Thermally Stimulated Luminescence in Powdered Soy Proteins Heating powder isolated soy proteins (ISPs) in a N2 environment produced thermally stimulated luminescence (TSL), in 2 major temperature regions, 50 to 250◦ C (region R1) and 250 to 350◦ C (region R2). In soy protein 7S fraction, strong TSL was detected in both regions with glow peak maximum (Tm ) at 150 ± 15◦ C and at 300 ± 10◦ C. Two additional satellite or shoulder peaks were detected from the ISP and 7S protein fraction within region R1 at Tm = 90◦ C and Tm = 210◦ C. The soy protein 11S fraction produced a broad, poorly defined TSL peak in the low-temperature region. Electron paramagnetic resonance spectroscopy data from the control ISP sample, deuterium sulfide-treated ISP, ISP stored in either N2 or O2 , and defatted soy flour, indicated that the trapped radicals present in ISP is associated with the production of the primary TSL peak at 150 ± 15◦ C. Activation energies required to release the trapped charges (for luminescence to occur) are approximately 0.70, 0.78, 1.50, and 1.8 eV for TSL at Tm = 100, 150, 200, and 300◦ C, respectively. The reaction mechanism that leads to the release of the trapped charges for TSL to occur followed a mixed order kinetic, between 1.5 and 1.8. The frequency factor varied between 107 /s and 1017 /s.
Abstract:
Keywords: electron paramagnetic resonance spectroscopy, free-radicals, soy protein, thermally stimulated luminescence
Free radicals are capable of catalyzing oxidative degradation of food components, and powdered soy proteins typically contain from 10 to 100 times more metastable radicals than other protein sources. The research described in this paper provides novel information about the nature of these radicals that can be used to develop processes that can minimize the content of free radicals in foods containing soy proteins.
Practical Application:
Introduction Thermally stimulated luminescence (TSL), also known as thermoluminescence (TL), has been described as a radiation-specific phenomenon in which a material emits light or luminescence (UV-visible region) when radiation-induced trapped electrons are thermally released from the traps and are recombined with holes or luminescent centers. In inorganic crystalline solids, TSL has been extensively used to study defects or impurities and color centers. Readers are referred to an introductory chapter on TSL by Horowitz (1984). In organic solids and polymers, TSL is attributed to the presence of impurities, molecular imperfections, voids within the polymer matrix, and a host of other factors including free radicals (Fleming 1990). Because of this, TSL can also be described as a defect or impurity-specific phenomenon. In 1989, a U.S. patent was issued for detection of surface impurities in high-temperature superconducting materials using TSL technique (Cooke and Jahan 1989; Jahan and others 1991), and, by employing the same technique, Jahan and others (1991) detected the surface impurity phases in high-temperature superconducting materials. In foods, herbs, and spices, TSL has been attributed to impurity contents, and it has been used as a diagnostic tool for determination of the radiation history or radiation processing results MS 20130979 Submitted 7/16/2013, Accepted 11/4/2013. Authors Abdi, Jahan, and Walters are with Dept. of Physics, Univ. of Memphis, 216 Manning Hall, Memphis, TN 38152, U.S.A. Authors Boatright and Lei are with Dept. of Animal and Food Sciences,Univ. of Kentucky, 412 W.P. Garrigus Building, Lexington, KY 40546-0215, U.S.A. Direct inquiries to author Boatright (E-mail: [email protected]).
R C 2013 Institute of Food Technologists
doi: 10.1111/1750-3841.12325 Further reproduction without permission is prohibited
(Glidewell and others 1993; Kiss and Kispeter 1995; Ziegelmann and others 1999; Carmichael and others 2000; Raffi and others 2000). Since radiation-induced radicals can remain trapped in a solid (organic material, polymer, or food), one would expect TSL from such a solid when radicals are thermally recombined or thermally released electrons recombine with radicals or radical ions. In the 1960s, Charlesby and Partridge (1963a, 1963b, 1965a, 1965b) proposed that upon capturing a thermally detrapped electron, a carbonyl group (present in irradiated hydrocarbons) moved to excited state, and subsequent de-excitation of which emitted a photon or luminescence (Charlesby and Partridge 1963a; Partridge 1972). The 2nd model, chemiluminescence (CL), suggests that luminescence is the result of peroxide or hydroperoxide decomposition into excited carbonyl groups which de-excite by emitting photons (Broska 2001; Corrales 2002). Jacobson (2001) used TSL technique to record luminescence by recording TSL at a constant temperature or time and attributed the observed luminescence (defined as chemiluminescence, CL) to the breakdown of hydroperoxy followed by de-excitation of the excited RC = O species. Thus, the TSL or CL was directly correlated to the presence of ROOH rather than to its precursor, the trapped radicals. In low-temperature studies by Serpi and others (1975) , Charlesby and Partridge (1963a) and Mele and others (1968), TSL could not be correlated to radical–radical recombination because the radicals were found to remain frozen (inactive) during (active) TSL emission. Kiss and Kispeter (1995) examined the TSL curves of intact gamma-irradiated milk protein, and reported activation energies of electron traps to be 0.65 and 1.6 eV for the glow peaks at 425 K (152◦ C) and 500 K (227◦ C), respectively. Without any
Vol. 79, Nr. 1, 2014 r Journal of Food Science C25
C: Food Chemistry
Dereje Abdi, Muhammad S. Jahan, William L. Boatright, Benjamin M. Walters, and Qingxin Lei
Thermally stimulated luminescence . . .
C: Food Chemistry
electron paramagnetic resonance (EPR) data, however, the role of milk protein radical in the production of thermoluminescence dosimetry (TSI) could not be ascertained. Mamoon (1995) reported glow curves of intact cornstarch, “Semilac” milk powder, and Klim milk powder both before and after irradiation treatment. Only the irradiated samples produced broad glow peaks around 175 to 200◦ C. Ahn and others (2013) compared the glow curves from irradiated and nonirradiated intact garlic powder. In other studies, TSL was accomplished on extracted inorganic components as an indicator or irradiation treatment. Raffi and others (2000) compared EPR and TSL results of extracted minerals of irradiated aromatic herbs, spices, and fruits, and Sanyal and others (2009) examined gamma-irradiated Basmati rice, which was found to produce strong TSL and EPR signals. In current work, we observed TSL in nonirradiated commercial soy protein containing metastable radicals. Isolated soybean proteins (ISPs) typically are not treated with ionizing radiation; however, the free radicals trapped in “dry” soy proteins produce EPR spectra similar to protein exposed to ionizing radiation (Boatright and others 2008; Boatright and others 2009). The free radical contents in commercial food products containing soy proteins ranged from 6.12 × 1014 to 9.10 × 1015 spins/g of soy protein, which is from 10 to 98 times greater than other food protein sources examined. Tritiated hydrogen sulfide has been used to specifically label the location of carbon-radicals within dry proteins (Riesz and others 1966; White and others 1967; Riesz and others 1968; Riesz and White 1970). Lei and others employed deuterium sulfide (D2 S) to quench the primary EPR signal at g = 2.005 in powdered soy proteins. The resulting isotopic labeling revealed that the metastable radicals were located on Ala, Gly, Leu, Ile, Asx (Asp+Asn), Glx (Glu+Gln), and Trp, while the results for tyrosine were inconclusive due to nonspecific deuterium—hydrogen exchange, which occurred during subsequent sample processing (Lei and others 2010). In the current investigation, TSL results were used in conjunction with EPR data to learn more about metastable (trapped) charges and radicals in soy proteins.
Materials and Methods ISPs were obtained from local markets or prepared in the laboratory according to the procedure of Stine and others (2004). Small-scale preparation of 7S and 11S protein fractions (henceforth, they are labeled as 7S and 11S) from defatted soy flour (DF) was achieved by the protocol described by Nagano and others (1992) with modification. DF (20 g), obtained from the Archer Daniels Midland Co. (Decatur, Ill., U.S.A.), was suspended with 300 mL of deionized water at 23◦ C. The pH of the slurry was adjusted to 7.5 with 1.0 N NaOH, followed by being stirred for 1 h. Then the slurry was centrifuged at 3000 × g for 20 min at 20◦ C and the supernatant was filtered with layers of cheese clothes. The filtrate was further centrifuged at 9000 × g for 30 min at 4◦ C. The pH was adjusted to 6.4 with 2 N HCl, and the mixture was kept in ice bath overnight. The 11S globulin fraction was obtained as slightly yellowish pellet from the centrifugation of above mixture at 6500 × g for 20 min at 4◦ C, which was resuspended with 50 mL of deionized water and the pH of the suspension was adjusted to 7.0 before being freeze dried. To the supernatant from 6500 × g centrifugation, solid NaCl was added to a final concentration of 0.25 M, followed by adjusting the pH to 5.0 with 2 N HCl. While keeping the temperature at 4◦ C with ice-water bath, the mixture was stirred for 1 h before being centrifuged at 9000 × g for 30 min at 4◦ C to remove the insoluble fraction. C26 Journal of Food Science r Vol. 79, Nr. 1, 2014
The supernatant was diluted 2-fold with ice-cold water and kept at ice-water bath for 30 min, then its pH was adjusted to 4.8 with 2 N HCl. The 7S globulin fraction, obtained as precipitate from centrifugation (6500 × g for 20 min at 4◦ C), was washed twice with distilled water, and resuspended with 50 mL of deionized water. After being adjusted pH to 7.0 with 2 N NaOH, the slurry was lyophilized.
Quenching the metastable radicals in ISP with D2 S Soy protein samples (approximately 1 g) were loosely wrapped with filter paper and introduced into the sample chamber. After being sealed, air was removed from the system with a high vacuum (
Abstract:
Keywords: electron paramagnetic resonance spectroscopy, free-radicals, soy protein, thermally stimulated luminescence
Free radicals are capable of catalyzing oxidative degradation of food components, and powdered soy proteins typically contain from 10 to 100 times more metastable radicals than other protein sources. The research described in this paper provides novel information about the nature of these radicals that can be used to develop processes that can minimize the content of free radicals in foods containing soy proteins.
Practical Application:
Introduction Thermally stimulated luminescence (TSL), also known as thermoluminescence (TL), has been described as a radiation-specific phenomenon in which a material emits light or luminescence (UV-visible region) when radiation-induced trapped electrons are thermally released from the traps and are recombined with holes or luminescent centers. In inorganic crystalline solids, TSL has been extensively used to study defects or impurities and color centers. Readers are referred to an introductory chapter on TSL by Horowitz (1984). In organic solids and polymers, TSL is attributed to the presence of impurities, molecular imperfections, voids within the polymer matrix, and a host of other factors including free radicals (Fleming 1990). Because of this, TSL can also be described as a defect or impurity-specific phenomenon. In 1989, a U.S. patent was issued for detection of surface impurities in high-temperature superconducting materials using TSL technique (Cooke and Jahan 1989; Jahan and others 1991), and, by employing the same technique, Jahan and others (1991) detected the surface impurity phases in high-temperature superconducting materials. In foods, herbs, and spices, TSL has been attributed to impurity contents, and it has been used as a diagnostic tool for determination of the radiation history or radiation processing results MS 20130979 Submitted 7/16/2013, Accepted 11/4/2013. Authors Abdi, Jahan, and Walters are with Dept. of Physics, Univ. of Memphis, 216 Manning Hall, Memphis, TN 38152, U.S.A. Authors Boatright and Lei are with Dept. of Animal and Food Sciences,Univ. of Kentucky, 412 W.P. Garrigus Building, Lexington, KY 40546-0215, U.S.A. Direct inquiries to author Boatright (E-mail: [email protected]).
R C 2013 Institute of Food Technologists
doi: 10.1111/1750-3841.12325 Further reproduction without permission is prohibited
(Glidewell and others 1993; Kiss and Kispeter 1995; Ziegelmann and others 1999; Carmichael and others 2000; Raffi and others 2000). Since radiation-induced radicals can remain trapped in a solid (organic material, polymer, or food), one would expect TSL from such a solid when radicals are thermally recombined or thermally released electrons recombine with radicals or radical ions. In the 1960s, Charlesby and Partridge (1963a, 1963b, 1965a, 1965b) proposed that upon capturing a thermally detrapped electron, a carbonyl group (present in irradiated hydrocarbons) moved to excited state, and subsequent de-excitation of which emitted a photon or luminescence (Charlesby and Partridge 1963a; Partridge 1972). The 2nd model, chemiluminescence (CL), suggests that luminescence is the result of peroxide or hydroperoxide decomposition into excited carbonyl groups which de-excite by emitting photons (Broska 2001; Corrales 2002). Jacobson (2001) used TSL technique to record luminescence by recording TSL at a constant temperature or time and attributed the observed luminescence (defined as chemiluminescence, CL) to the breakdown of hydroperoxy followed by de-excitation of the excited RC = O species. Thus, the TSL or CL was directly correlated to the presence of ROOH rather than to its precursor, the trapped radicals. In low-temperature studies by Serpi and others (1975) , Charlesby and Partridge (1963a) and Mele and others (1968), TSL could not be correlated to radical–radical recombination because the radicals were found to remain frozen (inactive) during (active) TSL emission. Kiss and Kispeter (1995) examined the TSL curves of intact gamma-irradiated milk protein, and reported activation energies of electron traps to be 0.65 and 1.6 eV for the glow peaks at 425 K (152◦ C) and 500 K (227◦ C), respectively. Without any
Vol. 79, Nr. 1, 2014 r Journal of Food Science C25
C: Food Chemistry
Dereje Abdi, Muhammad S. Jahan, William L. Boatright, Benjamin M. Walters, and Qingxin Lei
Thermally stimulated luminescence . . .
C: Food Chemistry
electron paramagnetic resonance (EPR) data, however, the role of milk protein radical in the production of thermoluminescence dosimetry (TSI) could not be ascertained. Mamoon (1995) reported glow curves of intact cornstarch, “Semilac” milk powder, and Klim milk powder both before and after irradiation treatment. Only the irradiated samples produced broad glow peaks around 175 to 200◦ C. Ahn and others (2013) compared the glow curves from irradiated and nonirradiated intact garlic powder. In other studies, TSL was accomplished on extracted inorganic components as an indicator or irradiation treatment. Raffi and others (2000) compared EPR and TSL results of extracted minerals of irradiated aromatic herbs, spices, and fruits, and Sanyal and others (2009) examined gamma-irradiated Basmati rice, which was found to produce strong TSL and EPR signals. In current work, we observed TSL in nonirradiated commercial soy protein containing metastable radicals. Isolated soybean proteins (ISPs) typically are not treated with ionizing radiation; however, the free radicals trapped in “dry” soy proteins produce EPR spectra similar to protein exposed to ionizing radiation (Boatright and others 2008; Boatright and others 2009). The free radical contents in commercial food products containing soy proteins ranged from 6.12 × 1014 to 9.10 × 1015 spins/g of soy protein, which is from 10 to 98 times greater than other food protein sources examined. Tritiated hydrogen sulfide has been used to specifically label the location of carbon-radicals within dry proteins (Riesz and others 1966; White and others 1967; Riesz and others 1968; Riesz and White 1970). Lei and others employed deuterium sulfide (D2 S) to quench the primary EPR signal at g = 2.005 in powdered soy proteins. The resulting isotopic labeling revealed that the metastable radicals were located on Ala, Gly, Leu, Ile, Asx (Asp+Asn), Glx (Glu+Gln), and Trp, while the results for tyrosine were inconclusive due to nonspecific deuterium—hydrogen exchange, which occurred during subsequent sample processing (Lei and others 2010). In the current investigation, TSL results were used in conjunction with EPR data to learn more about metastable (trapped) charges and radicals in soy proteins.
Materials and Methods ISPs were obtained from local markets or prepared in the laboratory according to the procedure of Stine and others (2004). Small-scale preparation of 7S and 11S protein fractions (henceforth, they are labeled as 7S and 11S) from defatted soy flour (DF) was achieved by the protocol described by Nagano and others (1992) with modification. DF (20 g), obtained from the Archer Daniels Midland Co. (Decatur, Ill., U.S.A.), was suspended with 300 mL of deionized water at 23◦ C. The pH of the slurry was adjusted to 7.5 with 1.0 N NaOH, followed by being stirred for 1 h. Then the slurry was centrifuged at 3000 × g for 20 min at 20◦ C and the supernatant was filtered with layers of cheese clothes. The filtrate was further centrifuged at 9000 × g for 30 min at 4◦ C. The pH was adjusted to 6.4 with 2 N HCl, and the mixture was kept in ice bath overnight. The 11S globulin fraction was obtained as slightly yellowish pellet from the centrifugation of above mixture at 6500 × g for 20 min at 4◦ C, which was resuspended with 50 mL of deionized water and the pH of the suspension was adjusted to 7.0 before being freeze dried. To the supernatant from 6500 × g centrifugation, solid NaCl was added to a final concentration of 0.25 M, followed by adjusting the pH to 5.0 with 2 N HCl. While keeping the temperature at 4◦ C with ice-water bath, the mixture was stirred for 1 h before being centrifuged at 9000 × g for 30 min at 4◦ C to remove the insoluble fraction. C26 Journal of Food Science r Vol. 79, Nr. 1, 2014
The supernatant was diluted 2-fold with ice-cold water and kept at ice-water bath for 30 min, then its pH was adjusted to 4.8 with 2 N HCl. The 7S globulin fraction, obtained as precipitate from centrifugation (6500 × g for 20 min at 4◦ C), was washed twice with distilled water, and resuspended with 50 mL of deionized water. After being adjusted pH to 7.0 with 2 N NaOH, the slurry was lyophilized.
Quenching the metastable radicals in ISP with D2 S Soy protein samples (approximately 1 g) were loosely wrapped with filter paper and introduced into the sample chamber. After being sealed, air was removed from the system with a high vacuum (
