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Sterilization and protection of protein in combinations of Camellia sinensis green tea extract and gamma irradiation
International Journal of Biological Macromolecules 48 (2011) 452–458
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Sterilization and protection of protein in combinations of Camellia sinensis green tea extract and gamma irradiation Kouass Sahbani Saloua a,b,∗ , Kouass Salah c , Benbettaieb Nasreddine d , Ayari Samia d , Saidi Mouldi e , Landoulsi Ahmed a a Unité de Recherche de Biochimie des Lipides et Interactions Avec les Macromolécules, 03/UR/0902, Laboratoire de Biochimie et de Biologie Moléculaire, Faculté des Sciences de Bizerte, 7021 Zarzouna, Tunisia b Groupe en Sciences des Radiations, Département de Médecine Nucléaire et de Radiobiology, Faculté de Médecine, Université de Sherbrooke, Québec, Canada J1H 5N4 c Laboratoire de Cristallochimie et Matériaux, Faculté des Sciences de Tunis, 1092 El Manar II, Tunisia d Laboratoire de Technologie Alimentaire, Unité de Radiotraitement, Centre National de la Recherche des Sciences et Technologies Nucléaires à Sidi Thabet, 2020, Tunisia e Laboratoire Radiopharmaceutique, Centre National de la Recherche des Sciences et Technologies Nucléaires à Sidi Thabet, 2020, Tunisia
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Article history: Received 12 November 2010 Received in revised form 18 December 2010 Accepted 4 January 2011 Available online 14 January 2011 Keywords: Caseins cow milk Camellia sinensis green tea extract Gamma-irradiation Sterilization
a b s t r a c t Sterilization of milk protein without heating is of great interest. Gamma irradiation is a very powerful method to decontaminated casein. Gamma-irradiation of proteins in aqueous media at doses higher than 5 kGy is known to induce their aggregation (without oxygen) or degradation (in presence of oxygen). Camellia sinensis green tea extract addition before irradiation of caseins cow milk proteins was examined. It was found that the presence of C. sinensis green tea extract during irradiation in the presence of oxygen conditions prevented the protein aggregation even at doses higher than 10 kGy, probably by scavenging oxygen radicals produced by irradiation. The protective role of C. sinensis green tea extract allowing the gamma-irradiation treatment of caseins cow milk proteins in solution, was asserted by sodium dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and by high performance liquid chromatography inverse phase (RP-HPLC). The total viable microorganisms content evaluated by Plate Count Agar (PCA) incubation for 12 h at 37 ◦ C, showed that caseins protein preparations gammairradiated remained sterile at a dose 2 kGy in absence of C. sinensis green tea extract and at a dose lower than 2 kGy in the presence of C. sinensis green tea extract. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Green tea is an important beverage with beneficial health attributes that have been described in many publications [1–5]. Tea (Camellia sinensis) is one of most popular beverages consumed worldwide and has a long history of consumption dating from ancient times. Among the various types of teas, green tea contains a relatively high level of polyphenols, which consist of flavanol monomers (flavan-3-ols), also referred to as catechins. Tea catechins have gained considerable attention as a result of demonstrated beneficial effects on health, such as their observed antioxidant [6] and anti-viral activity [7], as well as their ability to serve as an anti-plaque-forming agent [8]. Catechins were reported to have also anti-carcinogenic [9–12], anti-cardiovascular disease
∗ Corresponding author at: Groupe en Sciences des Radiations, Département de Médecine Nucléaire et de Radiobiology, Faculté de Médecine, Université de Sherbrooke, Québec, Canada J1H 5N4. Tel.: +1 819 346 1110x12103/+216 23 347283; fax: +1 819 564 5442/+216 72 590566. E-mail address: [email protected] (K.S. Saloua). 0141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2011.01.003
agent [13], hypotensive agent [14] and hypocholesterogenic properties [15]. They would also lower the amount of sugar in blood [16]. The way tea is consumed varies among populations. In the United Kingdom, Ireland, and Canada tea is consumed with a substantial amount of milk in it [17]. Milk contains various proteins, and interactions between polyphenols and proteins have been reported [18,19]. The caseins are the predominant milk proteins of almost all mammalian species [20]. The caseins are prolinerich, open-structured rheomorphic proteins (i.e. they assume any one of several energetically favorable conformations in solution), which have distinct hydrophobic and hydrophilic domains [21]. ␣s1 -CN, ␣s2 -CN and -CN have serine–phosphate residue centers for calcium sequestration. -CN is a glycoprotein, with 2 cysteines forming intermolecular disulfide bridges [21]. 95% of the caseins are naturally self-assembled into casein micelles which are spherical colloidal particles, 50–500 nm (average 150 nm) in diameter and molecular mass between 106 and 3 × 109 Da [21]. Milk proteins are widely available, inexpensive, natural and GRAS (generally recognized as safe) raw materials with high nutritional value and good sensory properties, and they have many structural
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Fig. 1. Combined effect of Camellia sinensis green tea extract (2.5% (2.5 g/100 ml); 5% (5 g/100 ml) and 10% (10 g/100 ml)) and gamma irradiation at 2 kGy, 4 kGy, 6 kGy, 8 kGy and 10 kGy on total aerobic plate counts of casein cow milk (n = 3; P < 0.05) the results were expressed as log(CFU/ml).
properties and functionalities which make them highly suitable as vehicles, or as components for the construction of vehicles for delivering various bioactives [21]. Milk proteins can bind a variety of molecules and ions at different degrees of affinity and specificity [21]. The major advances of the past year in harnessing milk proteins for novel health-promoting delivery applications were mainly in nanosizing, conjugation, crosslinking and targeting. Novel milk-protein nanoparticles were introduced for solubilizing and protecting hydrophobic nutraceuticals in clear systems, or for targeting gastric tumors, utilizing the natural digestibility of caseins [21]. Several interesting advancements were reported in the application of milk proteins for drug targeting [22]. Casein like all raw material of animal origin requires decontamination before using in the processing into consumable products or drug. The International Dairy Federation (IDF) Standard [23] specifies the following upper limits for bacterial contamination of edible casein: total count not more than 3 × 104 g−1 , coliforms negative in 0.1 g, mould and yeasts not more than 50 g−1 and thermophilic organisms not more than 5 × 103 g−1 . As for the microbial load of industrial caseins there is no legislation imposed yet [24]. The typical high microbial load of industrial raw products clearly limits the applications of contaminated casein. Sterilization of milk protein without heating is of great interest. Gamma irradiation is a very powerful method to decontaminated casein. Gamma-irradiation is known to cause irreversible alterations of protein conformation at the molecular level, such as fragmentation or aggregation [25]. Lee and Song showed via sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) study, that gamma-irradiation of myoglobin in solution, first caused the disruption and then aggregation due to the cross-linking of myoglobin fragments and that degradation and aggregation could be prevented by antioxidants such as ascorbic acid [26]. Protein damages due to indirect irradiation effects could be minimized by optimization of the irradiation parameters. One approach to achieve this goal is the addition of compounds that act as radioprotectors [27–29]. The aim of this study is to investigate the radioprotective effect of green tea extract towards casein in aqueous solution. It is known that the damages induced by ionising radiations to proteins in aqueous solution are much more important than to proteins in the solid state. However for some uses (medical purposes for instance) it would be much more convenient to be able to decrease the microbial contamination of solutions. This is the reason why we performed such an investigation.
2. Materials and methods 2.1. Casein preparation Casein was extracted from cow milk. The technique of Shahani and Sommer was used (isoelectric precipitation at pH 4.6) [30]. The degree of purification and the mass molecular of casein extracted were evaluated by SDS-PAGE, following [31] and RP-HPLC, following [32] and standard casein was used as control. 2.2. Preparation of tea extracts C. sinensis green tea extract was prepared using an aqueous extraction procedure (ultra-pure water). The 2.5%; 5% and 10% of C. sinensis green tea extract were freshly prepared by brewing respectively 2.5 g; 5 g and 10 g dried green tea leaves in 100 ml hot ultra pure water (80 ◦ C) for 5 min. After extraction, the infusions were filtered through a tea strainer (Tea infuser “Ball” green pincers INOX) [33]. 2.3. Microbian contamination evaluation The Plate Count Agar standard method based on counting of bacterial colonies on agar (15 g/l of water, autoclaved at 121 ◦ C during 20 min) was used to evaluate the microbian contamination of casein extracted. A volume of 100 ml of sample was plated into agar Petri dishes in duplicate and incubated aerobically for 12 h at 37 ◦ C. The colonies formed were quantified as CFU (colony forming units) and expressed as log(CFU/ml) and log(CFU/CFU0 ) of protein sample solution. 2.4. Gamma-irradiation Samples consisting of 3 ml of solutions of casein cow milk (0.22 mM) in 200 mM phosphate buffer (pH 7.2) were irradiated with and without additive C. sinensis green tea extract at the National Center for Nuclear Sciences and Technology, Sidi Thabet Tunisia using a Gamma cell 60 Co source (98,000 Ci) at a dose rate D• = 136.73 Gy/min. The doses were equal to 2 kGy, 4 kGy, 6 kGy, 8 kGy or 10 kGy. Irradiations were performed at room temperature (20 ◦ C). The irradiation dose was measured using PMMA dosimeters (PMMA Instruments, Harwell, UK). D10 (90% of total numbers
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Fig. 2. Combined effect of Camellia sinensis green tea extract (2.5% (2.5 g/100 ml); 5% (5 g/100 ml) and 10% (10 g/100 ml)) and gamma irradiation at 2 kGy, 4 kGy, 6 kGy, 8 kGy and 10 kGy on total aerobic plate counts of casein cow milk (n = 3; P < 0.05) the results were expressed as log(CFU/CFU0 ).
microorganisms was eliminated) was used to evaluate the microbian decontamination of casein samples irradiated. 2.5. Sodium dodecylsulfate gel electrophoresis (SDS-PAGE) The degree of homogeneity and the molecular integrity of casein irradiated in the absence or in the presence of C. sinensis green tea extract were evaluated by SDS-PAGE, following [31]. Samples were mixed with an electrophoresis sample buffer containing and boiled for 5 min. The conditions used were: staking and separation gel 4% and 12% acrylamide respectively and sample injection volume of 20 l in the gels. Estimation of molecular weights was done by comparison to SDS-PAGE Broad range protein molecular weight markers (V8491). 2.6. High performance liquid chromatography reverse phase (RP-HPLC) We used on-line HPLC following [32]. The chromatographic analysis was made with the help of the Shimadzu HPLC assembly. Two chromatographic columns C18 (250 mm × 4.6 mm) size was used: Hi-Pore RP-318 (Bio-Rad Laboratories GmbH) and Jupiter (Phenomenex). The Class-VP 6.0 software (Shimadzu) was used for data acquisition and processing. The protein separations were performed using the following solvents A was a mixture of acetonitrile–water–trifluoroacetic acid (TFA) in the ratio: 100:900:1 (v/v/v). Solvent B contained the same components in the ratio: 900:100:0.7 (v/v/v), respectively. Deionised water (MilliQsystem, Millipore), acetonitrile, and TFA for HPLC grade (Baker) were used to prepare the solutions. All solutions were filtered through a nylon filter of 0.45 m pore diameter. The separations were performed at 30 ◦ C. The flow rate was 0.8 ml min−1 . The gradients used Start, 72% of solvent A and 28% of solvent B; 57% solvent A and 43% solvent B after 20 min, 45% solvent A and 55% solvent B after 40 min. After the completion of the gradients, the concentration of solvent B increased to 80% over 3 min and was kept constant for 7 min (column washing). Then the column was equilibrated before the return to the initial conditions over the next 10 min.
test Mann–Whitney (U-test). The comparisons between the control and treated samples are indicated by the value of P < 0.05. Statistical analysis was performed with the Statistica software, version 5.1 (Statsoft, France). 3. Results 3.1. Role of C. sinensis green tea extract in the bacterial decontamination of casein solution after by radiotreatment Bacterial colonies formed were quantified as log(CFU/ml) of casein sample solution after irradiation (as summarized in Figs. 1 and 2). The total numbers of microorganisms in the untreated samples was 3.3 × 105 /mL. The treatment of casein with ionizing radiation in oxygen-free had a substantial effect on the total microbial loads. The decontamination of casein samples was quantified using D10 the dose that led to elimination of 90% of the microorganism colonies. In the absence of any additive D10 was equal to 2 kGy. When irradiation took place in the presence of green tea extract it was lower than 2 kGy (Figs. 1 and 2). 3.2. SDS-PAGE analysis of casein in denaturing conditions after irradiation SDS-PAGE profiles of irradiated casein showed the dosedependent breakdown the polypeptide chain and formation of
2.7. Statistical analysis The results presented in this study are the average of three independent measurements. The statistical analysis of data (mean ± standard deviation) was conducted by the non-parametric
Fig. 3. SDS/PAGE electrophoretic patterns of caseins (0.22 mM) non-irradiated and irradiated at increasing doses: STD: stands for standard molecular weights in kDa, (1) control whole casein prepared non-irradiated (0.22 mM), (2) whole casein cow’s milk (0.22 mM) irradiated to 2 kGy, (3) 4 kGy, (4) 6 kGy, (5) 8 kGy and (6) 10 kGy.
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Fig. 4. SDS/PAGE electrophoretic patterns of caseins (0.22 mM) non-irradiated and irradiated at increasing doses in the absence and in the presence of Camellia sinensis green tea extract. (a) Casein irradiated in the presence of Camellia sinensis green tea extract 2.5%, (b) casein irradiated in the presence of Camellia sinensis green tea extract 5%, (c) casein irradiated in the presence of Camellia sinensis green tea extract 10%. STD: stands for standard molecular weights in kDa, (1) control whole casein without green tea non-irradiated, (2) control whole standard casein non-irradiated, (3, 9 and 15) respectively control whole casein prepared non-irradiated with Camellia sinensis green tea extract: 2.5%, 5% and 10%, (4, 10 and 16) casein irradiated 2 kGy, (5, 11 and 17) 4 kGy, (6, 12 and 18) 6 kGy, (7, 13 and 19) 8 kGy and (8, 14 and 20) 10 kGy.
small molecular weight compounds as a result (Fig. 3). The formation of the high molecular weight aggregates was negligible at low doses, but increased significantly with increasing dose (Fig. 3). After irradiation with 10 kGy of dose the protein aggregates could not penetrate the separating gel. C. sinensis green tea extract protected the protein against aggregation (Fig. 4). Indeed the presence of C. sinensis green tea extract resulted in maintaining the intact bands of casein even after irradiation with 8 kGy and 10 kGy. These results were observed with C. sinensis green tea extract (5 and 10%) (Fig. 4, lanes II and III) but not with lower concentration of C. sinensis green tea extract (2.5%) (Fig. 4, lane I).
3.3. Analysis of casein solutions by reverse phase high-performance liquid chromatography The RP-HPLC elution patterns of total casein non irradiated and irradiated in the absence (Fig. 5) or in the presence of C. sinensis green tea extract (10%) (Figs. 6 and 7) were in agreement with the SDS-PAGE profiles, clearly supporting the finding that C. sinensis green tea extract prevented proteins aggregation. In fact, a decrease of the elution time for native total casein was found for the total casein irradiated with 6 kGy and 10 kGy under no protection (Fig. 5, clearly showing the protein aggregation). When the protein was irradiated
Fig. 5. Reversed-phase HPLC protein profile (280 nm) of native total casein cow’s milk (0.22 mM) non-irradiated and irradiated at the dose 6 kGy and 10 kGy.
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Fig. 6. Reversed-phase HPLC protein profile (280 nm) of irradiated casein (0.22 mM) in the absence and presence of Camellia sinensis green tea extract (10%) at the dose 6 kGy.
at the same dose (6 kGy and 10 kGy under C. sinensis green tea extract, see Figs. 6 and 7) protection the elution time was increased. 4. Discussion 4.1. Microbial decontamination of total cow’s milk caseins by gamma irradiation in the presence of C. sinensis green tea extract The reduction of the bacterial microflora in the casein depended on the concentration of green tea extract. Indeed, the concentration of 10% C. sinensis green tea extract was more effective in reducing the number of viable bacteria in the caseins without irradiation. Many studies have shown the antimicrobial effect of tea compounds: antibacterial, antiviral and antifungal [34–40]. Phenolic compounds known as catechins [41] are the major active ingredients of organic green tea. Catechins represent approximately 10% of the dry weight of tea [42]. The concentration 10% (10 g leaves of green tea/100 ml) has proven most effective in inhibiting the growth of bacteria. The radiotreatment of casein in the presence of C. sinensis green tea extract 10% was found to be able to lower D10 down to a value of 2 kGy decontaminate the proteins even with a dose of 2 kGy. This dose is lower than that set by the Joint F.A.O. 6 kGy which gives us the right to suggest that a decrease of the dose of bacterial decontamination D10 2 kGy in the absence of C. sinensis green tea extract at a dose lower than 2 kGy in the presence of C. sinensis green tea extract is due to the additive effect of green tea to the effect of gamma irradiation. This effect is comparable to synergistic combinations of compounds of green tea (catechins) and antibiotics [43–46]. 4.2. C. sinensis green tea extract protecting casein from irradiation-induced degradation It is known that two types of damage can be observed after irradiation of protein solutions: fragmentation and/or aggregation of
the protein gamma irradiated [25]. The electrophoretic profiles of casein irradiated with gamma rays in the absence of free radical scavengers in oxygen-free show that even for low doses, irradiation induces the rupture of the peptide chain. The result of this breakdown is the formation of low molecular weight peptides from the initially irradiated. Older studies with other proteins led to similar results [47–49], Schuessler and Schilling have proposed that proline residues are the main targets that cause fragmentation of the peptide chain [48]. Wolff et al. reported that cleavage of the peptide chain could be the result of the direct oxidation of proline residues [50]. The electrophoretic profiles of casein exposed to gamma rays show changes due to an increase in molecular mass of the protein. It seems that the caseins undergo aggregation when irradiated with high dose [51]. When caseins were irradiated with a dose greater than 6 kGy (Fig. 3), the two bands characteristic of total caseins were completely absent this disappearance is aggregation which prevents the proteins from migrating through the gel. Similar results were observed with another proteins like ceruloplasmin [49–51] and hemoglobin [52]. Indeed, irradiation induces fragmentation and aggregation of ovalbumin [49]. Stuart et al. suggested that when the protein is irradiated in solution most of the damage observed in chain peptide are due to the action of the hydroxyl radical OH generated by radiolysis of water [53]. This species is responsible for polymerization and also the fragmentation observed [54]. Increasing the molecular weight of proteins after aggregation is due to the formation of proteins on the reactions of cross-links and the formation of disulfide bridge [48–55], but in our study for probably increasing the molecular weight of casein is due to the reaction of cross-links. Aromatic amino acids are modified by oxidative action of free radicals (species generated by radiolysis of water). They are reactive and form covalent bonds (cross-linking) due to the formation of intermolecular bityrosine [55–57]. This represents a basic mechanism of the formation of crosslinking in proteins irradiated with gamma irradiation [58], which generates a high molecular mass after aggregation [48]. The aggre-
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Fig. 7. Reversed-phase HPLC protein profile (280 nm) of irradiated casein (0.22 mM) in the absence and presence of Camellia sinensis green tea extract (10%) at the dose 10 kGy.
gation of proteins after irradiation is negligible when the doses are low, but it increases remarkably with the increase in the dose of gamma irradiation. This process has also been observed with ovalbumin [49] and even to some interpretations are reported previously with egg white lysozyme and BSA when the damage caused by irradiation. The chromatographic analysis of irradiated casein solution showed a fall of elution time. The decrease in retention time is linked to the aggregation of proteins after irradiation with high doses of gamma irradiation. Irradiation of ceruloplasmin gave the same results [51]. C. sinensis green tea extract (5 and 10%) as shown in the electrophoretic profile is able to protect against protein aggregation probably by scavenging reactive oxygen radicals during the radiotreatment. Caseins that were irradiated with a dose of 10 kGy have migrated through the gel separation and intensities of two bands characteristic of these proteins are identical for all doses. Similar results were observed in other studies where the formation of organic radicals was decreased by addition of ascorbic acid [59]. According to the electrophoretic profile, green tea extract protected against caseins fragmentation and aggregation during the radiotreatment, but the protective role of green tea depends on its concentration. Indeed, green tea extract (2.5%) present during the duration of exposure of casein solution gamma radiation appears able to prevent aggregation and degradation of protein especially for a 6 kGy dose ≤ if we consider the intensity of the bands of casein. This shows that the extract of green tea (2.5%) effectively prevents the aggregation of casein in solution at low doses of gamma irradiation (2–6 kGy), but for high doses the effect of this is much less effective. For against, the green tea extract 5% added to the solution of casein appears more effective in preventing the aggregation of these proteins prepared. Indeed, the intensities of two bands characteristic of casein are almost identical to those of commercial casein and prepared unirradiated even high doses 6 kGy, 8 kGy and 10 kGy. This shows that the caseins have migrated through the gel separation. Fig. 4 is a powerful argument on the ability of the
extract of green tea (10%) to decrease the aggregation of casein irradiated solution even for doses of gamma-irradiation high. This result was verified by high performance liquid chromatography in reverse phase. Indeed, the retention time of the casein, irradiated in the presence of green tea extract (10%) gave a remarkable increase in comparison with those of casein without added tea extract green. Green tea has played a very important role in scavenging free radicals (species generated by radiolysis of water). This antioxidant effect is probably due to the wealth of green tea polyphenols and flavonoids and their ability to trap free radicals. Green tea is a powerful antioxidant that has neutralized during exposure the casein to gamma-irradiation the effect of hydroxyl radical. This antioxidant effect is certainly the wealth of green tea polyphenols and flavonoids and their ability to trap free radicals, especially for a dose of 6 kGy where the elution time of irradiated casein are very similar to the native state (not irradiated). Hasimoto et al. have evaluated the in vitro antioxidant effect of green tea polyphenols. Indeed, they have shown that polyphenols have antioxidant activity much greater than that of vitamin E [60]. 5. Conclusion The aim of this work was to elaborate protocols of radiosterilization of caseins extracted from cow’s milk in aqueous solution. Protection of proteins in aqueous solution from irradiation with doses compatible with an increase of hygienic quality is a challenge since the degradations occur by the reactions of water free radicals. However many food safety experts believe that irradiation can be an effective tool in helping to control foodborne pathogens and should be incorporated as part of a comprehensive program to enhance food safety. Irradiation, which involves exposing food briefly to radiant energy, can reduce or eliminate microorganisms that contaminate food or cause food spoilage and deterioration. The hygienic quality of our casein cow milk solutions prepared in our laboratory was obtained with doses of 2 kGy as expected
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(D10 = without additives). However for such doses the protein was damaged because the water free radicals are extremely reactive and induces protein aggregation (without oxygen) or degradation (in the presence of oxygen). After irradiation with 10 kGy of dose the protein aggregates could not penetrate the separating gel but the addition of C. sinensis green tea extract (5 and 10%) before irradiation in oxygen-free of caseins cow milk aqueous solutions prevented the protein aggregation even at doses higher than 10 kGy and maintaining the intact bands of casein even after irradiation with 8 kGy and 10 kGy. Recently many papers described protective effects of catechins from green tea in various cases related to oxidative stress [6]. Since the water free radicals created by irradiation with ionising radiations are the same as those formed in oxidative stress, it was of interest to test the radioprotection by these compounds. Irradiation of casein aqueous solutions in the presence of green tea extracts led to a concentration dependent protection. The results observed with addition of C. sinensis green tea extract 5 and 10% are not the same observed with lower concentration of C. sinensis green tea extract (2.5%). In addition the D10 was lowered to 2 kGy for all concentrations of green tea extract used. A protocol of radiosterilization using green tea extracts as radioprotectors would thus strongly improve the hygienic quality of the proteins and at the same time preserve its nutritional quality. Conflict of interest None declared. Acknowledgements The authors express their thanks National Center of the Research for the Sciences and Nuclear Technologies of Sidi Thabet and this work was supported by the Ministered of Higher Education, Tunisia. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Sterilization and protection of protein in combinations of Camellia sinensis green tea extract and gamma irradiation Kouass Sahbani Saloua a,b,∗ , Kouass Salah c , Benbettaieb Nasreddine d , Ayari Samia d , Saidi Mouldi e , Landoulsi Ahmed a a Unité de Recherche de Biochimie des Lipides et Interactions Avec les Macromolécules, 03/UR/0902, Laboratoire de Biochimie et de Biologie Moléculaire, Faculté des Sciences de Bizerte, 7021 Zarzouna, Tunisia b Groupe en Sciences des Radiations, Département de Médecine Nucléaire et de Radiobiology, Faculté de Médecine, Université de Sherbrooke, Québec, Canada J1H 5N4 c Laboratoire de Cristallochimie et Matériaux, Faculté des Sciences de Tunis, 1092 El Manar II, Tunisia d Laboratoire de Technologie Alimentaire, Unité de Radiotraitement, Centre National de la Recherche des Sciences et Technologies Nucléaires à Sidi Thabet, 2020, Tunisia e Laboratoire Radiopharmaceutique, Centre National de la Recherche des Sciences et Technologies Nucléaires à Sidi Thabet, 2020, Tunisia
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Article history: Received 12 November 2010 Received in revised form 18 December 2010 Accepted 4 January 2011 Available online 14 January 2011 Keywords: Caseins cow milk Camellia sinensis green tea extract Gamma-irradiation Sterilization
a b s t r a c t Sterilization of milk protein without heating is of great interest. Gamma irradiation is a very powerful method to decontaminated casein. Gamma-irradiation of proteins in aqueous media at doses higher than 5 kGy is known to induce their aggregation (without oxygen) or degradation (in presence of oxygen). Camellia sinensis green tea extract addition before irradiation of caseins cow milk proteins was examined. It was found that the presence of C. sinensis green tea extract during irradiation in the presence of oxygen conditions prevented the protein aggregation even at doses higher than 10 kGy, probably by scavenging oxygen radicals produced by irradiation. The protective role of C. sinensis green tea extract allowing the gamma-irradiation treatment of caseins cow milk proteins in solution, was asserted by sodium dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and by high performance liquid chromatography inverse phase (RP-HPLC). The total viable microorganisms content evaluated by Plate Count Agar (PCA) incubation for 12 h at 37 ◦ C, showed that caseins protein preparations gammairradiated remained sterile at a dose 2 kGy in absence of C. sinensis green tea extract and at a dose lower than 2 kGy in the presence of C. sinensis green tea extract. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Green tea is an important beverage with beneficial health attributes that have been described in many publications [1–5]. Tea (Camellia sinensis) is one of most popular beverages consumed worldwide and has a long history of consumption dating from ancient times. Among the various types of teas, green tea contains a relatively high level of polyphenols, which consist of flavanol monomers (flavan-3-ols), also referred to as catechins. Tea catechins have gained considerable attention as a result of demonstrated beneficial effects on health, such as their observed antioxidant [6] and anti-viral activity [7], as well as their ability to serve as an anti-plaque-forming agent [8]. Catechins were reported to have also anti-carcinogenic [9–12], anti-cardiovascular disease
∗ Corresponding author at: Groupe en Sciences des Radiations, Département de Médecine Nucléaire et de Radiobiology, Faculté de Médecine, Université de Sherbrooke, Québec, Canada J1H 5N4. Tel.: +1 819 346 1110x12103/+216 23 347283; fax: +1 819 564 5442/+216 72 590566. E-mail address: [email protected] (K.S. Saloua). 0141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2011.01.003
agent [13], hypotensive agent [14] and hypocholesterogenic properties [15]. They would also lower the amount of sugar in blood [16]. The way tea is consumed varies among populations. In the United Kingdom, Ireland, and Canada tea is consumed with a substantial amount of milk in it [17]. Milk contains various proteins, and interactions between polyphenols and proteins have been reported [18,19]. The caseins are the predominant milk proteins of almost all mammalian species [20]. The caseins are prolinerich, open-structured rheomorphic proteins (i.e. they assume any one of several energetically favorable conformations in solution), which have distinct hydrophobic and hydrophilic domains [21]. ␣s1 -CN, ␣s2 -CN and -CN have serine–phosphate residue centers for calcium sequestration. -CN is a glycoprotein, with 2 cysteines forming intermolecular disulfide bridges [21]. 95% of the caseins are naturally self-assembled into casein micelles which are spherical colloidal particles, 50–500 nm (average 150 nm) in diameter and molecular mass between 106 and 3 × 109 Da [21]. Milk proteins are widely available, inexpensive, natural and GRAS (generally recognized as safe) raw materials with high nutritional value and good sensory properties, and they have many structural
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Fig. 1. Combined effect of Camellia sinensis green tea extract (2.5% (2.5 g/100 ml); 5% (5 g/100 ml) and 10% (10 g/100 ml)) and gamma irradiation at 2 kGy, 4 kGy, 6 kGy, 8 kGy and 10 kGy on total aerobic plate counts of casein cow milk (n = 3; P < 0.05) the results were expressed as log(CFU/ml).
properties and functionalities which make them highly suitable as vehicles, or as components for the construction of vehicles for delivering various bioactives [21]. Milk proteins can bind a variety of molecules and ions at different degrees of affinity and specificity [21]. The major advances of the past year in harnessing milk proteins for novel health-promoting delivery applications were mainly in nanosizing, conjugation, crosslinking and targeting. Novel milk-protein nanoparticles were introduced for solubilizing and protecting hydrophobic nutraceuticals in clear systems, or for targeting gastric tumors, utilizing the natural digestibility of caseins [21]. Several interesting advancements were reported in the application of milk proteins for drug targeting [22]. Casein like all raw material of animal origin requires decontamination before using in the processing into consumable products or drug. The International Dairy Federation (IDF) Standard [23] specifies the following upper limits for bacterial contamination of edible casein: total count not more than 3 × 104 g−1 , coliforms negative in 0.1 g, mould and yeasts not more than 50 g−1 and thermophilic organisms not more than 5 × 103 g−1 . As for the microbial load of industrial caseins there is no legislation imposed yet [24]. The typical high microbial load of industrial raw products clearly limits the applications of contaminated casein. Sterilization of milk protein without heating is of great interest. Gamma irradiation is a very powerful method to decontaminated casein. Gamma-irradiation is known to cause irreversible alterations of protein conformation at the molecular level, such as fragmentation or aggregation [25]. Lee and Song showed via sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) study, that gamma-irradiation of myoglobin in solution, first caused the disruption and then aggregation due to the cross-linking of myoglobin fragments and that degradation and aggregation could be prevented by antioxidants such as ascorbic acid [26]. Protein damages due to indirect irradiation effects could be minimized by optimization of the irradiation parameters. One approach to achieve this goal is the addition of compounds that act as radioprotectors [27–29]. The aim of this study is to investigate the radioprotective effect of green tea extract towards casein in aqueous solution. It is known that the damages induced by ionising radiations to proteins in aqueous solution are much more important than to proteins in the solid state. However for some uses (medical purposes for instance) it would be much more convenient to be able to decrease the microbial contamination of solutions. This is the reason why we performed such an investigation.
2. Materials and methods 2.1. Casein preparation Casein was extracted from cow milk. The technique of Shahani and Sommer was used (isoelectric precipitation at pH 4.6) [30]. The degree of purification and the mass molecular of casein extracted were evaluated by SDS-PAGE, following [31] and RP-HPLC, following [32] and standard casein was used as control. 2.2. Preparation of tea extracts C. sinensis green tea extract was prepared using an aqueous extraction procedure (ultra-pure water). The 2.5%; 5% and 10% of C. sinensis green tea extract were freshly prepared by brewing respectively 2.5 g; 5 g and 10 g dried green tea leaves in 100 ml hot ultra pure water (80 ◦ C) for 5 min. After extraction, the infusions were filtered through a tea strainer (Tea infuser “Ball” green pincers INOX) [33]. 2.3. Microbian contamination evaluation The Plate Count Agar standard method based on counting of bacterial colonies on agar (15 g/l of water, autoclaved at 121 ◦ C during 20 min) was used to evaluate the microbian contamination of casein extracted. A volume of 100 ml of sample was plated into agar Petri dishes in duplicate and incubated aerobically for 12 h at 37 ◦ C. The colonies formed were quantified as CFU (colony forming units) and expressed as log(CFU/ml) and log(CFU/CFU0 ) of protein sample solution. 2.4. Gamma-irradiation Samples consisting of 3 ml of solutions of casein cow milk (0.22 mM) in 200 mM phosphate buffer (pH 7.2) were irradiated with and without additive C. sinensis green tea extract at the National Center for Nuclear Sciences and Technology, Sidi Thabet Tunisia using a Gamma cell 60 Co source (98,000 Ci) at a dose rate D• = 136.73 Gy/min. The doses were equal to 2 kGy, 4 kGy, 6 kGy, 8 kGy or 10 kGy. Irradiations were performed at room temperature (20 ◦ C). The irradiation dose was measured using PMMA dosimeters (PMMA Instruments, Harwell, UK). D10 (90% of total numbers
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Fig. 2. Combined effect of Camellia sinensis green tea extract (2.5% (2.5 g/100 ml); 5% (5 g/100 ml) and 10% (10 g/100 ml)) and gamma irradiation at 2 kGy, 4 kGy, 6 kGy, 8 kGy and 10 kGy on total aerobic plate counts of casein cow milk (n = 3; P < 0.05) the results were expressed as log(CFU/CFU0 ).
microorganisms was eliminated) was used to evaluate the microbian decontamination of casein samples irradiated. 2.5. Sodium dodecylsulfate gel electrophoresis (SDS-PAGE) The degree of homogeneity and the molecular integrity of casein irradiated in the absence or in the presence of C. sinensis green tea extract were evaluated by SDS-PAGE, following [31]. Samples were mixed with an electrophoresis sample buffer containing and boiled for 5 min. The conditions used were: staking and separation gel 4% and 12% acrylamide respectively and sample injection volume of 20 l in the gels. Estimation of molecular weights was done by comparison to SDS-PAGE Broad range protein molecular weight markers (V8491). 2.6. High performance liquid chromatography reverse phase (RP-HPLC) We used on-line HPLC following [32]. The chromatographic analysis was made with the help of the Shimadzu HPLC assembly. Two chromatographic columns C18 (250 mm × 4.6 mm) size was used: Hi-Pore RP-318 (Bio-Rad Laboratories GmbH) and Jupiter (Phenomenex). The Class-VP 6.0 software (Shimadzu) was used for data acquisition and processing. The protein separations were performed using the following solvents A was a mixture of acetonitrile–water–trifluoroacetic acid (TFA) in the ratio: 100:900:1 (v/v/v). Solvent B contained the same components in the ratio: 900:100:0.7 (v/v/v), respectively. Deionised water (MilliQsystem, Millipore), acetonitrile, and TFA for HPLC grade (Baker) were used to prepare the solutions. All solutions were filtered through a nylon filter of 0.45 m pore diameter. The separations were performed at 30 ◦ C. The flow rate was 0.8 ml min−1 . The gradients used Start, 72% of solvent A and 28% of solvent B; 57% solvent A and 43% solvent B after 20 min, 45% solvent A and 55% solvent B after 40 min. After the completion of the gradients, the concentration of solvent B increased to 80% over 3 min and was kept constant for 7 min (column washing). Then the column was equilibrated before the return to the initial conditions over the next 10 min.
test Mann–Whitney (U-test). The comparisons between the control and treated samples are indicated by the value of P < 0.05. Statistical analysis was performed with the Statistica software, version 5.1 (Statsoft, France). 3. Results 3.1. Role of C. sinensis green tea extract in the bacterial decontamination of casein solution after by radiotreatment Bacterial colonies formed were quantified as log(CFU/ml) of casein sample solution after irradiation (as summarized in Figs. 1 and 2). The total numbers of microorganisms in the untreated samples was 3.3 × 105 /mL. The treatment of casein with ionizing radiation in oxygen-free had a substantial effect on the total microbial loads. The decontamination of casein samples was quantified using D10 the dose that led to elimination of 90% of the microorganism colonies. In the absence of any additive D10 was equal to 2 kGy. When irradiation took place in the presence of green tea extract it was lower than 2 kGy (Figs. 1 and 2). 3.2. SDS-PAGE analysis of casein in denaturing conditions after irradiation SDS-PAGE profiles of irradiated casein showed the dosedependent breakdown the polypeptide chain and formation of
2.7. Statistical analysis The results presented in this study are the average of three independent measurements. The statistical analysis of data (mean ± standard deviation) was conducted by the non-parametric
Fig. 3. SDS/PAGE electrophoretic patterns of caseins (0.22 mM) non-irradiated and irradiated at increasing doses: STD: stands for standard molecular weights in kDa, (1) control whole casein prepared non-irradiated (0.22 mM), (2) whole casein cow’s milk (0.22 mM) irradiated to 2 kGy, (3) 4 kGy, (4) 6 kGy, (5) 8 kGy and (6) 10 kGy.
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Fig. 4. SDS/PAGE electrophoretic patterns of caseins (0.22 mM) non-irradiated and irradiated at increasing doses in the absence and in the presence of Camellia sinensis green tea extract. (a) Casein irradiated in the presence of Camellia sinensis green tea extract 2.5%, (b) casein irradiated in the presence of Camellia sinensis green tea extract 5%, (c) casein irradiated in the presence of Camellia sinensis green tea extract 10%. STD: stands for standard molecular weights in kDa, (1) control whole casein without green tea non-irradiated, (2) control whole standard casein non-irradiated, (3, 9 and 15) respectively control whole casein prepared non-irradiated with Camellia sinensis green tea extract: 2.5%, 5% and 10%, (4, 10 and 16) casein irradiated 2 kGy, (5, 11 and 17) 4 kGy, (6, 12 and 18) 6 kGy, (7, 13 and 19) 8 kGy and (8, 14 and 20) 10 kGy.
small molecular weight compounds as a result (Fig. 3). The formation of the high molecular weight aggregates was negligible at low doses, but increased significantly with increasing dose (Fig. 3). After irradiation with 10 kGy of dose the protein aggregates could not penetrate the separating gel. C. sinensis green tea extract protected the protein against aggregation (Fig. 4). Indeed the presence of C. sinensis green tea extract resulted in maintaining the intact bands of casein even after irradiation with 8 kGy and 10 kGy. These results were observed with C. sinensis green tea extract (5 and 10%) (Fig. 4, lanes II and III) but not with lower concentration of C. sinensis green tea extract (2.5%) (Fig. 4, lane I).
3.3. Analysis of casein solutions by reverse phase high-performance liquid chromatography The RP-HPLC elution patterns of total casein non irradiated and irradiated in the absence (Fig. 5) or in the presence of C. sinensis green tea extract (10%) (Figs. 6 and 7) were in agreement with the SDS-PAGE profiles, clearly supporting the finding that C. sinensis green tea extract prevented proteins aggregation. In fact, a decrease of the elution time for native total casein was found for the total casein irradiated with 6 kGy and 10 kGy under no protection (Fig. 5, clearly showing the protein aggregation). When the protein was irradiated
Fig. 5. Reversed-phase HPLC protein profile (280 nm) of native total casein cow’s milk (0.22 mM) non-irradiated and irradiated at the dose 6 kGy and 10 kGy.
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Fig. 6. Reversed-phase HPLC protein profile (280 nm) of irradiated casein (0.22 mM) in the absence and presence of Camellia sinensis green tea extract (10%) at the dose 6 kGy.
at the same dose (6 kGy and 10 kGy under C. sinensis green tea extract, see Figs. 6 and 7) protection the elution time was increased. 4. Discussion 4.1. Microbial decontamination of total cow’s milk caseins by gamma irradiation in the presence of C. sinensis green tea extract The reduction of the bacterial microflora in the casein depended on the concentration of green tea extract. Indeed, the concentration of 10% C. sinensis green tea extract was more effective in reducing the number of viable bacteria in the caseins without irradiation. Many studies have shown the antimicrobial effect of tea compounds: antibacterial, antiviral and antifungal [34–40]. Phenolic compounds known as catechins [41] are the major active ingredients of organic green tea. Catechins represent approximately 10% of the dry weight of tea [42]. The concentration 10% (10 g leaves of green tea/100 ml) has proven most effective in inhibiting the growth of bacteria. The radiotreatment of casein in the presence of C. sinensis green tea extract 10% was found to be able to lower D10 down to a value of 2 kGy decontaminate the proteins even with a dose of 2 kGy. This dose is lower than that set by the Joint F.A.O. 6 kGy which gives us the right to suggest that a decrease of the dose of bacterial decontamination D10 2 kGy in the absence of C. sinensis green tea extract at a dose lower than 2 kGy in the presence of C. sinensis green tea extract is due to the additive effect of green tea to the effect of gamma irradiation. This effect is comparable to synergistic combinations of compounds of green tea (catechins) and antibiotics [43–46]. 4.2. C. sinensis green tea extract protecting casein from irradiation-induced degradation It is known that two types of damage can be observed after irradiation of protein solutions: fragmentation and/or aggregation of
the protein gamma irradiated [25]. The electrophoretic profiles of casein irradiated with gamma rays in the absence of free radical scavengers in oxygen-free show that even for low doses, irradiation induces the rupture of the peptide chain. The result of this breakdown is the formation of low molecular weight peptides from the initially irradiated. Older studies with other proteins led to similar results [47–49], Schuessler and Schilling have proposed that proline residues are the main targets that cause fragmentation of the peptide chain [48]. Wolff et al. reported that cleavage of the peptide chain could be the result of the direct oxidation of proline residues [50]. The electrophoretic profiles of casein exposed to gamma rays show changes due to an increase in molecular mass of the protein. It seems that the caseins undergo aggregation when irradiated with high dose [51]. When caseins were irradiated with a dose greater than 6 kGy (Fig. 3), the two bands characteristic of total caseins were completely absent this disappearance is aggregation which prevents the proteins from migrating through the gel. Similar results were observed with another proteins like ceruloplasmin [49–51] and hemoglobin [52]. Indeed, irradiation induces fragmentation and aggregation of ovalbumin [49]. Stuart et al. suggested that when the protein is irradiated in solution most of the damage observed in chain peptide are due to the action of the hydroxyl radical OH generated by radiolysis of water [53]. This species is responsible for polymerization and also the fragmentation observed [54]. Increasing the molecular weight of proteins after aggregation is due to the formation of proteins on the reactions of cross-links and the formation of disulfide bridge [48–55], but in our study for probably increasing the molecular weight of casein is due to the reaction of cross-links. Aromatic amino acids are modified by oxidative action of free radicals (species generated by radiolysis of water). They are reactive and form covalent bonds (cross-linking) due to the formation of intermolecular bityrosine [55–57]. This represents a basic mechanism of the formation of crosslinking in proteins irradiated with gamma irradiation [58], which generates a high molecular mass after aggregation [48]. The aggre-
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Fig. 7. Reversed-phase HPLC protein profile (280 nm) of irradiated casein (0.22 mM) in the absence and presence of Camellia sinensis green tea extract (10%) at the dose 10 kGy.
gation of proteins after irradiation is negligible when the doses are low, but it increases remarkably with the increase in the dose of gamma irradiation. This process has also been observed with ovalbumin [49] and even to some interpretations are reported previously with egg white lysozyme and BSA when the damage caused by irradiation. The chromatographic analysis of irradiated casein solution showed a fall of elution time. The decrease in retention time is linked to the aggregation of proteins after irradiation with high doses of gamma irradiation. Irradiation of ceruloplasmin gave the same results [51]. C. sinensis green tea extract (5 and 10%) as shown in the electrophoretic profile is able to protect against protein aggregation probably by scavenging reactive oxygen radicals during the radiotreatment. Caseins that were irradiated with a dose of 10 kGy have migrated through the gel separation and intensities of two bands characteristic of these proteins are identical for all doses. Similar results were observed in other studies where the formation of organic radicals was decreased by addition of ascorbic acid [59]. According to the electrophoretic profile, green tea extract protected against caseins fragmentation and aggregation during the radiotreatment, but the protective role of green tea depends on its concentration. Indeed, green tea extract (2.5%) present during the duration of exposure of casein solution gamma radiation appears able to prevent aggregation and degradation of protein especially for a 6 kGy dose ≤ if we consider the intensity of the bands of casein. This shows that the extract of green tea (2.5%) effectively prevents the aggregation of casein in solution at low doses of gamma irradiation (2–6 kGy), but for high doses the effect of this is much less effective. For against, the green tea extract 5% added to the solution of casein appears more effective in preventing the aggregation of these proteins prepared. Indeed, the intensities of two bands characteristic of casein are almost identical to those of commercial casein and prepared unirradiated even high doses 6 kGy, 8 kGy and 10 kGy. This shows that the caseins have migrated through the gel separation. Fig. 4 is a powerful argument on the ability of the
extract of green tea (10%) to decrease the aggregation of casein irradiated solution even for doses of gamma-irradiation high. This result was verified by high performance liquid chromatography in reverse phase. Indeed, the retention time of the casein, irradiated in the presence of green tea extract (10%) gave a remarkable increase in comparison with those of casein without added tea extract green. Green tea has played a very important role in scavenging free radicals (species generated by radiolysis of water). This antioxidant effect is probably due to the wealth of green tea polyphenols and flavonoids and their ability to trap free radicals. Green tea is a powerful antioxidant that has neutralized during exposure the casein to gamma-irradiation the effect of hydroxyl radical. This antioxidant effect is certainly the wealth of green tea polyphenols and flavonoids and their ability to trap free radicals, especially for a dose of 6 kGy where the elution time of irradiated casein are very similar to the native state (not irradiated). Hasimoto et al. have evaluated the in vitro antioxidant effect of green tea polyphenols. Indeed, they have shown that polyphenols have antioxidant activity much greater than that of vitamin E [60]. 5. Conclusion The aim of this work was to elaborate protocols of radiosterilization of caseins extracted from cow’s milk in aqueous solution. Protection of proteins in aqueous solution from irradiation with doses compatible with an increase of hygienic quality is a challenge since the degradations occur by the reactions of water free radicals. However many food safety experts believe that irradiation can be an effective tool in helping to control foodborne pathogens and should be incorporated as part of a comprehensive program to enhance food safety. Irradiation, which involves exposing food briefly to radiant energy, can reduce or eliminate microorganisms that contaminate food or cause food spoilage and deterioration. The hygienic quality of our casein cow milk solutions prepared in our laboratory was obtained with doses of 2 kGy as expected
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(D10 = without additives). However for such doses the protein was damaged because the water free radicals are extremely reactive and induces protein aggregation (without oxygen) or degradation (in the presence of oxygen). After irradiation with 10 kGy of dose the protein aggregates could not penetrate the separating gel but the addition of C. sinensis green tea extract (5 and 10%) before irradiation in oxygen-free of caseins cow milk aqueous solutions prevented the protein aggregation even at doses higher than 10 kGy and maintaining the intact bands of casein even after irradiation with 8 kGy and 10 kGy. Recently many papers described protective effects of catechins from green tea in various cases related to oxidative stress [6]. Since the water free radicals created by irradiation with ionising radiations are the same as those formed in oxidative stress, it was of interest to test the radioprotection by these compounds. Irradiation of casein aqueous solutions in the presence of green tea extracts led to a concentration dependent protection. The results observed with addition of C. sinensis green tea extract 5 and 10% are not the same observed with lower concentration of C. sinensis green tea extract (2.5%). In addition the D10 was lowered to 2 kGy for all concentrations of green tea extract used. A protocol of radiosterilization using green tea extracts as radioprotectors would thus strongly improve the hygienic quality of the proteins and at the same time preserve its nutritional quality. Conflict of interest None declared. Acknowledgements The authors express their thanks National Center of the Research for the Sciences and Nuclear Technologies of Sidi Thabet and this work was supported by the Ministered of Higher Education, Tunisia. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
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