Food Chemistry 154 (2014) 230–237

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Gamma radiation effects on microbiological, physico-chemical and antioxidant properties of Tunisian millet (Pennisetum Glaucum L.R.Br.) Maha Ben Mustapha a,⇑, Mehrez Bousselmi b, Taïeb Jerbi b, Nasreddine Ben Bettaïeb b, Sami Fattouch a a b

LIP-MB, National Institute of Applied Sciences and Technology (INSAT), University of Carthage, Tunisia National Center of Sciences and Nuclear Technologies of Tunis (CNSTN), Pôle Technologique, B.P. 72, Sidi Thabet 2020, Tunisia

a r t i c l e

i n f o

Article history: Received 22 June 2013 Received in revised form 6 January 2014 Accepted 7 January 2014 Available online 13 January 2014 Keywords: Gamma irradiation Millet D10-value GC-FID Ochratoxin A Antioxidant activities

a b s t r a c t Hygienic quality of Tunisian pearl millet flour is always of major concern to consumers as well as all involved in the production, processing and distribution sectors. In the present study, the microbiological and biochemical properties of this food were examined following gamma-radiation. The D10-values for the Total Aerobic Plate Count, yeasts and moulds were respectively 1.5 and 3.7 kGy. Furthermore, millet flour is commonly susceptible to mycotoxin contaminations, so the Ochratoxin A residues were also investigated; a reduction of 74% was observed with 10 kGy. Moreover, the radiation process did not significantly alter fatty acids composition of the millet flour as obtained with Gas chromatography-flame ionisation detector technic. The peroxide value had increased from 26.16 to 34.43 meq O2/kg with 3 kGy. At 1 kGy, we noticed an important loss of vitamin A of about 88.6%. In contrast, the total phenolic content, the ABTS-RSA and the DPPH-RSA of the radiated millet flour exhibited non-significant changes (p < 0.05). Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Although Africa has only 9% of the totality of production of coarse grains, it presents the monopoly of production of the Millet and the Sorghum with 49% and 38.4% respectively of the world production of these two cereals (FAO, 2004). Despite the most part of the millet (Pennisetum Glaucum L.R.Br.) available in Tunisia comes from the imports, the country produces small quantities as a secondary cereal after wheat and barely. The region of Kairouan, in the centre of Tunisia, is the main contributor to the local production (50%), followed by Medenine (26%) in the south, Nabeul (15.6%) and Bizerte 4.7% in the north and Mahdia 3.4% in the Sahel area (FAO, 2004). The nutritional values of millet indicate that it is a good source of energy (Gassem & Osman, 2008), that’s why it’s used to prepare some Tunisian dishes (Drôo, Bouza, Cake, Traditional sweets, Berkoukech . . .). Nevertheless, hygienic quality of this food is always of major concern to consumers as well as all involved in the production, processing and distribution sectors. Particularly, storage in

Abbreviations: TAPC, Total Aerobic Plate Count; OTA, Ochratoxin A; GC-FID, Gas chromatography-flame ionisation detector; PV, peroxide value; TPC, total phenolic content; RSA, Radical Scavenger Activity. ⇑ Corresponding author. Tel.: +216 71718288; fax: +216 71704532. E-mail address: [email protected] (M. Ben Mustapha). 0308-8146/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2014.01.015

bulk or in small packed quantities of millet during the seasonally massive agricultural production of cereals is still raising awareness of public authorities and industrialists due to fungal contaminations and mycotoxin problems (ISO Standard 6322-1, 1996). Therefore, there is continuously a need for innovative preservative technologies and sustainable trade and commercialisation practices. Several methods have been tried for microbial decontamination of various foods and feed products such as fumigation with ethylene oxide or methyl bromide and radiation with gamma rays. This method, among the safer and promising effective processes, is used to achieve some beneficial effects including disinfestations, improvement of shelf life and safety of millet flour by reducing the microbial load (Codex Committee on Food Additives & Contaminants, 2001) which requires doses in the range of 1–10 kGy (Aziz Nagy, Souzan, & Shahin Azza, 2006). It consists in exposing for a determined time period the packed or in bulk food products to control and optimise amounts of ionising radiations. This process prevents the growth of microorganisms and slows down the maturation of some fruits and vegetables by inhibiting biochemical reactions in the physiological processes of maturation (Singh & Pal, 2009). The radiation process should not have toxicological side effects such as the increase of the normal level of the radioactivity or the degradation of the organoleptic properties and the nutritional contents of the food (International Atomic Energy Agency, 1999). Thus, to the best of our knowledge, the purpose of this

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physical treatment applied to the food field is to improve the nutritional, organoleptic quality and lengthen the shelf life of various products. Hence, the exposure time and the absorptive radiation amount must be sufficient to reach the required biological effects without deterioration of the food qualities, being suitable for human use and commercialisation (International Atomic Energy Agency, 1999; Khattak, Simpson, & Ihasnullah, 2009). In the current study, to evaluate the potential industrial application of the radiotreatment, yeasts and moulds, as principal contaminants of cereals, were followed to guarantee a weak microbial load and confirm the effectiveness of this process for a long-term storage (Hsiao-Wei, Hsiao-Ping, Fong-In, I-hsin, & Po-Chow, 2006). In addition, the influence of the ionising radiations’ amount (1–10 kGy) on Ochratoxin A (OTA), a mycotoxin synthesized and released by Aspergillus and Penicillium fungi, was investigated to determine whether this process can bring a solution to this toxicological problem for the Tunisian millet and, subsequently, for other cereals and derivatives. Moreover, the effect of various radiation doses on the biochemical properties was also achieved to bear out the nutritional quality preservation of the radiated samples. This study is part of a national program (RAF/8/024) designed to investigate food products preservation using innovative technologies. 2. Materials and methods 2.1. Millet samples Millet (Pennisetum Glaucum L.R.Br.) samples were hand harvested from local farmers in the Kairouan governorship (Marguellil area). Collected grains were powdered vigorously using an electric grinder to get the millet’s flour. The obtained farina was packed hermetically in polyethylene bags (500 g each lot) prior to radiation treatments and storage. 2.2. Gamma-radiation process The millet flour samples were radiated in a Cobalt-60 irradiator with an activity of 47.582 Curie (CNSTN, National Center of Sciences and Nuclear Technologies – The unit of radiotreatment, Sidi Thabet, Tunis) which is a pilot scheme carried out within the framework of the cooperation of Tunisia with the IAEA and the CEA (France). Two protocols of gamma-radiation were adopted and realised at 25 °C and at 1 atm. The first protocol was undergoing for microbiological and biochemical analyses. Samples were irradiated with 1, 2, 3 and 5 kGy which needs respectively 38min49sec, 77min38sec, 116min27sec and 194min5sec. With a first cartography performed using Gammachrome dosimeters, the index of heterogeneity was 1.09 and the dose rate was 43.3 Gy/ min. The second protocol which undergoes the follow-up of the presence of the OTA in the millet flour was done with a second cartography using the Amber Perpex Batch NR dosimeters (index of heterogeneity 1.18 and dose rate 25.76 Gy/min). Samples were irradiated with 1, 3, 5 and 10 kGy which needs respectively 23min5sec, 69min17sec, 115min28sec and 230min56sec. 2.3. Microbiological analyses 2.3.1. Total mesophile flora, yeasts and moulds This analysis is carried out referring to the standard norms for the Total Aerobic Bacteria (TAPC) (ISO 4833, 1991) and for yeasts and moulds (NF ISO 7954, 1987). The counting of the colony formation units (CFU) per milliliter of suspension was performed after incubation at 30 °C during 72 h for the TAPC or at 25 °C during 5 days for yeasts and moulds.

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2.3.2. Microbial growth during storage Stored radiated (1, 2, 3 and 5 kGy) and non-radiated samples for 30 days at room temperature (25 °C) with a relative humidity of 60% were investigated. The TAPC, yeasts and moulds were determined following the above described protocols. During the storage process the water activity (Aw) of the different samples was measured. 2.3.3. Determination of radiation D10-values The radiation D10-value was defined as the dose required to achieve a 90% reduction in viable microorganisms, so to inactivate one logarithmic cycle of the initial population. To calculate the radiation D10-values, the kinetic of bacteria destruction by irradiation was evaluated by derived linear regression whose equation is:

Log10 ðCFUÞ ¼

 

 1  Radiation dose þ b; for the type y D10

¼ ax þ b D10-value was determined by calculating the negative reciprocal of the survivor curve slope (Ayari, Dussault, Millette, Hamdi, & Lacroix, 2009). 2.4. Water content and Aw Referring to the standard norm (NT 51-21, 2002 and ISO 712, 1998), the water content in the examined samples was deduced from the difference of the initial weigh (5 g) and the final weight after drying at 130 °C during 90 min. The water activity (Aw) was measured using a calibrated Aquaspector-1-NAGY (Multilab, Ariana). 2.5. Extraction and quantification of Ochratoxin A (OTA) The effects of gamma radiation on the OTA mycotoxin were studied. The extraction and quantification of the OTA was done from 20 g of Millet flour. Different samples were examined (in triplicate): (i) non-radiated samples which were doped with 1 mL of a 250 ng/mL solution of pure OTA; (ii) radiated (1, 3, 5 and 10 kGy) samples which were doped with the same amount of pure OTA as (i); (iii) control non-radiated and non-doped samples to check for intrinsic OTA amount. Referring to standard norm NT ISO 15141–1, the Immuno-affinity ‘‘OchraPrep IAC’’ columns were used for the purification of OTA. The OTA was quantified by chromatographic reverse phase HPLC. This quantification was performed with a fluorescence Varian Prostar detector at kexcitation = 334 nm and kemission = 465 nm by comparison with reference standards. 2.6. Fat content and peroxide value In accordance with the standard norm NF V04-402 (1968), the extraction of the total fat content of 5 g millet flour was carried out using the Soxhlet method. The determination of the peroxide value (PV) was carried out referring to the standard norm NF T60-220 (10-1995). The peroxide value is expressed in meq O2/ kg. The profiles of the fatty acids were analysed by gas chromatography (GC) in accordance to the standard COI T20 DOC (N° 24). The fatty acids were detected at 250 °C with FID detector according to their retention times and quantified in reference to standards calibration curves. 2.7. Vitamin A extraction and quantification According to the method of ‘‘Analytical methods for vitamins’’ (AOAC, 1984), 40 g of millet flour were supplemented by 150 mg

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of hydroquinone and 120 mL of ethanol 95%. Extraction of vitamin A with diethylic ether was monitored by addition of KOH 50% (w/v) in a rotavap at 80 °C during 30 min three times to be finally recuperated in ethanol. A volume of 50 lL of the vitamin A extract was analysed by HPLC using Nucleosil 100-5C18 column (flow: 1 mL/ min and solvent phase: Methanol/H2O (98/2)) and detected at k = 326 nm with a retention time of 6 min. The results were expressed in UI/kg of flour as mentioned in the referred method knowing that 1 UI of vitamin A corresponds to the activity of 0.3 lg of retinol. 2.8. Amino-acids analysis The qualitative and quantitative analysis of Phenylalanine, Proline, Glutamic acid and Valine amino-acids is done by reverse phase HPLC according to the Agriculture Materials ‘‘Determination of amino-acids in feed’’. This analysis was carried out with C18 Hypercil ODS column. A Derivatization step of these amino acids was done by phenylisothiocyanate (PITC). The purified aminoacids were eluted with Na2HPO4 pH 6.5 and H2O/Acetonitrile/ Methanol (10/45/45) buffers and then detected by fluorescence detector using kexcitation = 340 nm and kemission = 450 nm. 2.9. Total polyphenols content Each sample (1 g) was thoroughly mixed with 10 mL of cold acetone/water (7:3 v/v). The mixture was sonicated for 20 min and centrifuged at 10000g for 15 min at room temperature. The supernatants were collected, pooled, and concentrated using a rotary evaporator (60 °C) to a final volume of 3 mL. To prevent oxidation of the polyphenols, extraction was achieved rapidly and extracts were immediately used or stored at 20 °C until further use. The total phenolic content (TPC) of millet extracts was evaluated using the Folin–Ciocalteu colorimetric method according to Singleton, Orthofer, and Lamuela-Raventos (1999) and expressed as mg/g catechol equivalent. Absorbances at 725 nm were recorded after 1 h incubation at 25 °C. 2.10. Antioxidant activity The antioxidant activity has been evaluated for its free radical scavenging activity with two assays; DPPH and ABTS+. The free radical scavenging activity using the free radical DPPH was evaluated by measuring the decrease in absorbance at 517 nm as previously reported (Espin, Soler-Rivas, & Wichers, 2000). The reaction started by adding 25 ll of the corresponding homogenate to the cuvette containing 80 lM (methanolic solution) (975 ll) of the free radical (DPPH). The resulting violet coloured mixture was read at 517 nm after baseline correction with methanol with a Boeco S22 UV–Visible spectrophotometer (Germany). Radical scavenging activity was expressed as the inhibition percentage and was calculated using the following formula:

  Abssample  100 Radical scavenging activity ð%Þ ¼ 1  Abscontrol where Abscontrol refers to the absorbance of DPPH without sample added at 517 nm. The first reaction of ABTS+ assay started by adding 50 ll of the corresponding phenolic millet extracts to the cuvette containing 32 lM (950 ll) of the free radical (methanolic solution) (ABTS+). The disappearance of ABTS2+ was determined by measuring the decrease in absorbance at 734 nm for 10 min at room temperature in the above described spectrophotometer. The experiments were always performed on freshly made up solutions. In addition, calibration curves were made for each assay

using Trolox as standard. The antioxidant activity (DPPH and ABTS+) was expressed as Trolox equivalent antioxidant activity (TEAC). 2.11. Statistical analysis All determinations were conducted in triplicate of independent experiments. All results were calculated as mean ± SD (standard deviation). Statistical analyses were carried out using StatGraphics 4.1 version software, using one way ANOVA-analysis of variance. Significant differences between values were presented at a significance level of p < 0.05. 3. Results and discussion 3.1. Effect on the microbiological properties Microbial decontamination was performed with several methods for various foods and feed products. Irradiation has been found a promising method for this purpose and has been approved by international organisations, namely FAO, IAEA and WHO (Ferreira-Castro et al., 2007). The viability of microorganisms in millet flour, following irradiation with different gamma-ray doses of 0, 1, 2, 3 and 5 kGy, was evaluated. In this work, irradiation reduced the bacterial, yeast and moulds populations in a dose-dependent manner and during storage time (Fig. 1). TAPC were generally much higher than counts for moulds and yeasts in all examined samples. The TAPC was initially 8.975 ± 0.05 log CFU/ml and the yeast and moulds was 3.823 ± 0.13 log CFU/ml on non-radiated millet flour sample. It decreased with increase of radiation dose as demonstrated in Khattak et al. (2009) report. Radiation dose of 5 kGy appeared to be sufficient for decreasing bacterial, yeast and moulds counts; TAPC and TYM show a decrease of 54.62% and 63% respectively, compared to the samples non-radiated. The D10-value, the decimal reduction dose, calculated for the first 90% micro-organisms reduction with radiation process, is calculated over the linear part of the death logarithmic curve (log N = log N0  a  Dose). The D10-value is equal to 1.27 kGy for TAPC and to 2.08 kGy for TYM. After one month of storage, millet which had received 1 kGy showed a TAPC decrease of 0.993 log CFU/ml, while the decrease was about 1.18 log CFU/ml following 5 kGy. However, it was observed that a period of one month after gamma radiation was adequate to reduce fungal counts (moulds and yeasts) from 2 to 0.301 log CFU/ml respectively from 1 to 5 kGy. These results were in agreement with earlier observations (Maity, Kar, Banerjee, Chakraborty, & Santra, 2009) showing that irradiation reduce the counts of aerobic mesophilic bacteria and fungi and prolonged the storage shelf-life of samples. The ionising radiations involve important metabolic dysfunctions which can kill microbial cells. Their action can also appear on progressive loss of the multiplication capacity. The Aw parameter could provide longer storage life knowing that the absolute limit for microbial growth is >0.6 (Adams & Moss, 1995). 3.2. Ochratoxin A (OTA) analyses Ochratoxin A, produced mainly by Aspergillus ochraceus or Penicillium verrucosum, is detected in several cereals as corn, barley, millet, etc. Many reports show that radiation treatment is a suitable method for decontaminating foods from fungi and mycotoxins. The effect of the ionising radiations on OTA appears very important (Herzallah, Alshawabkeh, & Al Fataftah, 2008). In our study, gamma radiation has effectively inhibited fungal growth

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9 log CFU (TAPC)

8

log CFU (TAPC) aer 1 month

y1 = -0.788x + 7.7465 R² = 0.9527 D10 = 1.27 kGy

7

log CFU (TYM) log CFU (TYM) aer 1 month

log CFU

6 5 4 y3 = -0,4787x + 3,6579 R² = 0.9444 D10 = 2.08 kGy

3

y2 = -0,6803x + 6,3445 R² = 0.9939 D10 = 1.47 kGy

2 y4 = -0,4537x + 2,4923 R² = 0.9767 D10 = 2.2 kGy

1 0 0

1

2

3

4

5

6

Radiaon dose (kGy) Fig. 1. Total Aerobic Plate Counts (TAPC), Total Yeast and Moulds (TYM) plate counts in un-irradiated and irradiated millet flour (log CFU/ml ± SD).

Table 1 Content of the residual OTA after irradiation of the flour of millet and the percentage of its amount-dependent elimination.

* **

Samples

Radiation dose (kGy)

Ochratoxin A (lg/kg)

A* B** C** D**

0 1 3 10

651.7 568.25 367.77 170.5

Contaminated (with 1 mL of pure OTA solution 250 ng/ml) and not irradiated. Contaminated with the same solution and irradiated.

and reduced the concentrations of ochratoxin A in millet flour samples (Table 1). The residual OTA concentration in the different samples, unirradiated and irradiated, was measured by reverse phase HPLC. The control sample, non-radiated and non-contaminated, had a concentration of 484.12 lg/kg OTA as a result of existing moulds in the sample which could be responsible of mycotoxin production. According to the FAO (2003), which precise that the maximal limit is 5 mg/kg, this concentration remains endurable. To assess the effect of c-radiation on OTA in millet flour, samples were contaminated by 1 mL of pure Ochratoxin solution 250 ng/ ml. The artificial contaminated and unirradiated sample shows higher concentration, 651.7 lg/kg OTA, but always under the maximal limit of tolerable OTA in cereals. The concentrations of OTA found in the irradiated samples were inversely proportional to the radiation dose, and shows a dose-dependent effect. The levels of OTA were reduced approximately by 13% and 44% with 1 kGy and 3 kGy radiation dose respectively. With 10 kGy, the levels of OTA were reduced by 74%. According to FAO (2003) and World Health Organization (1999), a dose of 10 kGy is the maximum dose to apply for cereals. However, Ferreira-Castro et al. (2007) reports that 10 kGy was not sufficient for a complete elimination of fumonisins. In this work, a radiation dose of 10 kGy is not sufficient for complete elimination of OTA in millet flour. Obviously, the radiolysis of water has an important role in the destruction of mycotoxin. This phenomenon produces highly reactive free radicals which could interact with OTA by producing molecules with lower biological activity or degrade OTA. The water content remains roughly stable after gamma radiation, with an average of 10.7%, but, the water activity Aw was above 0.65 and decreases after one month storage to 0.55. Thus, our result could offer a very important solution for mycotoxins decontamination in Tunisian cereals without toxicological hazard or special nutritional or microbiological problems.

3.3. Effect on physical properties After gamma radiation and during one month of storage, the temperature, the relative humidity (RH), the pH and electric conductivity were controlled. These parameters have an important effect on microbiological analyses, especially for fungi (ISO Standard 6322-1, 1996). The temperature of the storing local and of the millet flour samples was stable about 25 °C. The RH decreases slightly from 50% to 46% in the millet flour samples, whereas it increases from 56% to 65% after one month of storage. The pH measurements were carried out to evaluate the radio-induced acidity in the millet flour. The initial hydrogen potential of the unirradiated sample was 6.54. When, the millet flour samples were irradiated, a non-significant decrease was observed from 6.54 to 6.47 with a dose of 1 kGy and to 6.42 with a dose of 5 kGy. The pH values remained unaffected by gamma irradiation treatment up to 5 kGy. The results are in good agreement with the results earlier reported by Khattak (2012). Any modifications could be observed due to the slight water radiolysis which releases the protons H+ and/or to the oxidation of the lipids which releases the fatty acids, or microorganism development. The electric conductivity (EC) increases from 0.61‰ to 0.65‰ equivalent NaCl and shows an average of 0.64‰ after gamma radiation. Increase in EC values may be due to rise in the ions levels resulted from the mineralisation phenomenon; breakdown of bigger molecules into smaller ions. In accordance to our results, Khattak (2012) reported that the EC of radiated samples were higher than that of the non-radiated ones. 3.4. Effect on biochemical properties 3.4.1. Peroxide value and fatty acids composition The peroxide value (PV) is determined to quantify the produced free peroxides (especially hydroxylated radicals and hydroperoxides). The Table 2 provides results for PV (meq O2/kg) determined on lipids extracted from millet flour samples exposed to gamma radiation. The data show the mean values and the standard deviations for each sample in triplicate. PV increased from an initial value of 26.16 to a value of 34.43 meq O2/kg respectively from the non-irradiated sample to a sample radiated with 3 kGy dose. It is obvious that increasing doses of irradiation enhanced lipid peroxidation and represented peroxide formation. With a dose of 5 kGy, the PV show a decrease to 17.8 meq O2/kg which could be related to the simultaneous formation and destruction of hydroperoxides for the production of secondary oxidation products at intermediate

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Table 2 Changes of major fatty acids (mean percentages) of total lipid extracted from radiated millet flour and peroxide value after radiation at 0, 1, 3 and 5 kGy. Radiation dose (kGy)

Peroxide value (103 meq O2/kg)

Palmitic acid (C16:0)

Stearic acid (18:0)

Oleïc acid (C18:1)

Linoleïc acid (C18:2)

Linolenic acid (C18:3)

SFA

MUFA

PUFA

0 1 3 5

26.16 27.6 34.43 17.8

17.64599 18.20708 18.94643 19.22372

3.8964 4.05033 4.21997 4.55029

30.82598 27.87688 27.53217 27.43247

45.78596 46.89893 47.0119 47.09483

1.84567 2.18188 2.46682 2.30631

21.54239 22.25741 23.1664 23.77401

30.82598 27.87688 27.53217 27.43247

47.63163 49.08081 49.47872 49.40114

SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid.

Table 3 Effect of gamma radiation doses on vitamin A content in millet flour. Radiation dose (kGy)

Vitamin A (lg retinol/kg)

0 1 2 3 5

34.2 3.9 40.2 15 0.6

radiation doses (Anwei et al., 2011). It could be also attributed to breakdown of primary initiation products of oxidation including peroxides (Harrison & Were, 2007). It has been reported that non-radiated samples showed lower PV than those of irradiated at 5 and 10 kGy, and PV in radiated samples of chilled pork was dose-dependent and increased with the irradiation dose (Anwei et al., 2011), whereas other investigators (Hampson, Fox, Lakritz, & Thayer, 1996) said that peroxide values show no dependence on radiation doses.Indeed, the polyunsaturated fatty acids (PUFA), contained in the lipidic fraction of the millet flour, when exposed to the gamma radiations, react with radio-induced free radicals and produce lipidic peroxides. This lipid peroxidation phenomenon is a complicated autocatalytic process which leads the production of aldehydes, ketones, cyclic peroxides and volatile fatty acids (Bakalivanova, Grigorova, & Kaloyanov, 2009). The major fatty acids (FA) extracted from the lipidic fraction of the millet flour samples were palmitic acid (C16:0) and stearic acid (C18:0) as saturated fatty acid (SFA), oleic acid (C18:1) as monounsaturated fatty acid (MUFA) and linoleic acid (C18:2) and linolenic acid (C18:3) as polyunsaturated fatty acid (PUFA). The results of the GC analysis shown in Table 2 represent an abundance of PUFA, particularly of linoleïc acid with an average of 46.7%, while the SFA represented only 22.7%.Linolenic, linoleic, stearic, palmitic acids increase very slightly with increasing of gamma radiation dose with no significant differences between the values of control and radiated samples. It is possibly due to partial triglycerides degradation

35

which releases some fatty acids with radiation process. The free radicals OHand/or H fit in the glycerol-fatty acid connections and cause its rupture and the release of the corresponding fatty acid (Morehouse, Kiesel, & Ku, 1993). Whereas, oleic acid showed a relatively notable reduction with 11% at 5 kGy compared to the non-radiated sample. 3.4.2. Vitamin A determination The results of HPLC analysis of vitamin A as trans-retinol form according to its retention time (5–5.5 min) were shown in Table 3. An important loss of 88.6% of vitamin A was observed in the millet flour radiated with 1 kGy compared to the non-radiated sample, from 34.2 to 3.9 lg retinol/kg. Vitamin A is found to be among the very radiosensitive vitamins (Konopacka & Rzeszowska-wolny, 2001). As can be seen, radiated sample with 2 kGy showed an increase to 40.2 lg retinol/kg of the vitamin A content, and those radiated with 3 and 5 kGy, showed again a decrease to 15 and 0.6 lg retinol/kg respectively. In this case, the observed increase in vitamin A with 2 kGy could be due to the radioinduced degradation of b-carotene, as it was reported in the studies of Lutfullah, Zeb, Ahmad, Atta, and Bangash (2003). 3.4.3. Gamma radiation effects on amino acids (AA) The HPLC analysis of phenylalanine, an aromatic amino acid, proline as heterocyclic amino acid (imino acid), valin as aliphatic amino acid and glutamic acid as dicarboxylic amino acid was done to deduce their residual concentrations generated by ionising radiations (Fig. 2). As described in ElShazali, Isam, Abu ElGasim, and Elfadil (2010), most of the amino acids of pearl millet flour were stable against radiation process except leucine, glutamic acid and phenylalanine. In our study, we report a decrease on phenylalanine concentrations in a dose-dependent manner, from 32 to 23 mg/g millet flour with 5 kGy. The structure of this aromatic amino acid could be selectively modified by radiation process and may leads the decarboxylation or hydroxylation to the formation of free amine in aqueous solutions (Cataldo, Angelini, Iglesias-Groth, &

0KGy

1KGy

2KGy

3KGy

5KGy

30

mg/g millet flour

25 20 15 10 5 0

Phenylalanine

Proline

Glutamic acid

Valine

Fig. 2. Phenylalanine, proline, glutamic acid and valine content of irradiated and unirradiated millet flour.

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Manchado, 2011) or the formation of complex products like dipeptides or oligopeptides in solid state (Wang, Yuan, Wang, & Yu, 2007). This loss of 28.1% might be also a result of a radiation-induced splitting of peptide bonds with a formation of free radicals (Aziz Nagy et al., 2006). On the contrary, radiation of millet flour to 5 kGy did not cause any significant decrease in the concentrations of proline, glutamic acid and valin and showed respectively losses of 3.84%, 1.90% and 2.17%. So, as mentioned in Aziz Nagy et al. (2006) reports, the amino acid content of irradiated millet flour to 5 kGy is generally very similar to non-radiated samples and, therefore, is roughly equivalent to them nutritionally.

(A)

These amino acids appear to be radio-stable without radio-induced modifications compared to the other amino acids as demonstrated in previous works (Cataldo et al., 2011). Future work might include specific structural studies by Mass Spectrometry or by Optical Rotatory Dispersion Spectroscopy as reported in other researches to identify the radio-induced modifications by the gamma radiations. 3.4.4. Total phenolic content The effect of gamma radiation on total phenolic compounds (TPC) in all millet flour samples (unirradiated and irradiated) is

180 160

Phenolic content (mg/g)

140 120 100 80 60 40 20 0 0

(B)

1

2

3

5

Radiaon dose (kGy) 120

ABTS-RSA (%)

100

80

60

40

20

0

(C)

Trolox 1mM

Trolox 0,8mM

Trolox 0,5mM

Trolox 1mM

Trolox 0,8mM

Trolox 0,5mM

Trolox 0,2mM

Trolox Trolox 0,1mM 0,005mM

0 kGy

1 kGy

2 kGy

3 kGy

5 kGy

120

DPPH-RSA (%)

100

80

60

40

20

0

Trolox 0,2mM

Trolox Trolox 0 kGy 0,1mM 0,005mM

1 kGy

2 kGy

3 kGy

5 kGy

Fig. 3. Gamma radiation effects on phenolic content (A) and ABTS (B) and DPPH (C) radical scavenging activity (RSA) of Trolox (black bars) and non-radiated–radiated (1, 2, 3 and 5 kGy) millet flour aqueous acetone extracts (gray bars). Data presented are averages with a standard deviation of 0.05) in TPC from 168.37 mg/g to 145.92 mg/g was observed in the 1, 2, 3 and 5 kGy irradiated millet flour, in comparison with non-irradiated control samples. This result is in correlation with data published previously in Harrison and Were (2007) study which evaluated that phenolic compounds in radiated plants, showed no significant loss of these compounds. Similar observations were obtained by ElShazali, Nahid, Salma, and Elfadil (2011) when they studied the effect of radiation process on antinutrients of pearl millet flour. They reported that radiation process alone had no effect on tannin content. However, for other plant materials, diverse effects of radiation on the phenolic content have been reported. Zhu, Cai, Bao, and Corke (2010) reported a decrease in the amount of TPC in soybean and in black rice after radiation doses when compared to controls. Conversely, it was reported that irradiation of black rice at 8 kGy achieved the highest TPC compared with the control (Zhu et al., 2010). The difference in the effect of radiation on TPC may be due to geographical and environmental conditions, plant type, state of the sample (solid or dry), phenolic content composition, extraction solvent, extraction procedures, temperature, dose of gamma irradiation, etc. 3.4.5. DPPH and ABTS activities To appraise the antioxidant potential of the prepared polyphenolic extracts of non-radiated and radiated millet flour, we used the ABTS and the DPPH scavenging tests. Antioxidative activities were expressed as ABTS radical scavenging activity and DPPH radical scavenging activity as presented in Fig. 3(B) and (C) respectively. From these results, application of radiation at different levels exhibited different but non-significant (p < 0.05) antioxidative activities. For ABTS radical-scavenging activity, samples radiated with 1, 2, 3 and 5 kGy showed non-significant changes in the activity (p < 0.05). The application of radiation doses of 2, 3 and 5 kGy does not affect the RSA activity since the differences of measured values increase from 57.89% to 67% and are not significant (p < 0.05). Antioxidative activity of radiated sample with 1 kGy was lower by 8% than its of the control (non-radiated sample). The differences in antioxidative activity were mostly due to the differences in behaviors of polyphenolic compounds following exposure to gamma rays, types and amounts of antioxidative components as reported by Tongnuanchan, Benjakul, and Prodpran (2013). To better understand the radical scavenging properties of polyphenolic millet flour extracts, stable DPPH radicals were also employed in this research.Fig. 3(C) shows the results of DPPHradical scavenging activity changes of irradiated samples of millet flour, measured in polyphenolic extracts. Irradiation resulted in a non-significant tendency to decreasing of DPPH radical-scavenging activity of millet flour extracts, mainly immediately after irradiation at 2 kGy. Irradiation at 1, 2, 3 and 5 kGy resulted in a slight decrease with values ranging from 19.75% to 25.86% in the DPPH-RSA of the millet flour acetonic extract, which was found to be non-significant when compared to the non-radiated sample. Despite few results have been found earlier regarding the antioxidative changes after gamma radiation on millet seeds or flour, previous research studies on other plant materials showed different results for the effect of gamma irradiation on the free radical scavenging properties. Our present study was well in accordance with the previous studies of Murcia, Egea, and Romojaro (2004) who exhibited that water extracts of the irradiated spices of anise, cinnamon, ginger,

licorice, mint, nutmeg, and vanilla at 1, 3, 5, and 10 kGy did not show significant differences of the antioxidant activity in the radical-scavenging assays with respect to the nonirradiated samples. On the other hand, Kim et al. (2009) reported that irradiation resulted in a slight increase in the DPPH radical-scavenging ability of the cumin ethanolic extract at 1, 3, 5 and 10 kGy which was found to be non-significant when compared to the nonirradiated sample. Likewise, Harrison and Were (2007) found that greatest scavenging ability of Almond skin extracts corresponded with the higher total phenolic content of ASE irradiated at the higher dose levels.For the irradiated samples of Nelumbo Nucifera Rhizome, the pattern of change in scavenging activity as a function of concentration was similar in all the extracts to that in the control. Acetone extract showed increase in the DPPH scavenging activity, following exposure to gamma rays as reported by Khattak et al. (2009). In contrast to the previously, the work of Kim et al. (2009) showed that radiation resulted in a significant tendency to decreasing of DPPH radical-scavenging ability of pepper ethanol extracts after application of doses of 20 and 30 kGy. 4. Conclusion In conclusion, this work contributes to the knowledge of the beneficial application of gamma radiation on millet flour. Thus, this investigation is among few reports available on gamma rays effect on microbiological and biochemical properties of millet flour, which demonstrate the success of Tunisian pearl millet decontamination by means of a gamma-radiation technique at a low dose (2–3 kGy) with non-significant losses on biochemical properties. Our findings lead to conclude that gamma radiation exhibited an important dose-dependent reduction of the bacterial, yeast and moulds species, ensuring good microbiological quality, lengthen shelf-life and storage period. The levels of OTA were reduced by 44% and 74% with 3 and 10 kGy radiation doses respectively showing a dose-dependent effect without toxicological hazard or special nutritional or microbiological problems, so to offer a promising solution for our country regarding mycotoxin problems. Following radiation at 3 kGy, PV was increased from 26.16 to 34.43 meq O2/ kg compared to the non-radiated sample, an indication to lipid peroxidation phenomena. Linolenic, linoleic, stearic, palmitic acids did not reveal important modifications with increasing gamma radiation dose. An important loss from 34.2 to 3.9 lg retinol/kg of vitamin A was observed in the millet flour radiated with 1 kGy compared to the non-radiated sample. In contrast, with 2 kGy, vitamin A was significantly increased highlighting a radioinduced degradation of b-carotene. There was no apparent dose effects observed with prolin, valin and glutamic amino acids. However, phenylalanine decreases in a dose-dependent manner, from 32 to 23 mg/g millet flour with 5 kGy. A non significant decrease (p > 0.05) in TPC from 168,37 mg/g to 145,92 mg/g was observed in the 1, 2, 3 and 5 kGy radiated millet flour, in comparison with control sample. The same observation was found for antioxidant activities. The data indicated a non-significant increase of ABTSRSA and DPPH-RSA at applied dose levels. Consequently, considering the increasing demand and benefit of the high quality of the plant materials, gamma irradiation could be the efficient method to decontaminate millet and other cereal seeds or flour.Future studies are planned to develop a standard protocol to control the microbiological and biochemical properties of all cereals seeds and flour. Conflict of interest The authors declare no conflict of interest.

M. Ben Mustapha et al. / Food Chemistry 154 (2014) 230–237

Acknowledgements We acknowledge financial support from the Ministry of Higher Education and Scientific Research of Tunisia through CNSTN. Authors would like to thank Pr. Hassen Bacha and his team, particularly Salwa Abid-Essefi and Chiraz Zaied, from Laboratory for Research on Biologically Compatible Compounds, Faculty of Dentistry (Monastir University), for their kind help. The main author gratefully acknowledges Mr Bechir Dridi from MULTILAB laboratories for the technical assistance.

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