Food Chemistry 171 (2015) 200–205
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Evaluation of non-thermal effects of electricity on anthocyanin degradation during ohmic heating of jaboticaba (Myrciaria cauliflora) juice Giovana Domeneghini Mercali a,⇑, Poliana Deyse Gurak b, Frederico Schmitz c, Ligia Damasceno Ferreira Marczak c a
Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Av. Bento Gonçalves, 9500, Porto Alegre, RS 91501-970, Brazil Nutrition Department, Federal University of Health Sciences of Porto Alegre, Rua Sarmento Leite, 245, Porto Alegre, RS 90050-170, Brazil c Chemical Engineering Department, Federal University of Rio Grande do Sul, Rua Engenheiro Luiz Englert s/n, Porto Alegre, RS 90040-040, Brazil b
a r t i c l e
i n f o
Article history: Received 16 June 2014 Received in revised form 29 August 2014 Accepted 3 September 2014
Keywords: Thermal treatment Electric field strength Flavonoid compounds
a b s t r a c t This study investigated the non-thermal effects of electricity on anthocyanin degradation during ohmic heating of jaboticaba juice. For this, temperature profiles during conventional and ohmic heating processes were matched, and the degradation kinetics of anthocyanins were compared at temperatures ranging from 70 to 90 °C. The monomeric anthocyanin content was quantified by UV–Visible spectroscopy using the pH-differential method. Anthocyanin degradation was fitted to a first-order model. The rate constants ranged from 1.7 to 7.5 103 min1 and from 1.8 to 7.6 103 min1 for ohmic and conventional heating, respectively. The analysis of variance (a = 0.05) showed no significant differences between rate constants of the ohmic and conventional heating at the same temperatures. All kinetic and thermodynamic parameters evaluated showed similar values for both technologies. These results indicate that the presence of the oscillating electric field did not affect the degradation rates of anthocyanins during ohmic heating. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Jaboticaba (Myrciaria cauliflora) is a juicy spherical berry, characterised as a non-climacteric fruit. Commonly found in Brazilian markets, the fresh fruit is widely consumed. However, it is highly perishable with a shelf life of only 2–3 days at ambient temperature (Reynertson et al., 2006; Wu, Long, & Kennelly, 2013). Due to its high potential for industrial applications, derived products such jelly, pulp, juice, jam, ice cream, peel flour, fermented and liqueur are also gaining economic importance (Costa, GarciaDiaz, Jimenez, & Silva, 2013). Jaboticaba is considered an emerging fruit crop from Brazil known for its high content of phenolic compounds, mainly anthocyanins (Alezandro, Granato, & Genovese, 2013; Borges, Conceição, & Silveira, 2014). Leite-Legatti et al. (2012) identified and quantified jaboticaba anthocyanins by HPLC-PDA and LC–MS/MS and the results showed the presence of two majority anthocyanins: delphinidin 3-glucoside (567 mg 100 g1, d.w.) and cyanidin 3-glucoside (186.6 mg 100 g1, d.w.). Reynertson (2007) quantified ⇑ Corresponding author. Tel.: +55 51 3308 7862; fax: +55 51 3308 7048. E-mail address: [email protected] (G.D. Mercali). http://dx.doi.org/10.1016/j.foodchem.2014.09.006 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
anthocyanins of the whole fruit by HPLC–PDA and found values of 433 and 81 mg 100 g1 (d.w.) for cyanidin 3-glucoside and delphinidin 3-glucoside, respectively. These compounds were also confirmed by other studies using TLC (Santos, Veggi, & Meireles, 2010) and HPLC–TOF-MS (Wu, Dastmalchi, Long, & Kennelly, 2012). Heat treatments are commonly used to extend shelf life of jaboticaba products for longer periods and to maintain its quality during storage and commercialisation. Nowadays, new alternative technologies have been studied to substitute conventional methods of heat treatment. Ohmic heating technology has been drawing attention because it provides uniform and rapid heating of foods, with greater preservation of nutritional and organoleptic properties of processed fruit and vegetables products (Bozkurt & Icier, 2012; Guida et al., 2013; Moreno et al., 2013). Initially, it was assumed that destruction of microorganisms and biological cells during ohmic heating was due solely to thermal effects. Nevertheless, it has recently been observed that the nonthermal effects occur when moderate electric field (MEF) treatments are applied. Some studies have investigated the non-thermal effects of electricity on microorganisms’ destruction such as vegetative bacterial cells and bacterial spores (Lee, Sagong, Ryu,
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& Kang, 2012; Somavat, Mohamed, Chung, Yousef, & Sastry, 2012; Somavat, Mohamed, & Sastry, 2013). However, the interaction of electric fields with bioactive compounds of foods has not been always considered. Mercali, Schwartz, Marczak, Tessaro, and Sastry (2014) evaluated the effects of electric field frequency on ascorbic acid degradation and colour changes in acerola pulp during ohmic heating and observed that the ascorbic acid molecule predisposition for hydrogen donation in redox reactions was not affected by the rapidly varying electric field. According to the authors, further studies are needed to better understand and exploit the precise mechanism of MEF-induced changes on nutritional components. From this standpoint, the present study aimed to investigate non-thermal effects of electricity on anthocyanin degradation during ohmic heating of jaboticaba juice. Even though ohmic heating is considered a thermal processing technology, the non-thermal effects associated with the passage of the electrical current thought the food exists and it can influence the degradation of nutritional compounds. One of the techniques to evaluate the non-thermal effects during the heating process consists of matching the time– temperature histories during conventional and ohmic heating processes. As the thermal histories of both heating technologies are the same, any difference in the results can be associated with the non-thermal effects of the electricity. In the present study the thermal histories of ohmic and conventional were matched during the heat treatment of jaboticaba juice, and the degradation kinetics of anthocyanins were evaluated and compared to get a better understanding of the interaction between electric fields and the bioactive compounds of foods. 2. Materials and methods 2.1. Jaboticaba juice Jaboticaba fruits (Myrciaria jaboticaba) were obtained from a local market (Porto Alegre, RS, Brazil). Extraction of the juice was performed on a laboratory-scale hot steam drag followed by pressing of the fruits. This technology has also been used for grape juice production (Frankel, Bosanek, Meyer, Silliman, & Kirk, 1998). After extraction, the juice was packed in 300 mL bags and frozen at 18 °C. Two hours before each experiment, the samples were thawed in water bath at room temperature and homogenised. 2.2. Ohmic and conventional heating processes The ohmic heating setup used to conduct the experiments comprised a power supply, a variable transformer (Sociedade Técnica Paulista LTDA, model Varivolt, São Paulo, SP, Brazil), a stabiliser (Forceline, model EV 1000 T/2-2, São Paulo, SP, Brazil), a data acquisition system, a computer and an ohmic cell. A more complete description of the ohmic heating setup can be found elsewhere (Sarkis, Mercali, Tessaro, & Marczak, 2013). The ohmic cell was made of a 350 mL Pyrex glass vessel with a water jacket. The electrodes were made of titanium (5 cm of height) and were curved to conform the reactor dimensions. The inter-electrode gap was between 5.5 cm and 7.0 cm. Kinetic experiments were conducted at 70, 80, 85 and 90 °C. The product was stirred during heating by means of a magnetic stirrer plate (Ika, Model HS10, Germany). Samples were withdrawn at various heating times (0, 15, 30, 45, 60, 75 and 90 min). This heating time will not be used in practice. In the food industry the product is usually heated up to 80–90 °C for few minutes for achieving commercial sterilisation. As experiments of kinetics require about 5 point of concentration against time to have an appropriate fit of the degradation model, it was chosen 90 min to have a significant
decrease of concentration after each time analysed. Heating just for few minutes would make it impossible to perform such evaluation and get information such as rate constants, order of the reaction, and energy of activation, among others. The conventional heating was carried out in the ohmic cell without the application of the electric field. Samples were heated up using just hot water through the jacket. The ohmic heating experiments were conducted using 25 V (60 Hz of frequency) and controlled-temperature cooling water in the jacket simultaneously to match the time–temperature histories between electric field treatments and thermal controls. The water in the jacket was always set at a temperature lower than the temperature of the experiment. This procedure decreased the heating rate of the ohmic heating and helped to cool the sample in order to match time–temperature histories of both heating technologies. This strategy was necessary to ensure that any differences observed between both technologies would be solely due to the electric field. Fig. 1 shows the temperature profiles during the first 20 min of ohmic and conventional heating for the experiments conducted at 85 °C. The time–temperature histories at 70, 80 and 90 °C showed a similar plot behaviour. 2.3. Determination of monomeric anthocyanin content Monomeric anthocyanin content of the samples was assayed by UV–Visible spectroscopy using the pH-differential method (Lee, Durst, & Wrolstad, 2005). Samples were centrifuged (Cientec, model 500R, Piracicaba, SP, Brazil) at 5 °C (10 min, 3000g) and two sample dilutions were prepared using the supernatant: one with potassium chloride buffer, pH 1.0 and the other with sodium acetate buffer, pH 4.5. These dilutions were sat out at room temperature for 20 min. The absorbance of the samples was calculated using Eq. (1):
A ¼ ðA520 A700 ÞpH1:0 ðA520 A700 ÞpH4:5
ð1Þ
where A520 and A700 are the absorbance at the wavelength 520 and 700 nm, respectively. The concentration of monomeric anthocyanins was expressed as cyanidin 3-glucoside and was obtained using Eq. (2):
Anthocianins ðmg=LÞ ¼
A MW FD 103 el
ð2Þ
where MW is the molar weight (g mol1), DF is the dilution factor, e is the molar absorptivity (26900 L mol1 cm1) and l is pathlength of the cuvette (cm).
Fig. 1. Temperature profiles of jaboticaba juice during ohmic and conventional heating treatments.
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2.4. Determination of kinetic parameters
3. Results and discussion
Data of monomeric anthocyanin content against time were fitted to the first-order degradation kinetics, according to the following equation:
3.1. Monomeric anthocyanin degradation
C ¼ C 0 expðk tÞ
ð3Þ
where t is the time (min); C0 and C are the anthocyanin content (mg/L) at time zero and time t, respectively; and k is the first-order rate constant (min1). The decimal reduction time (D-value) and the half-life value of degradation (t1/2) were calculated from Eqs. (4) and (5), respectively:
D¼
lnð10Þ k
ð4Þ
lnð2Þ k
ð5Þ
t 1=2 ¼
The thermal resistance coefficient (z-value) is the temperature needed to vary D-value one log unit, and it was obtained by plotting the D-values on a log scale versus the corresponding temperatures.
2.5. Thermodynamic analysis The effect of temperature on the reaction rate constant was evaluated using the Arrhenius equation:
k ¼ kref exp
Ea 1 1 R T T ref
ð6Þ
where Tref is the reference temperature (80 °C), kref is the rate constant at Tref (min1), Ea is the activation energy (J/mol), R is the ideal gas constant (8.314 J mol1 K1) and T is the temperature (K). The enthalpy of activation (DH–) and the free energy of inactivation (DG–) at each temperature were obtained using Eqs. (7) and (8), respectively:
DH – ¼ E a R T
DG– ¼ R T ln
ð7Þ
kh kB T
ð8Þ
The average concentration of monomeric anthocyanins in the jaboticaba juice (fresh sample) was 14.8 ± 0.78 mg 100 g1. Reynertson (2007) quantified the total anthocyanin content of jaboticaba fruit by pH-differential method and the results showed values of 278 ± 17 mg 100 g1 (dry basis). Rufino, Alves, Fernandes, and Brito (2011), using the same analytical method, found 58.1 mg 100 g1 fresh matter for the whole fruit. Results reported by other authors are higher than those found in the present study. This could be attributed to the fact that anthocyanin content of the whole fruit was quantified in other studies while in the present research only the juice obtained from a hot extraction process was evaluated. The hot extraction process extracts anthocyanins from the whole fruit but a great amount of the compound still remains in the pomace. Anthocyanin degradation of jaboticaba juice was evaluated at different temperatures (70, 80, 85 and 90 °C) during ohmic and conventional heating treatment. Ohmic and conventional treatments at 60 °C were also conducted and a high stability of the monomeric anthocyanins was observed (degradation of 1–2% after 60 min). Studies conducted by Wang and Xu (2007) evaluated anthocyanin degradation in blackberries using temperatures between 60 and 90 °C. Their results showed very low degradation after 60 min of heating at 60 °C. They also found approximately 10% of anthocyanin degradation after 150 min at 60 °C. Figs. 2 and 3 show anthocyanin degradation in a dimensionless scale plotted against time for experiments carried out using ohmic and conventional heating process, respectively. It appears that both heating technologies showed similar effects on anthocyanin degradation. For a proper comparison between the two heating technologies, results were fitted to the first-order kinetic equation. The determination coefficients (R2) presented values higher than 0.95 for all treatments. Table 1 shows the kinetic parameters obtained for each temperature evaluated. The kinetic rate constants increased with increasing temperature, with values ranging from 0.0017 to 0.0075 min1 for ohmic heating and from 0.0018 to 0.0076 min1 for conventional heating. Other studies reporting values for the kinetic rate constants of anthocyanin degradation during heat treatment of jaboticaba products were not found. Moreover the values found in this study are in the range of those found for anthocyanin degradation in other fruits and vegetables (0.006–0.09 min1) (Aurelio, Edgardo, & Navarro-Galindo, 2008; Kirca, Özkan, &
where h is the is the Planck’s constant (6.6262 1034 J s) and kB is the Boltzmann’s constant (1.3806 1023 J K1). The entropy of activation (DS–) was calculated using Eq. (9):
DS– ¼
DH– DG– T
ð9Þ
2.6. Statistical analysis Two independent experiments were carried out for each temperature and type of heating technology evaluated. Monomeric anthocyanin analyzes were performed in duplicate. The kinetic and thermodynamic parameters of all experiments were compared using ANOVA, Tukey test and Student’s t-test (95% of confidence level). Statistical analyses were performed using Statistica 7.0 (Statsoft @, Tulsa, OK, USA) and Microsoft Excel 2011 (MapInfo, Troy, NY, USA).
Fig. 2. Degradation of anthocyanins during the conventional heating process. Results are the average values of two independent experiments. Plotted lines correspond to the values estimated by the first-order model.
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and t1/2 values (p > 0.05). The thermal resistance coefficient (z-value) describes the influence of temperature on D-values over the range of temperatures used in the experimental measurements (Heldman, 2011a). The z-values for ohmic and conventional processes were of 31.6 and 32.4 °C, respectively. A Student’s t-test was performed to compare z-values of both heating technologies and the results showed that no significant differences existed between them (p > 0.05). These results corroborate the finding that the presence of the electric field is not affecting the way anthocyanins react in the medium. 3.2. Thermodynamic analysis
Fig. 3. Degradation of anthocyanins during the ohmic heating process. Results are the average values of two independent experiments. Plotted lines correspond to the values estimated by the first-order model.
Cemerog˘lu, 2007; Wang & Xu, 2007). Mercali, Jaeschke, Tessaro, and Marczak (2013) evaluated the degradation kinetics of monomeric anthocyanins in acerola pulp during thermal treatment by ohmic and conventional heating (75–90 °C) and it was reported that the kinetic rate constants ranged from 0.0059 to 0.0199 min1. The rate constants for acerola pulp were approximately three times higher than the ones found for jaboticaba juice. This difference may be due to the presence of distinct phytochemical compounds, such as ascorbic acid, that negatively influences anthocyanin stability (De Rosso & Mercadante, 2007). As shown in Table 1, there were no significant differences between the rate constants of the ohmic and conventional heating when same temperatures were compared. The results reported by Mercali et al. (2013) are in agreement with those found in this study. The phytochemical compounds of jaboticaba juice, including the anthocyanins, are subjected to an electric field distortion when the ohmic heating technology is applied. It was theorised that the alignment of the molecules with the oscillating electric field could have influenced its reactivity. However, anthocyanin degradation is happening in the same rate for both heating processes at the same temperatures. This indicates that the orientational movements of the molecules stimulated by the oscillating electric field, phenomenon known by polarisation, did not affect the degradation rates of anthocyanins during ohmic heating. The decimal reduction (D-values) and the half-time (t1/2) values for each experiment are shown in Table 1. For the ohmic heating process, D-values ranged between 309 and 1397 min and t1/2values ranged from 93 to 420 min. For the conventional heating technology, D-values varied from 303 to 1317 min and t1/2-values ranged from 91 to 396 min. Both parameters decreased as the temperature of the process increased. When compared in the same temperature, ohmic and conventional heating showed similar D
The activation energy values (Ea) for ohmic and conventional heating were of 75.5 and 73.9 kJ mol1, respectively (Table 2). The statistical analysis (Student’s t-test) showed no significant difference between the values aforementioned (p > 0.05). The magnitude of the activation energy is an excellent indicator of the sensitivity of the reaction to temperature. According to Heldman (2011b), the activation energy constants for anthocyanin degradation range from 35 to 125 kJ mol1. Higher values indicate strong temperature dependence: the reaction runs slowly at low temperature but relatively fast at high temperatures. The activation energy constants for anthocyanin degradation have relatively low magnitudes as compared to the constants for inactivation of spores and vegetative cells (250–625 kJ/mol) (Heldman, 2011b). The thermodynamic parameters can give valuable information when studying a wide variety of physical, chemical, and biochemical phenomena. The change of free energy (DG–), enthalpy (DH–) and entropy (DS–) of the degradation reactions at all temperatures during ohmic and conventional heating are presented in Table 3. DH– shows the energy difference between the reagent and activated complex; if DH– is small, the formation of the activated complex is favored because the potential energy barrier is low (Georgieva, Zvezdova, & Vlaev, 2012). As shown in Table 3, DH– values were similar for all conditions evaluated in this study (p > 0.05), varying between 70.9 and 72.6 kJ mol1. This indicates that the energy barrier that must be overcome in order to achieve the transition state is similar for both heating technologies. The positive sign of DH– means that anthocyanin degradation is an endothermic reaction, accompanied by the adsorption of heat. DG– is the criteria for equilibrium and spontaneity; the positive sign means that anthocyanin degradation is a nonspontaneous reaction. DG– showed very close values for all conditions evaluated in this study (102.5–104.3 kJ mol1). This suggests that the total energy increase of the system at the approach of the reagents and the formation of the activated complex was similar for both heating technologies. DS– measures the disorder change of molecules in the system and it ranged from 85.1 to 92.3 J mol1 K1. The negative entropy values found in this study suggest that the transition state
Table 1 Parameters of the first-order model for anthocyanin degradation in jaboticaba juice during ohmic and conventional heating. Process
T (°C)
R2
k (min1)d
D (min)d a
t1/2 (min)d a
z (°C)d
a
31.6 ± 0.9a
32.4 ± 0.1a
Ohmic heating
70 80 85 90
0.98 0.98 0.99 0.98
0.0017 ± 0.0001 0.0048 ± 0.0001b 0.0049 ± 0.0001b 0.0075 ± 0.0001c
1397 ± 60 480 ± 14b 470 ± 0b 309 ± 3c
420 ± 18 144 ± 4b 141 ± 0b 93 ± 1c
Conventional heating
70 80 85 90
0.95 0.99 0.97 0.99
0.0018 ± 0.0001a 0.0046 ± 0.0004b 0.0051 ± 0.0001b 0.0076 ± 0.0004c
1317 ± 53a 503 ± 46b 456 ± 6b 303 ± 17c
396 ± 16a 151 ± 14b 137 ± 2b 91 ± 5c
d Results are the average values of two independent experiments (mean of two replicates ± standard error); means in the same column with different lowercase letters are significantly different (p < 0.05).
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G.D. Mercali et al. / Food Chemistry 171 (2015) 200–205 Table 2 The activation energy values (Ea) obtained for anthocyanin degradation during ohmic and conventional heating. Ea (kJ mol1)b
Process
kref (min1)b
a
R2 a
0.94 0.96
Ohmic heating
75.5 ± 0.02
0.0038 ± 0.0001
Conventional heating
73.9 ± 0.06a
0.0039 ± 0.0002a
b
Results are the average values of two independent experiments (mean of two replicates ± standard error); means in the same column with different lowercase letters are significantly different (p < 0.05).
Table 3 Thermodynamic parameters obtained for anthocyanin degradation during ohmic and conventional heating. Process
T (°C)
DH– (kJ mol1)c
DG– (kJ mol1)c
a
a
DS– (J mol1 K1)c
Ohmic heating
70 80 85 90
72.6 ± 2.1 72.6 ± 2.1a 72.5 ± 2.1a 72.5 ± 2.1a
102.7 ± 0.1 102.6 ± 0.1a 104.1 ± 0.1b 104.3 ± 0.1b
87.6 ± 6a 85.1 ± 6a 88.1 ± 6a 87.6 ± 6a
Conventional heating
70 80 85 90
71.0 ± 0.6a 71.0 ± 0.6a 70.9 ± 0.6a 70.9 ± 0.6a
102.5 ± 0.1a 102.7 ± 0.3a 104.0 ± 0.1b 104.2 ± 0.2b
91.7 ± 1a 90.1 ± 1a 92.3 ± 2a 91.9 ± 1a
c Results are the average values of two independent experiments (mean of two replicates ± standard error); means in the same column with different lowercase letters are significantly different (p < 0.05).
is more structurally organised than the reactants and, therefore, the formation of activated complex is associated with decrease of entropy. According to Georgieva et al. (2012), a lower activation entropy means that the state of the material is close to its own thermodynamic equilibrium. As a consequence, the material shows little reactivity, increasing the time necessary to form the activated complex. Oppositely, when the activation entropy values are higher, as observed in this study, the material is far from its own thermodynamic equilibrium. In this situation, the system can react faster to produce the activated complex because of the higher reactivity. Table 3 shows that DS– was similar for both heating technologies in all temperatures, which indicates similar reactivity and reaction times. Ohmic and conventional heating technologies showed similar values for all kinetic and thermodynamic parameters (when compared in the same temperature), indicating that anthocyanins have similar degradation pathways during ohmic and conventional heating. These results reinforce the finding that the presence of the electric field is not affecting the mechanism and the rate of anthocyanin degradation.
4. Conclusion This study evaluated the non-thermal effects of electricity on anthocyanin degradation kinetics during ohmic heating of jaboticaba juice. The kinetic rate constants ranged from 0.0017 to 0.0075 min1 and from 0.0018 to 0.0076 min1 for ohmic heating and conventional heating, respectively. Statistical analyses showed no significant differences between the rate constants of the ohmic and conventional heating at the same temperatures. Moreover, D-values, t1/2-values and z-values showed similar values (p > 0.05) for both heating methods. Ohmic and conventional heating also showed similar values for all thermodynamic parameters when compared in the same temperatures. These results indicate that the presence of the electric field strength did not affect the degradation kinetics of anthocyanins. Therefore the anthocyanin molecule is reacting the same way and in the same rate in the medium when both heating methods are applied. This demonstrates similar mechanisms of degradation. Although more studies are required to elucidate the effect of this technology on bioactive compounds stability, these results reveal that ohmic heating
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Evaluation of non-thermal effects of electricity on anthocyanin degradation during ohmic heating of jaboticaba (Myrciaria cauliflora) juice Giovana Domeneghini Mercali a,⇑, Poliana Deyse Gurak b, Frederico Schmitz c, Ligia Damasceno Ferreira Marczak c a
Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Av. Bento Gonçalves, 9500, Porto Alegre, RS 91501-970, Brazil Nutrition Department, Federal University of Health Sciences of Porto Alegre, Rua Sarmento Leite, 245, Porto Alegre, RS 90050-170, Brazil c Chemical Engineering Department, Federal University of Rio Grande do Sul, Rua Engenheiro Luiz Englert s/n, Porto Alegre, RS 90040-040, Brazil b
a r t i c l e
i n f o
Article history: Received 16 June 2014 Received in revised form 29 August 2014 Accepted 3 September 2014
Keywords: Thermal treatment Electric field strength Flavonoid compounds
a b s t r a c t This study investigated the non-thermal effects of electricity on anthocyanin degradation during ohmic heating of jaboticaba juice. For this, temperature profiles during conventional and ohmic heating processes were matched, and the degradation kinetics of anthocyanins were compared at temperatures ranging from 70 to 90 °C. The monomeric anthocyanin content was quantified by UV–Visible spectroscopy using the pH-differential method. Anthocyanin degradation was fitted to a first-order model. The rate constants ranged from 1.7 to 7.5 103 min1 and from 1.8 to 7.6 103 min1 for ohmic and conventional heating, respectively. The analysis of variance (a = 0.05) showed no significant differences between rate constants of the ohmic and conventional heating at the same temperatures. All kinetic and thermodynamic parameters evaluated showed similar values for both technologies. These results indicate that the presence of the oscillating electric field did not affect the degradation rates of anthocyanins during ohmic heating. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Jaboticaba (Myrciaria cauliflora) is a juicy spherical berry, characterised as a non-climacteric fruit. Commonly found in Brazilian markets, the fresh fruit is widely consumed. However, it is highly perishable with a shelf life of only 2–3 days at ambient temperature (Reynertson et al., 2006; Wu, Long, & Kennelly, 2013). Due to its high potential for industrial applications, derived products such jelly, pulp, juice, jam, ice cream, peel flour, fermented and liqueur are also gaining economic importance (Costa, GarciaDiaz, Jimenez, & Silva, 2013). Jaboticaba is considered an emerging fruit crop from Brazil known for its high content of phenolic compounds, mainly anthocyanins (Alezandro, Granato, & Genovese, 2013; Borges, Conceição, & Silveira, 2014). Leite-Legatti et al. (2012) identified and quantified jaboticaba anthocyanins by HPLC-PDA and LC–MS/MS and the results showed the presence of two majority anthocyanins: delphinidin 3-glucoside (567 mg 100 g1, d.w.) and cyanidin 3-glucoside (186.6 mg 100 g1, d.w.). Reynertson (2007) quantified ⇑ Corresponding author. Tel.: +55 51 3308 7862; fax: +55 51 3308 7048. E-mail address: [email protected] (G.D. Mercali). http://dx.doi.org/10.1016/j.foodchem.2014.09.006 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
anthocyanins of the whole fruit by HPLC–PDA and found values of 433 and 81 mg 100 g1 (d.w.) for cyanidin 3-glucoside and delphinidin 3-glucoside, respectively. These compounds were also confirmed by other studies using TLC (Santos, Veggi, & Meireles, 2010) and HPLC–TOF-MS (Wu, Dastmalchi, Long, & Kennelly, 2012). Heat treatments are commonly used to extend shelf life of jaboticaba products for longer periods and to maintain its quality during storage and commercialisation. Nowadays, new alternative technologies have been studied to substitute conventional methods of heat treatment. Ohmic heating technology has been drawing attention because it provides uniform and rapid heating of foods, with greater preservation of nutritional and organoleptic properties of processed fruit and vegetables products (Bozkurt & Icier, 2012; Guida et al., 2013; Moreno et al., 2013). Initially, it was assumed that destruction of microorganisms and biological cells during ohmic heating was due solely to thermal effects. Nevertheless, it has recently been observed that the nonthermal effects occur when moderate electric field (MEF) treatments are applied. Some studies have investigated the non-thermal effects of electricity on microorganisms’ destruction such as vegetative bacterial cells and bacterial spores (Lee, Sagong, Ryu,
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& Kang, 2012; Somavat, Mohamed, Chung, Yousef, & Sastry, 2012; Somavat, Mohamed, & Sastry, 2013). However, the interaction of electric fields with bioactive compounds of foods has not been always considered. Mercali, Schwartz, Marczak, Tessaro, and Sastry (2014) evaluated the effects of electric field frequency on ascorbic acid degradation and colour changes in acerola pulp during ohmic heating and observed that the ascorbic acid molecule predisposition for hydrogen donation in redox reactions was not affected by the rapidly varying electric field. According to the authors, further studies are needed to better understand and exploit the precise mechanism of MEF-induced changes on nutritional components. From this standpoint, the present study aimed to investigate non-thermal effects of electricity on anthocyanin degradation during ohmic heating of jaboticaba juice. Even though ohmic heating is considered a thermal processing technology, the non-thermal effects associated with the passage of the electrical current thought the food exists and it can influence the degradation of nutritional compounds. One of the techniques to evaluate the non-thermal effects during the heating process consists of matching the time– temperature histories during conventional and ohmic heating processes. As the thermal histories of both heating technologies are the same, any difference in the results can be associated with the non-thermal effects of the electricity. In the present study the thermal histories of ohmic and conventional were matched during the heat treatment of jaboticaba juice, and the degradation kinetics of anthocyanins were evaluated and compared to get a better understanding of the interaction between electric fields and the bioactive compounds of foods. 2. Materials and methods 2.1. Jaboticaba juice Jaboticaba fruits (Myrciaria jaboticaba) were obtained from a local market (Porto Alegre, RS, Brazil). Extraction of the juice was performed on a laboratory-scale hot steam drag followed by pressing of the fruits. This technology has also been used for grape juice production (Frankel, Bosanek, Meyer, Silliman, & Kirk, 1998). After extraction, the juice was packed in 300 mL bags and frozen at 18 °C. Two hours before each experiment, the samples were thawed in water bath at room temperature and homogenised. 2.2. Ohmic and conventional heating processes The ohmic heating setup used to conduct the experiments comprised a power supply, a variable transformer (Sociedade Técnica Paulista LTDA, model Varivolt, São Paulo, SP, Brazil), a stabiliser (Forceline, model EV 1000 T/2-2, São Paulo, SP, Brazil), a data acquisition system, a computer and an ohmic cell. A more complete description of the ohmic heating setup can be found elsewhere (Sarkis, Mercali, Tessaro, & Marczak, 2013). The ohmic cell was made of a 350 mL Pyrex glass vessel with a water jacket. The electrodes were made of titanium (5 cm of height) and were curved to conform the reactor dimensions. The inter-electrode gap was between 5.5 cm and 7.0 cm. Kinetic experiments were conducted at 70, 80, 85 and 90 °C. The product was stirred during heating by means of a magnetic stirrer plate (Ika, Model HS10, Germany). Samples were withdrawn at various heating times (0, 15, 30, 45, 60, 75 and 90 min). This heating time will not be used in practice. In the food industry the product is usually heated up to 80–90 °C for few minutes for achieving commercial sterilisation. As experiments of kinetics require about 5 point of concentration against time to have an appropriate fit of the degradation model, it was chosen 90 min to have a significant
decrease of concentration after each time analysed. Heating just for few minutes would make it impossible to perform such evaluation and get information such as rate constants, order of the reaction, and energy of activation, among others. The conventional heating was carried out in the ohmic cell without the application of the electric field. Samples were heated up using just hot water through the jacket. The ohmic heating experiments were conducted using 25 V (60 Hz of frequency) and controlled-temperature cooling water in the jacket simultaneously to match the time–temperature histories between electric field treatments and thermal controls. The water in the jacket was always set at a temperature lower than the temperature of the experiment. This procedure decreased the heating rate of the ohmic heating and helped to cool the sample in order to match time–temperature histories of both heating technologies. This strategy was necessary to ensure that any differences observed between both technologies would be solely due to the electric field. Fig. 1 shows the temperature profiles during the first 20 min of ohmic and conventional heating for the experiments conducted at 85 °C. The time–temperature histories at 70, 80 and 90 °C showed a similar plot behaviour. 2.3. Determination of monomeric anthocyanin content Monomeric anthocyanin content of the samples was assayed by UV–Visible spectroscopy using the pH-differential method (Lee, Durst, & Wrolstad, 2005). Samples were centrifuged (Cientec, model 500R, Piracicaba, SP, Brazil) at 5 °C (10 min, 3000g) and two sample dilutions were prepared using the supernatant: one with potassium chloride buffer, pH 1.0 and the other with sodium acetate buffer, pH 4.5. These dilutions were sat out at room temperature for 20 min. The absorbance of the samples was calculated using Eq. (1):
A ¼ ðA520 A700 ÞpH1:0 ðA520 A700 ÞpH4:5
ð1Þ
where A520 and A700 are the absorbance at the wavelength 520 and 700 nm, respectively. The concentration of monomeric anthocyanins was expressed as cyanidin 3-glucoside and was obtained using Eq. (2):
Anthocianins ðmg=LÞ ¼
A MW FD 103 el
ð2Þ
where MW is the molar weight (g mol1), DF is the dilution factor, e is the molar absorptivity (26900 L mol1 cm1) and l is pathlength of the cuvette (cm).
Fig. 1. Temperature profiles of jaboticaba juice during ohmic and conventional heating treatments.
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2.4. Determination of kinetic parameters
3. Results and discussion
Data of monomeric anthocyanin content against time were fitted to the first-order degradation kinetics, according to the following equation:
3.1. Monomeric anthocyanin degradation
C ¼ C 0 expðk tÞ
ð3Þ
where t is the time (min); C0 and C are the anthocyanin content (mg/L) at time zero and time t, respectively; and k is the first-order rate constant (min1). The decimal reduction time (D-value) and the half-life value of degradation (t1/2) were calculated from Eqs. (4) and (5), respectively:
D¼
lnð10Þ k
ð4Þ
lnð2Þ k
ð5Þ
t 1=2 ¼
The thermal resistance coefficient (z-value) is the temperature needed to vary D-value one log unit, and it was obtained by plotting the D-values on a log scale versus the corresponding temperatures.
2.5. Thermodynamic analysis The effect of temperature on the reaction rate constant was evaluated using the Arrhenius equation:
k ¼ kref exp
Ea 1 1 R T T ref
ð6Þ
where Tref is the reference temperature (80 °C), kref is the rate constant at Tref (min1), Ea is the activation energy (J/mol), R is the ideal gas constant (8.314 J mol1 K1) and T is the temperature (K). The enthalpy of activation (DH–) and the free energy of inactivation (DG–) at each temperature were obtained using Eqs. (7) and (8), respectively:
DH – ¼ E a R T
DG– ¼ R T ln
ð7Þ
kh kB T
ð8Þ
The average concentration of monomeric anthocyanins in the jaboticaba juice (fresh sample) was 14.8 ± 0.78 mg 100 g1. Reynertson (2007) quantified the total anthocyanin content of jaboticaba fruit by pH-differential method and the results showed values of 278 ± 17 mg 100 g1 (dry basis). Rufino, Alves, Fernandes, and Brito (2011), using the same analytical method, found 58.1 mg 100 g1 fresh matter for the whole fruit. Results reported by other authors are higher than those found in the present study. This could be attributed to the fact that anthocyanin content of the whole fruit was quantified in other studies while in the present research only the juice obtained from a hot extraction process was evaluated. The hot extraction process extracts anthocyanins from the whole fruit but a great amount of the compound still remains in the pomace. Anthocyanin degradation of jaboticaba juice was evaluated at different temperatures (70, 80, 85 and 90 °C) during ohmic and conventional heating treatment. Ohmic and conventional treatments at 60 °C were also conducted and a high stability of the monomeric anthocyanins was observed (degradation of 1–2% after 60 min). Studies conducted by Wang and Xu (2007) evaluated anthocyanin degradation in blackberries using temperatures between 60 and 90 °C. Their results showed very low degradation after 60 min of heating at 60 °C. They also found approximately 10% of anthocyanin degradation after 150 min at 60 °C. Figs. 2 and 3 show anthocyanin degradation in a dimensionless scale plotted against time for experiments carried out using ohmic and conventional heating process, respectively. It appears that both heating technologies showed similar effects on anthocyanin degradation. For a proper comparison between the two heating technologies, results were fitted to the first-order kinetic equation. The determination coefficients (R2) presented values higher than 0.95 for all treatments. Table 1 shows the kinetic parameters obtained for each temperature evaluated. The kinetic rate constants increased with increasing temperature, with values ranging from 0.0017 to 0.0075 min1 for ohmic heating and from 0.0018 to 0.0076 min1 for conventional heating. Other studies reporting values for the kinetic rate constants of anthocyanin degradation during heat treatment of jaboticaba products were not found. Moreover the values found in this study are in the range of those found for anthocyanin degradation in other fruits and vegetables (0.006–0.09 min1) (Aurelio, Edgardo, & Navarro-Galindo, 2008; Kirca, Özkan, &
where h is the is the Planck’s constant (6.6262 1034 J s) and kB is the Boltzmann’s constant (1.3806 1023 J K1). The entropy of activation (DS–) was calculated using Eq. (9):
DS– ¼
DH– DG– T
ð9Þ
2.6. Statistical analysis Two independent experiments were carried out for each temperature and type of heating technology evaluated. Monomeric anthocyanin analyzes were performed in duplicate. The kinetic and thermodynamic parameters of all experiments were compared using ANOVA, Tukey test and Student’s t-test (95% of confidence level). Statistical analyses were performed using Statistica 7.0 (Statsoft @, Tulsa, OK, USA) and Microsoft Excel 2011 (MapInfo, Troy, NY, USA).
Fig. 2. Degradation of anthocyanins during the conventional heating process. Results are the average values of two independent experiments. Plotted lines correspond to the values estimated by the first-order model.
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and t1/2 values (p > 0.05). The thermal resistance coefficient (z-value) describes the influence of temperature on D-values over the range of temperatures used in the experimental measurements (Heldman, 2011a). The z-values for ohmic and conventional processes were of 31.6 and 32.4 °C, respectively. A Student’s t-test was performed to compare z-values of both heating technologies and the results showed that no significant differences existed between them (p > 0.05). These results corroborate the finding that the presence of the electric field is not affecting the way anthocyanins react in the medium. 3.2. Thermodynamic analysis
Fig. 3. Degradation of anthocyanins during the ohmic heating process. Results are the average values of two independent experiments. Plotted lines correspond to the values estimated by the first-order model.
Cemerog˘lu, 2007; Wang & Xu, 2007). Mercali, Jaeschke, Tessaro, and Marczak (2013) evaluated the degradation kinetics of monomeric anthocyanins in acerola pulp during thermal treatment by ohmic and conventional heating (75–90 °C) and it was reported that the kinetic rate constants ranged from 0.0059 to 0.0199 min1. The rate constants for acerola pulp were approximately three times higher than the ones found for jaboticaba juice. This difference may be due to the presence of distinct phytochemical compounds, such as ascorbic acid, that negatively influences anthocyanin stability (De Rosso & Mercadante, 2007). As shown in Table 1, there were no significant differences between the rate constants of the ohmic and conventional heating when same temperatures were compared. The results reported by Mercali et al. (2013) are in agreement with those found in this study. The phytochemical compounds of jaboticaba juice, including the anthocyanins, are subjected to an electric field distortion when the ohmic heating technology is applied. It was theorised that the alignment of the molecules with the oscillating electric field could have influenced its reactivity. However, anthocyanin degradation is happening in the same rate for both heating processes at the same temperatures. This indicates that the orientational movements of the molecules stimulated by the oscillating electric field, phenomenon known by polarisation, did not affect the degradation rates of anthocyanins during ohmic heating. The decimal reduction (D-values) and the half-time (t1/2) values for each experiment are shown in Table 1. For the ohmic heating process, D-values ranged between 309 and 1397 min and t1/2values ranged from 93 to 420 min. For the conventional heating technology, D-values varied from 303 to 1317 min and t1/2-values ranged from 91 to 396 min. Both parameters decreased as the temperature of the process increased. When compared in the same temperature, ohmic and conventional heating showed similar D
The activation energy values (Ea) for ohmic and conventional heating were of 75.5 and 73.9 kJ mol1, respectively (Table 2). The statistical analysis (Student’s t-test) showed no significant difference between the values aforementioned (p > 0.05). The magnitude of the activation energy is an excellent indicator of the sensitivity of the reaction to temperature. According to Heldman (2011b), the activation energy constants for anthocyanin degradation range from 35 to 125 kJ mol1. Higher values indicate strong temperature dependence: the reaction runs slowly at low temperature but relatively fast at high temperatures. The activation energy constants for anthocyanin degradation have relatively low magnitudes as compared to the constants for inactivation of spores and vegetative cells (250–625 kJ/mol) (Heldman, 2011b). The thermodynamic parameters can give valuable information when studying a wide variety of physical, chemical, and biochemical phenomena. The change of free energy (DG–), enthalpy (DH–) and entropy (DS–) of the degradation reactions at all temperatures during ohmic and conventional heating are presented in Table 3. DH– shows the energy difference between the reagent and activated complex; if DH– is small, the formation of the activated complex is favored because the potential energy barrier is low (Georgieva, Zvezdova, & Vlaev, 2012). As shown in Table 3, DH– values were similar for all conditions evaluated in this study (p > 0.05), varying between 70.9 and 72.6 kJ mol1. This indicates that the energy barrier that must be overcome in order to achieve the transition state is similar for both heating technologies. The positive sign of DH– means that anthocyanin degradation is an endothermic reaction, accompanied by the adsorption of heat. DG– is the criteria for equilibrium and spontaneity; the positive sign means that anthocyanin degradation is a nonspontaneous reaction. DG– showed very close values for all conditions evaluated in this study (102.5–104.3 kJ mol1). This suggests that the total energy increase of the system at the approach of the reagents and the formation of the activated complex was similar for both heating technologies. DS– measures the disorder change of molecules in the system and it ranged from 85.1 to 92.3 J mol1 K1. The negative entropy values found in this study suggest that the transition state
Table 1 Parameters of the first-order model for anthocyanin degradation in jaboticaba juice during ohmic and conventional heating. Process
T (°C)
R2
k (min1)d
D (min)d a
t1/2 (min)d a
z (°C)d
a
31.6 ± 0.9a
32.4 ± 0.1a
Ohmic heating
70 80 85 90
0.98 0.98 0.99 0.98
0.0017 ± 0.0001 0.0048 ± 0.0001b 0.0049 ± 0.0001b 0.0075 ± 0.0001c
1397 ± 60 480 ± 14b 470 ± 0b 309 ± 3c
420 ± 18 144 ± 4b 141 ± 0b 93 ± 1c
Conventional heating
70 80 85 90
0.95 0.99 0.97 0.99
0.0018 ± 0.0001a 0.0046 ± 0.0004b 0.0051 ± 0.0001b 0.0076 ± 0.0004c
1317 ± 53a 503 ± 46b 456 ± 6b 303 ± 17c
396 ± 16a 151 ± 14b 137 ± 2b 91 ± 5c
d Results are the average values of two independent experiments (mean of two replicates ± standard error); means in the same column with different lowercase letters are significantly different (p < 0.05).
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Process
kref (min1)b
a
R2 a
0.94 0.96
Ohmic heating
75.5 ± 0.02
0.0038 ± 0.0001
Conventional heating
73.9 ± 0.06a
0.0039 ± 0.0002a
b
Results are the average values of two independent experiments (mean of two replicates ± standard error); means in the same column with different lowercase letters are significantly different (p < 0.05).
Table 3 Thermodynamic parameters obtained for anthocyanin degradation during ohmic and conventional heating. Process
T (°C)
DH– (kJ mol1)c
DG– (kJ mol1)c
a
a
DS– (J mol1 K1)c
Ohmic heating
70 80 85 90
72.6 ± 2.1 72.6 ± 2.1a 72.5 ± 2.1a 72.5 ± 2.1a
102.7 ± 0.1 102.6 ± 0.1a 104.1 ± 0.1b 104.3 ± 0.1b
87.6 ± 6a 85.1 ± 6a 88.1 ± 6a 87.6 ± 6a
Conventional heating
70 80 85 90
71.0 ± 0.6a 71.0 ± 0.6a 70.9 ± 0.6a 70.9 ± 0.6a
102.5 ± 0.1a 102.7 ± 0.3a 104.0 ± 0.1b 104.2 ± 0.2b
91.7 ± 1a 90.1 ± 1a 92.3 ± 2a 91.9 ± 1a
c Results are the average values of two independent experiments (mean of two replicates ± standard error); means in the same column with different lowercase letters are significantly different (p < 0.05).
is more structurally organised than the reactants and, therefore, the formation of activated complex is associated with decrease of entropy. According to Georgieva et al. (2012), a lower activation entropy means that the state of the material is close to its own thermodynamic equilibrium. As a consequence, the material shows little reactivity, increasing the time necessary to form the activated complex. Oppositely, when the activation entropy values are higher, as observed in this study, the material is far from its own thermodynamic equilibrium. In this situation, the system can react faster to produce the activated complex because of the higher reactivity. Table 3 shows that DS– was similar for both heating technologies in all temperatures, which indicates similar reactivity and reaction times. Ohmic and conventional heating technologies showed similar values for all kinetic and thermodynamic parameters (when compared in the same temperature), indicating that anthocyanins have similar degradation pathways during ohmic and conventional heating. These results reinforce the finding that the presence of the electric field is not affecting the mechanism and the rate of anthocyanin degradation.
4. Conclusion This study evaluated the non-thermal effects of electricity on anthocyanin degradation kinetics during ohmic heating of jaboticaba juice. The kinetic rate constants ranged from 0.0017 to 0.0075 min1 and from 0.0018 to 0.0076 min1 for ohmic heating and conventional heating, respectively. Statistical analyses showed no significant differences between the rate constants of the ohmic and conventional heating at the same temperatures. Moreover, D-values, t1/2-values and z-values showed similar values (p > 0.05) for both heating methods. Ohmic and conventional heating also showed similar values for all thermodynamic parameters when compared in the same temperatures. These results indicate that the presence of the electric field strength did not affect the degradation kinetics of anthocyanins. Therefore the anthocyanin molecule is reacting the same way and in the same rate in the medium when both heating methods are applied. This demonstrates similar mechanisms of degradation. Although more studies are required to elucidate the effect of this technology on bioactive compounds stability, these results reveal that ohmic heating
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