorbed doses of 2, 4, 6, 8 and 10 kGy, were analyzed for the total bacterial and total fungal count according to the method described by Arici et al., (2007).In each of the three replicate experiments, 1.0 mL oil sample was mixed with 10 mL of 2% autoclaved peptone water.The samples were diluted and plated on agars.nutritional agar (DIFCO, USA) was used for bacterial counts and potato dextrose agar (Oxoid, UK), acidified with 10% tartaric acid, was used for yeast.Total bacterial counts were performed after incubation at 37 °C for 24 hrs and yeast colonies of the non-irradiated and irradiated oil samples were counted after 3 days of incubation at 28 °C.These microbial analyses were performed at the Bioassay Section, Protein Molecular Biology Lab., Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad.

Statistical analysis
Almond seeds (irradiated & unirradiated) were analyzed in triplicate and the results were reported as means ± SD.Statistical significance of the difference between mean values was assessed by AnOvA Staistix 8.1 version (Steel et al., 1992).(AOAC, 1990).For determination of ash contents, 2 g residue, left after oil extraction, were carborized and ashed in a muffle furnace at 600 °C until a constant mass was reached (ISO, 1977).

Physico-chemical characteristics of oils
The physiocochemical parameters such as density, refractive index, iodine value, saponification value, acid value, peroxide value and unsaponifiable matter of the oils extracted form un-irradiated and irradiated almond seeds were measured by IUPAC methods (IUPAC, 1987).

Oxidative status of oils
The oxidative status of the oils extracted from non-irradiated and irradiated almond seeds was evaluated spectrophotometrically.The oil samples were diluted using iso-octane and the absorbance which corresponded to conjugated dienes and trienes was recorded at λ max 232 nm and λ max 268 nm, respectively.Using the absorbance data the extinction coefficients were calculated following the IUPAC method (1987).For the measurement of para-anisidine value, the oil samples were dissolved in iso-octane and made to react with 5 mL of a P-anisidine solution (0.25% w/v in acetic acid) for 10 min (IUPAC, 1987).A colored complex was formed; its absorbance was measured at a wavelength of 350 nm using a double beam spectrophotometer (Cecil, 7200, UK).

Fatty acid profile of oils
The almond kernel oils were analyzed for fatty acid profiles according to the standard method (IUPAC, 1987).The oils extracted (0.2 g) were transmethylated with potassium methoxide by refluxing at 50 °C in a round bottom flask resulting in fatty acid methyl esters (FAMEs).After the transesterification, the mixture was cooled to room temperature and the contents transferred into a separating funnel.A small volume of n-hexane was added into the funnel and the mixture was shaken and then centrifuged for phase separation.The upper FAMEs layer was decanted,washed with distilled water and further dried with anhydrous sodium sulphate.Finally after filtration, the FAMEs recovered were ready for the gas chromatographic analysis.FAMEs were analysed on a SHIMADZU gas chromatograph model 17-A, fitted with an SP-2330 (Supelco) methyl-lignocerate-coated (film thickness 0.20 µm) polar capillary column (30 m × 0.32 mm) and a flame ionizing detector (FID).Oxygen-free nitrogen was used as carrier gas at a flow rate of 3 mL/min.Other analytical conditions were as follow: initial oven temperature, 180 °C; ramp rate, 5 °C min -1 ; final temperature, 220 °C; injector temperature, 230 °C; detector temperature, 250 °C.The FAMEs were identified by comparing their relative and absolute retention times with QUALITY CHARACTERISTICS AnD MICROBIOLOGICAL SAFETY EvALUATIOn OF OILS ExTRACTED FROM GAMMA… the absorbed doses from 2-10 kGy, are given in Table 2.There was no difference for the values of refractive index and density between the irradiated (1.4600-1.4700,0.89-0.90mg mL -1 ) and nonirradiated oil samples (1.4599-1.4700,0.90-0.91 mg/ mL), respectively, indicating that γ-radiation up to doses of 10 kGy did not exert a significant negative effect on these parameters of the oils.Our results are in accordance with the previously reported findings of Bhatti et al., (2010) and Yaqoob et al., (2010) who also did not observe any significant change in refractive indices and densities between the controls and irradiated peanut, sunflower and maize oils.The refractive index and density values depend on thermal degradation and percentage of polar compounds formed during oxidation and hydrolytic reactions (Benedito et al., 2007).
As a result of exposure to gamma radiation, the iodine values of the control almond oils (105.7-110.7 g of I 100g -1 oil ) were found to be substantially decreased to the levels as low as 61.3-79.3g of I 100g -1 oil (the values for samples exposed to 10 kGy dose).Generally, at higher doses the decline in the oils' iodine value was more remarkeable.The decreasing trend in the oil iodine value upon irradiation in this study might refer to the saturation of the oil as a result of the breakdown of double bonds due to oxidative deterioration in the fatty acids.A similar decreasing trend in iodine value has already been seen (Al-Bachir, 2004;Anjum et al., 2006;Bhatti et al., 2010;Yaqoob et al., 2010).The oils saponification values increased upon irradiation (an increase from 185-187 to 204-231 mg KOH g -1 of oil) which indicated that large original molecules of oils containing long-chain fatty acids degraded to smaller molecules as a result of oxidation and cleavage of bonds (Agatemor,

RESULTS AND DISCUSSION
The proximate composition (oil, fiber, moisture, ash and protein contents) of unirradiated and irradiated almond (var.Mission and Price) seeds is given in Table 1.The irradiation up to 10 kGy did not show any significant (p < 0.05) effect on the proximate composition (oil, fiber, moisture, ash and protein contents) of almond (var.Mission and Price) seeds (Table 1).The contents of oil and protein of the unirradiated (control) and irradiated almond seeds, with contributions of 40.0-40.1,39.8-40.4% and 22.7-24.8,22.4-24.7%,respectively did not vary significantly between the treated and untreated samples.Similarly, the levels of moisture (7.3-8.2%),ash (4.2-4.8%) and fiber (5.3-5.6%) in the unirradiated seeds was comparable with those (7.3-8.3%,4.1-4.8% and 5.2-5.6%) of the irradiated ones revealing no considerable vartaions between the two types.
Our results, related to the proximate analyses, are consistent with previous reports which also reveal non significant difference in moisture, fat, ash and protein contents between irradiated and unirradiated almond seeds (Al-Bachir, 2004).Similarly, Bela et al., (2008) also reported that the protein and crude fiber contents of almonds did not change after irradiation.In agreement with our present results, Bhatti et al., (2010) and Yaqoob et al., (2010) also determined that gamma irradiation (2-10 kGy) did not affect the lipids, protein, fiber and ash contents of either sunflower nor maize seeds significantly (p < 0.05).
The refractive index, density, iodine value, saponification value, unsaponifiable matter and free fatty acid contents determined for the oils produced from unirradiated and irradiated almond seeds, to irradiation was observed in the Pv of different lipids (Hampson et al., 1996, Al-Bachir, 2004;Bhatti et al., 2010) which might be linked to the breakdown of primary oxidation products including hydroperoxide into smaller stable fragments such as carbonyl compounds, alcohols and hydrocarbons.Peroxide value characterizes the quantity of peroxides formed in the oils as intermediates of oxidative reactions after irradiation (Uquiche et al., 2008).Peroxide value characterizes the quantity of peroxides formed in the oils as intermediates of oxidative reactions after irradiation (Uquiche et al., 2008).The effect of γ irradiation (up to 10 kGy) on the para-anisidine value (an increase from 20.10 to 23.50) in this study was found to be significant in accordance with the findings of Yaqoob et al., (2010).The extinction coefficients corresponding to λ max 232 nm and λ max 268 are related to the conjugated diene and trienes, respectively.The magnitude of these oxidation products is reflected as purity index and depicts the oxidative degradation of oil.These values were affected slightly at low radiation doses but increased rapidly at doses higher than 6 kGy.The oxidative stability of oils predicts their resistance to the formation of conjugated dienes, trienes and peroxides.Conjugated diene and triene values correspond to bond shifting as a result of oxidation (Deiana et al., 2002).The increase in the conjugated diene and triene contents for both varieties of almond seed oils observed in the present investigation might be linked to the lipid oxidation caused by irradiation (Bhatti et al., 2010).Table 4 depicts the values of tocopherol homologues of the oils extracted from unirradiated 2006).Similarly, in the case of unsaponifiable matter, an increasing trend was observed with gamma radiation absorbed doses.These results are in accordance with Yaqoob et al., (2010) who reported an increase in unsaponifiable matter in sunflower and maize oil extracted form gamma irradiated seeds.In our experiments, the highest unsaponifiable matter (0.52-0.76%) was observed for the samples irradiated to 10 kGy, which might be due to the existence of high contents of hydrocarbons, sterols and triterpenols at this stage (Uquiche et al., 2008).The increase in free fatty acids from 1.11-1.61%(for control oils) to 1.34-1.90%for oils extracted from γ-irradiated almond seeds, to a final dose of 10 kGy, might be due to slight and random hydrolysis of triglycerol molecules to free fatty acids and diacylglycerols (Al-Bachir, 2004;Boonchoo et al., 2005;Anjum et al., 2006;Badr, 2006).
The results regarding oxidative status of the oils produced from γ-irradiated and control almond seeds of both varieties are shown in Table 3. Irradiation significantly increased (an increase from 2.26-3.46 to 5.18-6.21meq O 2 kg -1 oil) the peroxide value (Pv) of the oils tested which might be attributed to the excessive formation of hydroperoxide as a result of oxidation, dehydration and polymerization reactions due to the interaction of γ radiation with fat molecules (Evren and Gulden, 2008).Our results are in accordance with Badr, ( 2006) and Yusof et al., (2007) who also observed an increase in the peroxide values in gamma irradiated egg yolks and coconut oil samples.However, few previous reports are available where no significant increase upon Mean ± SD calculated from three replicates.The means with different superscript letters within the same row vary significantly (P < 0.05) among radiation doses.Control (non-irradiated sample).
QUALITY CHARACTERISTICS AnD MICROBIOLOGICAL SAFETY EvALUATIOn OF OILS ExTRACTED FROM GAMMA… the amounts of total tocopherols of irradiated sunflower, maize and peanut oil up to absorbed doses of 10 kGy.The decreasing trend in the tocopherol contents of irradiated oil samples might be linked to the degradation of these antioxidant componenets during irradiation.Furthermore, due to the thermal oxidation of the oil, the tocopherol values may also decrease (Yaqoob et al., 2010).
The effect of gamma radiations on the tocopherol contents of the oils from both almond varieties was similar.Previous studies conducted by Lalas et al., (2007), Lakritz and Thayer (1994), and Lakritz et al., (1995) showed that there was a slight decrease in tocopherol contents of seed oils, fresh chicken breast muscle, and red meat, respectively with an increase in gamma radiation doses.While another study on cooked minced chicken showed that there was no considerable and γ-irradiated almond seeds to the absorbed doses of 2-10 kGy.There was a noticeable difference in tocopherol contents (α, γ and δ) of the oil samples derived from irradiated verses non-irradiated (control) seeds.The values of α-tocopherol in unirradiated almond oils were predominant (436 mg kg -1 and 485 mg kg -1 ) in Mission and Price variety, respectively).Alphatocopherol contents were higher in the case of the non-irradiated Price verity as compared to Mission.Meanwhile, γ-and δ-tocopherols were detected in small amounts with contributions of 8.81-8.95mg kg -1 and 2.32-2.80mg kg -1 , respectively in both the varieties of almond oils.Overall, the tocopherol values were decreased by increasing the irradiation dose and the trend was in accordance with the studies of Bhatti et al. (2010) and Yaqoob et al., (2010) who studied a decreasing trend in   (2010) who reported that the effects of irradiation on the fatty acid composition of sunflower oil showed a significant (p < 0.05) change in the amounts of stearic, oleic and linoleic acids, while the concentration of palmitic acid was unaffected even at 10 kGy.
The effect of gamma irradiation on the microbial inactivation of oils extracted from irradiated and unirradiated seeds of both varieties of almond are shown in Table 6.The bacterial populations in nonirradiated oil samples were 4.30 × 10 3 CFU/g and 3.87 × 10 3 CFU/g while the fungal spores 3.87 × 10 2 /mL and 3.87 × 10 2 mL -1 for Mission and Price varieties, respectively.After radiation treatment of 2 kGy the bacterial load was reduced to 3.91 × 10 2 CFU g -1 for Mission variety and 3.21 × 10 2 CFU/g (Price) and the fungal count levels were 3.45 × 10 1 spores mL -1 (Mission) and 2.84 × 10 1 spores mL -1 for Price variety.A significant reduction of microbes was observed at an absorbed dose of 4 kGy whereas at 6 kGy irradiation increasingly hampered the microbial growth and no population wentundetected.Similar results as observed in the present investigation were reported by Thomas et al. (2008), who studied colony formation in black tea irradiated up to 10 kGy absorbed dose.Likewise, Alighourchi et al., (2008) reported a progressive decrease in the microbial load of pomegranate juice irradiated to 0.5-10 kGy.

CONCLUSIONS
The results of this study showed that gamma irradiation up to an absorbed dose of 6 kGy did effect of irradiation on the tocopherol contents up to 4 kGy (Galvin et al., 1998).Furthermore, the concentration of tocopherols depends on genotype, cultivar traits as well as agroclimatic conditions of the harvest (Kodad et al., 2011).It is known that α-tocopherol along with tocotrienols (vitamin E) are important liposoluble metabolites and due to their strong anti-oxidative effects, they retard the oxidation of unsaturated fatty acids in foods and biological systems.The most active form of vitamin E in vivo is α-tocopherol, while γ-tocopherol is an active form of vitamin E in vitro (Gimeno et al., 2000;Uquiche et al., 2010).Suhaj et al., (2006) reported that antioxidant activity was influenced by radiation treatment which can be attributed to the degradation and peroxidation of unsaturated fatty acid in the oils (Hassanein et al., 2003).Lalas et al., (2007) also reported a decrease in the total tocopherol contents of sunflower and soybean oils after irradiation at higher doses, while no significant change for tocopherol concentration was observed at lower doses.Similar results have been reported earlier in case of irradiated peanut, sunflower and maize seeds oils (Bhatti et al., 2010;Yaqoob et al., 2010).The contents of α-, γ-and δ-tocopherols were slightly affected in the oils extracted from peanut, sunflower and maize seeds irradiated up to 6 kGy, however, the effect was comparatively pronounced at higher dosages.
Radiation-induced changes in the fatty acid profile for Mission and Price varieties of almond seeds are shown in Table 5.The effect of γ radiation (dose 2-10 kGy) on fatty acid composition was found to be almost insignificant for palmitic acid (16:0), the content of stearic acid (18:0) and oleic acid (18:1) increased, while the concentration of linoleic acid (18:2) decreased significantly (p < 0.05) by increasing the absorbed dose.The increasing trend for stearic acid (an increase from 2.18-2.37 to 2.88-3.00%)and oleic acid (an increase from 69.51-70.00 to 70.25-71.5%)not significantly alter the routine physiochemical characteristics of the almond (Mission and Price verity) oils, while the microbial load was nullified completely at this treatment level.The fatty acid and tocopherol profiles as well as the oxidation status of the irradiated oils were negatively affected especially at higher irradiation doses (> 6 kGy).It can be concluded that higher gamma irradiation doses might lead to the deterioration of some valuable components such as tocopherols and essential fatty acids in the oils.In order to preserve the almond seed oil from disinfection as well as from some other quality-oriented deteriorative effects, an appropriate gamma irradiation treatment should be sought.

Table 1 Effect of gamma irradiation on proximate composition (%) a of almond seeds of different varieties Contents Variety Radiation doses
Mean ± SD calculated from three replicates.a The mean values of oil, moisture, protein, fiber and ash within the same row are non-significantly (P < 0.05) varied among radiation doses.Control (non-irradiated sample).I.A. BHATTI, M. IQBAL, F. AnWAR, S.A.SHAHID AnD M. SHAHID

Table 4 Effect of gamma irradiation on tocopherols content of oils extracted from almond seeds of different varieties Tocopherols (mg/Kg) Variety Radiation doses
.A. BHATTI, M. IQBAL, F. AnWAR, S.A.SHAHID AnD M. SHAHID and decreasing trend of linoleic acid (a decrease from 22.0-22.10 to 20.05-20.90%)mightbe due to the preferential cleavage of double bonds.Radiation treatment caused the saturation of double bonds of linoleic acid which increased with the absorbed dose.Our findings are in agreement withYaqoob et al., I

Table 5 Effect of gamma irradiation on the fatty acidS profile (g/100 g FA) of oils extracted from almond seeds of different varieties
a Mean ± SD calculated from three replicates.The means with different superscript letters within the same row vary significantly (P < 0.05) among radiation doses.Control (non-irradiated sample).