Kinetics for the distribution of acrylamide in French fries, fried oil and vapour during frying of potatoes

Accepted Manuscript Kinetics for the distribution of acrylamide in French fries, fried oil and vapour during frying of potatoes Hui-Tsung Hsu, Ming-Jen Chen, Tzu-Ping Tseng, Li-Hsin Cheng, Li-Jen Huang, Tai-Sheng Yeh PII: DOI: Reference:

S0308-8146(16)30814-7 http://dx.doi.org/10.1016/j.foodchem.2016.05.125 FOCH 19274

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

9 December 2015 16 May 2016 18 May 2016

Please cite this article as: Hsu, H-T., Chen, M-J., Tseng, T-P., Cheng, L-H., Huang, L-J., Yeh, T-S., Kinetics for the distribution of acrylamide in French fries, fried oil and vapour during frying of potatoes, Food Chemistry (2016), doi: http://dx.doi.org/10.1016/j.foodchem.2016.05.125

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Kinetics for the distribution of acrylamide in French fries, fried oil and vapour during frying of potatoes Hui-Tsung Hsu a, Ming-Jen Chen*,b, Tzu-Ping Tsengb, Li-Hsin Chengb, Li-Jen Huangb, Tai-Sheng Yehc a

Department of Health Risk Management, China Medical University, 91 Hsueh-Shih

Road, Taichung 40402, Taiwan b

Department of Occupational Safety and Hygiene, Fooyin University, 151

Chin-Hsueh Rd., Ta-Liao Dist., Kaohsiung 83102, Taiwan c

Department of Food Science and Nutrition, Meiho University, 23 Ping-Kuang Rd.,

Nei-Pu, Pingtung 91202, Taiwan

*Corresponding author. Tel.: +886 7 7811151 ext. 5111; fax: +886 7 7826735 E-mail address: [email protected] (Ming-Jen Chen) Department of Occupational Safety and Hygiene, Fooyin University, 151 Chin-Hsueh Rd., Ta-Liao Dist., Kaohsiung 831, Taiwan

1

Abstract Kinetic analysis for the formation of acrylamide in heated foods has been typically performed using only measured data of acrylamide in foods; however, its possible loss caused by release from heated foods into fried oil and air has seldom been considered. The results obtained from the monitoring of acrylamide by frying French fries indicated that acrylamide is distributed in three phases: French fries, frying oil, and air. From the evolved gas analysis of acrylamide and the measured concentration profile of the total acrylamide amount present in these phases, the kinetic behaviour for acrylamide formation does not obey the commonly used model of two-step consecutive reactions during frying, while a lumped kinetic model was proposed for the total acrylamide amount. Moreover, a high acrylamide level in air was observed, implying that, apart from consumers of French fries, fast-food restaurant workers are potentially subject to occupational hazards from acrylamide inhalation.

Keywords: Acrylamide; French fries; Fried oil, Air, Kinetic model; Evolved gas analysis.

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1. Introduction Acrylamide (AA) has been reported to be a neurotoxin, causing degenerative nerve changes via chronic oral intake (Parzefall, 2008), and a possible carcinogen to humans, classified in Group 2A by the International Agency for Research on Cancer. Moreover, the formation of AA has been previously reported in a wide variety of fried and baked potato products (WHO, 2002). From a mechanistic standpoint, the formation pathways of AA during the frying of potatoes are very complex and not well understood: the Maillard reaction between amino acids and carbohydrates, mainly asparagine and reducing sugars, has been recognised as the most probable process for producing AA at frying temperatures greater than 120°C (Mottram, et al., 2002; Yaylayan & Stadler, 2005). The factors affecting the amount of AA formed in French fries mainly depend on the composition of raw potatoes, such as asparagine, reducing sugars and moisture content, as well as frying process conditions such as temperature, time and pH (De Vleeschouwer et al., 2006; Mustafa et al., 2008; Romani et al., 2009). Among these factors, cooking temperature and time have been recognised as the two most significant operating variables for AA formation during frying (Romani et al., 2008). Some statistical regression models using the data gathered under a specified cooking condition or from different investigators were established to predict the concentration of AA in foods as a function of frying temperature and time (Knol et al., 2008; Ghasemian et al., 2011; Chen et al., 2012); however, the main drawback is that marginal information was obtained with respect to AA formation due to the interactive effects of various frying conditions. On the other hand, the knowledge of kinetics with associated influences of the frying conditions could be used to manipulate AA formation levels in foods because kinetic models provide a conceptual framework for explaining the generation of AA during frying (Palazo lu & Gökmen, 2008a). 3

During the last decade, as compared with complex mechanistic models, several relatively simple, kinetic models have been proposed for AA formation under isothermal conditions with a view of describing probable mechanisms for AA formation and elimination (Knol et al., 2005; Claeys et al., 2005; Gökmen and Senyuva, 2006; Gökmen and Palazo lu, 2008). Reducing sugars are the limiting reactant in the Maillard reaction (Morales et al., 2008). Accordingly, in the kinetic studies of AA formation in French fries, most researchers have suggested that kinetic models supposedly obey the hypothesis of two successive irreversible first-order reactions, where reactants asparagine and reducing sugars react to form AA, which then partially decomposes to degradation products (Knol et al., 2005; Claeys et al., 2005; De Vleeschouwer et al., 2006; Gökmen and Senyuva, 2006; Gökmen and Palazo lu, 2008; Palazo lu and Gökmen, 2008b). Meanwhile, the kinetic analysis of two-step consecutive reactions (TSCR) could also be conducted to determine the rate constants associated with AA formation and elimination, using experimental data obtained from frying. In general, kinetic studies of AA formation under isothermal conditions showed that AA formation noticeably increases at a short frying time with frying temperature, while it subsequently decreases at frying temperatures greater than 160°C (Corradini and Peleg, 2006). This result is partly attributed to the enhanced evaporation of AA at elevated frying temperatures (Rydberg et al., 2003), as well as to the thermal degradation of AA in subsequent reactions (Gökmen & Senyuva, 2006; Gökmen & Palazo lu, 2008). Currently, few studies have reported that evaporation or pyrolysis, or both, is the predominant pathway resulting in decreased AA levels during frying. However, when reviewing these kinetic approaches, most kinetic studies focus on the data utilised in the chemical model, i.e., basically using a defined mole ratio of asparagine and reducing sugar, rather than considering actual food processing 4

conditions. In addition, even in real food tests, the AA formation rate has been estimated only using the measurement data from the profile of AA concentration versus frying time in heated foods, such as French fries and baked cut potatoes, without considering the potential amounts of AA released from heated foods into both frying oil and vapour phases. Moreover, non-uniform temperature distribution within the heated potato strip caused by non-isothermal heating can give rise to questions associated with the measurement accuracy of the AA concentration based on unit mass of potato. Previous studies by Gökmen and colleagues (Gökmen et al., 2006; Gökmen & Palazo lu, 2009) examined the geometrical effect of surface area to volume ratio (A/V) of potato strip on the surface and core temperature profiles of potato sample during frying, and indicated that both A/V and frying temperature profile in the potato strip appeared to be critical factors, influencing the concentration of AA formed in French fries and the amount of AA evaporated into the air. These aforementioned results imply that the kinetic information of AA formation around the frying system still needs to be validated. For improving our understanding about the intrinsic kinetics of the formation of AA in French fries during frying, this study aimed to systematically investigate the distribution of AA between French fries, fried oil and air by quantitatively measuring AA in the individual fractions under actual frying conditions. Also, a kinetic model for the formation of AA, considering the total mass of AA in three phases, was proposed on the basis of the lumped-parameter model. Meanwhile, the evolved gas analysis (EGA) of AA was conducted to verify whether thermal decomposition of AA occurred throughout the frying temperature ranging from 135 to 190 ƒC during frying.

2. Materials and methods 2.1. Preparation for sample collection of French fries, fried oil, and vapour 5

Kennebec potatoes (Solanum tuberosum) grown in the Dounan town of central Taiwan were chosen as the target species to produce French fries, as they are predominantly used for making deep-fried or baked potatoes. Potatoes were cut into cylindrical slices using a cork borer, in which the length and diameter of each potato slice was 20 mm and 7 mm, respectively, and the average mass was 0.846 ± 0.032 g, as measured using an electronic balance with an accuracy of 0.0001 g (EL204-IC; Mettler Toledo, Switzerland). The surface area to volume ratio (A/V) of each potato slice was 0.67 mm௅1. A preheated oven was employed to dry the sliced potatoes at 70 °C for 2 h, corresponding to an average drying ratio of 69.83 ± 5.23%, before they were fried. A specially designed dry heat bath consisting of insertion holes, with the diameter and depth of each hole being 52 mm and 60 mm, respectively, was employed for frying potato slices; a glass test tube (50 mm outer diameter × 150 mm length) was vertically fitted in the hole. Fifty millilitres of palm oil (Chang Guann Co., Taiwan) were poured into the test tube. The height of the fried oil in the test tube (40 mm) was lower than the depth of the insertion hole (60 mm) so as to ensure uniform heating of the fried oil. Once the fried oil was heated to a certain temperature, five potato slices were immersed into the fried oil. French fries were prepared under different conditions of temperature (135, 160, 175, and 190 °C) and frying time (1, 2, 3, 5, 7, 9, and 12 min). A 4-channel thermometer, equipped with a real-time data logger, at a resolution of 0.1°C (Lutron TM-947SD, Taiwan) was used to record the profile of fried oil temperatures during frying (Fig. 1a). Meanwhile, the core temperature of French fries was measured by inserting a tip digital probe thermometer with a diameter of 1.7 mm (Lutron TM-947SD, Taiwan) into the centre of the potato slice (Fig. 1b). A vapour sampling system consisted of a 5 cm Teflon tube, a syringe filter holder for 13-mm membrane filters (SX0001300, Millipore, Billerica, MA), silica-gel 6

sorbent tubes (Cat. No. 226-10; SKC Inc., Eighty Four, PA), Tygon tubing (E-3603; Saint Gobain Performance Plastics, Corby, UK), and an air sampling pump (Model 224-PCXR8; SKC Inc.), arranged in series. The other end of the Teflon tube was inserted into the glass test tube through aluminium foil used to tightly cover the top of the glass test tube; the total vapour amount was collected in the glass test tube by the air sampling pump at a rate of 1 L/min during a complete period of frying under a certain defined condition. After each specified length of frying time, French fries were removed from the glass test tube and then rapidly stored in a −15 °C freezer to prevent further formation of AA by residual heat. Meanwhile, 20 mL of frying oil were collected from the glass test tube, the membrane filters were removed from the syringe filter holder, and the silica-gel sorbent tubes separated from the vapour sampling system were also immediately stored in a 4°C refrigerator and tightly sealed for further analysing the AA content.

2.2. Analysis of AA in French fries, fried oil, and vapour AA in French fries, fried oil, and the vapour phase was determined by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). Sample preparation for the analysis of AA in French fries and fried oil was adopted from that reported by Tateo et al. (2010). The extraction procedure of AA from the vapour phase was carried out using the standard method of NIEA A742.10B developed by the Environmental Analysis Laboratory of the Taiwan Environmental Protection Administration at which AA was extracted from the filter by anhydrous ethanol (EAL-TWEPA, 2015). The title of Taiwan EPA NIEA A742.10B method was “Analysis of acrylamide, caprolactam, dimethyl formamide, dimethyl sulfoxide in air by GC-FID”. 7

First, portions of the homogenised French fries or fried oil samples were accurately weighed (1.0 g) and transferred into 50-mL centrifuge tubes. Second, 20 13

µL of internal standard C3-AA (1 µg/mL) (Sigma-Aldrich, St. Louis, MO) and 10 mL ACN (J. T. Baker, Philipsburg, NJ) were added, and the mixture was vortexed for 5 min. Third, defatting was carried out using 5 mL of n-hexane (Tedia, Fairfield, OH), and the centrifuge tubes were vortexed for 30 s and centrifuged at 7000 rpm for 5 min. Next, the n-hexane layer was removed. Defatting was performed two times. After defatting, ACN was evaporated to dryness using nitrogen, followed by reconstitution with 1 mL ACN. Finally, the sample solution was filtered using a 0.2-µm filter before LC-MS/MS analysis. All solutions were prepared using 18 M! -cm deionised water. HPLC analysis was carried out on an Agilent 1260 series HPLC system (Agilent Technologies, Santa, Clara, CA). Positive ion electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) data were acquired on an AB SCIEX QTRAP 5500 (Applied Biosystems, Foster City CA) triple quadrupole tandem MS system. An Agilent SB-C18 (4.6 × 150 mm, 5 µm) column was used. The flow rate and injection volume were 0.8 mL/min and 10 "L, respectively. The complete run time was 13 min. Mobile phase A was 0.01% acetic acid in water, and mobile phase B was 0.01% acetic acid in ACN. Gradient elution was programmed to start with 2% B, increased to 5% B in 2 min, to 10% B in 3 min, to 50% B in 2 min, held for 2 min, decreased to 2% B in 1 min, and held for 3 min before the next injection. The mass spectrometer operatiing conditions were as follows: curtain gas, 20 psi; collision gas, medium; ion spray voltage, 5500 V; temperature, 550 °C; ion source gas 1, 55 psi; ion source gas 2, 55 psi; dwell time, 100 ms; scan type, multiple-reaction monitoring mode. For acrylamide, three transitions were monitored using the settings shown in parentheses: acrylamide 71.8 > 54.6 (DP: 51.5 V, CE: 14.2 eV), 71.8 > 43.7 8

(DP: 51.5 V, CE: 31.5 eV), and 71.8 > 26.7 (DP: 51.5 V, CE: 32.1 eV). For the 13

internal standard acrylamide- C3, one transition was monitored using the settings in parenthesis: 75.0 > 57.7 (DP: 58.9 V, CE: 16.9 eV). The limit of quantification for potato chips, fried oil, silica-gel sorbent and membrane filter was 10 ppb based on the signal to noise ratio (S/N) being greater than 10. At 10 ppb, the S/N ratios for the sample matrices of potato chips, fried oil, silica-gel sorbent and membrane filter were 866, 1600, 2170 and 1750, respectively. The recovery test was performed by spiking 50 ng/mL of standard solution with both 13

acrylamide and C-labelled acrylamide to sample matrices in triplicate runs. The average recovery rates for potato chips, fried oil, silica-gel sorbent and membrane filter were 100.8, 113.3, 113.1 and 117.1 %, respectively. The acrylamide was quantified by matrix-matched calibration curve with standard solution containing 10, 20, 50, 100, 200 µg/mL

13

C3-AA internal standard. Four

matrix-matched calibration curves were constructed for potato chips, fried oil, silica-gel sorbent and membrane filter. The respective r-values for the calibration curves of potato chips, fried oil, silica-gel sorbent and membrane filter were 0.999, 0.998, 0.998 and 0.998.

2.3. Py-GC-MS Analysis of AA For analysing the thermal degradation of AA at different temperatures, pyrolysis–gas chromatography-mass spectrometry (Py-GC-MS) in the evolved gas analysis (EGA) mode was performed using a double-shot pyrolyser-type PY-2010iD (Frontier Lab, Fukushima, Japan) coupled to a 6890N GC instrument and 5975 MSD (Agilent Technologies, Santa Clara, USA). A deactivated stainless steel capillary tube (UADTM, 0.15 mm internal diameter × 2.5 m length; Frontier Labs., Fukushima, Japan) was used to connect the GC injection port to the MS ion source. The injection 9

mode was set to a split ratio of 1/50, with helium as the carrier gas flowing at a constant linear velocity of 50 mL/min. The ion source was controlled at 230 °C with a constant pyrolysis–GC interface temperature of 320 °C. For ionisation, the GC-MS instrument employed electron ionisation (EI) mode at an energy of 70 eV. The EGA curve of AA was obtained when the desorption temperature was increased from 60 to 660 °C at a heating rate of 20 °C/min using selected ion monitoring (SIM) acquisition (m/z 71).

3. Results and discussion 3.1. Mass distribution of AA in French fries, fried oil, and vapor phases Figs. 2a-c shows the concentration profiles of AA formation under different frying oil temperatures (T); the AA concentration was distributed in three phases: French fries (Mf), fried oil (Mo), and vapour (Mv). As stated before, temperature and time (θ) are the two most important parameters affecting AA formation levels during frying (Williams, 2005). As anticipated, Mf in French fries rapidly reached a peak value at θ ≤ 2 min at T ≥ 160 ƒC, whereas Mf did not exhibit a peak at T = 135 ƒC (Fig. 2a). These results are in agreement with those observed previously (Knol et al., 2005; Claeys et al., 2005; Granda & Moreira, 2005; Gökmen & enyuva, 2006; De Vleeschouwer et al., 2006), where kinetic studies for AA formation were conducted for heated foods either using a laboratory model system or under realistic food processing, indicating that higher temperatures, often at T ≥ 160 ƒC, result in a higher peak level of AA produced at a shorter frying time. However, shortly after the peak measurement of AA in French fries, notably, Mf measured at T ≥ 160 ƒC abruptly decreased, and a stable decrease in Mf was observed by factors of 8.2, 5.6, and 1.5 at 190, 175, and 160 ƒC, respectively, for θ ≥ 7 min. At T ≥ 160 ƒC, the AA concentration profile for fried oil (Mo) appeared to be the same as that for French 10

fries, albeit with a slight delay in the decrease of Mo at θ ≥ 2 min. This delay is attributed to the mass-transfer resistance of AA from the inside of French fries to oil phase. On the other hand, at T = 135 ƒC, Mo smoothly increased, which was also similar to the concentration profile of Mf. The decrease in AA formation in both French fries and fried oil at T ≥ 160 °C is attributed to the fact that the chemical structure of AA contains an amide group, which exhibits a high boiling point at around 175–300 ƒC at normal pressure (OSHA, 1992); hence, more AA is vaporised into air from French fries and fried oil at frying temperatures greater than 160ƒC. This hypothesis can also be confirmed by the observations that the AA concentration profile in the vapour phase (Mv) exponentially increased, and the exponential rate of increase became more rapid with frying temperature (Fig. 2c). Furthermore, notably, a time lag existed before the emission of the AA vapour from the oil phase to air by vaporisation: the lower the temperature, the longer the lag time. A similar result has been reported from the online monitoring of the gas-phase generation of AA in food-based models, suggesting that the rate of AA vaporisation is related to that of the internal heat transfer in foods, which is enhanced at higher temperature; this AA vaporization rate in turn rapidly dries it (Cook and Taylor, 2005). As can be observed in Figs. 2a-c, the decreased AA concentration in French fries and fried oil is supposedly attributed partly to the release of AA from French fries to fried oil and its subsequent vaporisation from fried oil to air. Figs. 2d-f shows the mass fraction of AA distributed in French fries (φf), fried oil (φo), and vapour (φv). From Fig. 2d, at θ ≥ 7 min, φf measured gradually tended to attain stable values of approximately 0.02, 0.04, 0.08, and 0.10 at T values of 190, 175, 160, and 135 °C, respectively, indicating that higher frying temperatures can lead to lower φf values. Moreover, based on the profile of the mass fraction of AA present in fried oil (Fig. 2e), higher frying temperature resulted in a greater rate of decrease of 11

φo, where decrease in φo at a θ of 12 min was estimated to be approximately 3.4-, 3.3-, 1.8-, and 1.2-fold corresponding to T values of 190, 175, 160, and 135 °C, respectively, as compared to their individual peak values. As can be observed in Fig. 2f, during frying, φf and φo clearly decreased, while φv increased. The φv values at θ = 12 min were 0.75, 0.68, 0.50, and 0.26 corresponding to T = 190, 175, 160, and 135 °C, respectively, demonstrating that high values of φv are observed at high frying temperatures. Notably, a near-linear increase in φv with frying time was observed; the rates of φv were estimated as 0.065, 0.048, 0.037, and 0.015 θ−1 (r2 range 0.79–0.98) at frying temperatures of 190, 175, 160, and 135 °C, respectively. These results support those of Gökmen and Palazo÷lu (2009) who found that there was a linear relationship between the amount of evaporated AA and the frying time. Moreover, significant attention has been focused on the dietary exposure of AA in French fries; however, in contrast, few studies have been reported on the possible exposure to AA vapour via inhalation of fast-food workers who prepare French fries. The fried oil temperature, constituting typical frying conditions for French fries, generally ranges from 170 to 200 °C for approximately 3–5 min of frying (Cummins et al., 2009; Vinci et al., 2012); notably, when using the data pool obtained under typical frying conditions, the average value of φv was estimated to be 0.19 ± 0.05, nearly 2 times of magnitude higher than that of AA in French fries. This result implies that the health effects associated with occupational exposure among fast food workers are alarming, caused by the inevitable exposure to AA vapour via inhalation under typical conditions for frying French fries. For this reason, it may be noteworthy to investigate the health risk associated with workers exposed to AA from cooking fumes during French fries. In addition, in terms of food consumers and food-service providers, amounts of AA accumulated in fried oil appeared to be significant because of the rather high AA amounts in fried oil under typical frying conditions, estimating 12

that the average φo was 0.71 ± 0.03, i.e., almost 7 times as high as the average φf. Furthermore, the frying process is a continuous heating process, in which AA is first formed within potato slices and subsequently diffuses into oil and eventually evaporates into air. Therefore, there exist no equilibrium partition coefficients between French fries, oil and vapour. It can be seen that there will be more AA being vaporised into the air with the increase of frying time and temperature (Figs. 2c/f), while small amounts of AA may remain bound in potato slices during thermal process (Fig. 2b). From the foregoing discussion, it is apparent that for conducting kinetic analysis of AA formation during frying, the AA concentration profile data should consider not only the content of AA formed in French fries but also the release of AA from French fries into both fried oil and vapour phases. Hence, according to the law of mass conservation of AA formation in the frying test system, the data measured for the kinetic analysis of AA formation should be based on the total concentration of AA (MTAA) estimated by the summation of Mf, Mo, and Mv, as shown in Fig. 3a.  3.2. Kinetics of AA pyrolysis For clarifying whether AA during frying underwent possible thermal decomposition, EGA was conducted for examining the characteristics of AA pyrolysis using pure AA powder. The major consideration of using pure AA powder in oil phase was to reduce the probable interference of a complex series of Maillard browning reaction in potato matrix and to avoid the difficulties of interpretation of the test data because of much more thermal fragments produced from potato matrix in the pyrolysis experiment. Fig. 4a shows the normalised signal intensity (λ) of m/z 71 measured by SIM for AA during pyrolysis. Under nitrogen gas, dry AA powder started to decompose at 80 °C, and it reached nearly 100% degradation at 140 °C, 13

while the degradation of AA powder mixed with fried oil initially occurred at 310 °C, and it eventually decomposed completely at nearly 500 °C. Fig. 4b shows the pyrolysis kinetics of AA using the data obtained from EGA (Fig. 4a), in which the fractional conversion of AA (α) during thermal decomposition at a certain temperature Tn can be defined as Tn

³ Į= ³

i=n

T0 T∞

¦(

ȜdT

T0

i =∞

¦( i =1

Here, λ T

i

and λ T

i −1

i −1

i

i =1

≈ ȜdT

λT +λ T λT

i

2 +λ T

i −1

2

) ∆T (1) ) ∆T

represent the measured signals with respect to heating

temperatures Ti and Ti−1, respectively, and ∆T represents the change in the heating temperature between two consecutive measurements at recording temperatures, defined by (Ti − Ti−1). According to the measured signal profiles of SIM, measured α can be calculated using the discrete form shown in the right term of Eq. (1). Fig. 4b shows the profiles of α versus T. The α value represents the fraction of AA converted into thermal decomposition products. Assuming a first-order reaction (Claeys et al., 2005; Gökmen & enyuva, 2006; Gökmen & Palazo lu, 2008), the pyrolysis rate of AA ( rDP ) can, therefore, be expressed as follows (Kitahara et al., 2010): rDP = k d C AA =

Because the heating rate (

dĮ = k d (1 − Į) dθ

(2)

dT ) was maintained constant, 20 ƒC/min, during the dθ

pyrolysis of AA, and the reaction rate constant ( k d ) was well represented by the Arrhenius law, Eq. (2) can be rewritten as E dĮ = k d∗ exp(− D )(1 − Į) dT RT

(3)

Here k d∗ and E D represent the frequency factor and activation energy of the thermal decomposition of AA, respectively, and R is the universal gas constant (8.314 14

J/mol-K). By taking natural logarithms of both sides of Eq. (3) and fitting the data 2

obtained from Fig. 4b, in Fig. 4c, a strong linear relationship (r = 0.98) was observed dĮ between ln[ dT ] and T −1 at temperatures ranging from 80 to 140 °C for dry AA (1 − Į)

powder in N2 and 310 to 500 °C for AA powder mixed with fried oil. The slope of the straight line was equal to the activation energy divided by the universal gas constant; hence, E D values were estimated to be 91.8 ± 0.1 kJ/mol for dry AA powder in N2 and 181.8 ± 10.5 kJ/mol for AA powder in fried oil, suggesting that a higher ED in AA powder mixed with fried oil means a larger temperature dependence. So, a reaction with a high ED goes much faster and gives a less stable reactant, i.e., AA, at high temperature than a reaction with a low ED. Theoretically, the degree of thermal decomposition depends on the temperature and free volume of the system. As a rule, the thermal decomposition rate in the gas phase is considerably higher than in the liquid phase, whereas the thermal decomposition rate in the solid matrix is the minimal one (Manelis et al., 2003). As expected, the results indicated that dry AA powder in N2 gas and in oil phase started to decompose at 80 °C and 310 °C, respectively. Accordingly, although not direct evidence from the potato matrix, it can be reasonably inferred from the test results that the thermal decomposition of AA in potato matrix should occur above 310 °C. This implies that the thermal decomposition of AA formed in French fries did not occur within the operating temperature during the frying process.

3.3. Kinetic model for AA formation in French fries

In the light of so many factors and their interactions, the common simple assumption for the modelling of the formation of AA in heated foods is not likely to be applicable to actual food processing conditions. Despite these difficulties, several 15

kinetic models of AA formation, based on the considerations of overall reactions, have been constructed to describe the AA formation rate with simultaneous elimination during frying (Knol et al., 2005; Claeys et al., 2005; De Vleeschouwer et al., 2006; Gökmen & enyuva, 2006; De Vleeschouwer et al., 2008; De Vleeschouwer et al., 2009). Among these models, to account for the net result of both AA formation and elimination, the TSCR model has been typically employed to analyse kinetic data because the model gives better fits to experimental data (Claeys et al., 2005; Knol et al., 2005; De Vleeschouwer et al., 2006; Gökmen & enyuva, 2006). By considering the reaction between the reducing sugars and asparagine and AA formation, followed by decomposition into degradation products, the kinetic model of TSCR may be written as follows: k

kd f CGlu + C Asp → C AA → C DP

(4)

Here, CGlu, CAsp, CAA, and CDP represent the concentrations of reducing sugars, asparagine, AA, and degradation products, respectively, and kf and kd represent the rate constants associated with the formation and degradation of AA, respectively. The results from previous studies indicated that the rate of the net formation of AA can be expressed by a second-order reaction of both reactants CGlu and CAsp for AA formation and a first-order reaction of CAA for AA elimination (De Vleeschouwer et al., 2006; Gökmen & Palazo lu, 2008). The observed rate equation for the net formation of AA ( rNet − AA ) is, therefore, given by rNet − AA = rAA − rDP = k f C Glu C Asp − k d C AA

(5)

Here, rAA and rDP represent the rates of AA formation and elimination, respectively. As several studies on the kinetics of AA decomposition have employed the TSCR mechanism, some questions arise about the decomposition of the formed AA, the produced degradation product, and if subsequent decomposition of AA 16

occurs. For instance, based on a real-food investigation, Granda and Moreira (2005) have measured the concentration profiles of AA in potato chips and have reported that a counteracting effect exists, in which the AA formation rate rapidly increased at the early stage of frying and then gradually decreased after some time, albeit AA levels did not decrease in the observed data. Their results, however, are not very conclusive for answering the above questions because of limited available data. Moreover, in a separate study, Knol et al. (2010) have reported experimental evidence for AA formation using a fructose–asparagine model system that AA degradation is not significant during heating, while this result is different from their earlier observation that AA formation using a glucose–asparagine model system follows a degradation route (Knol et al., 2005). Gökmen and enyuva (2006) have also conducted a study using the same fructose–asparagine model system and found that the experimental data fit well with the TSCR model for frying temperatures greater than 150 °C, suggesting that AA decomposition is subject to thermal degradation. Although the contradicting results from different studies are attributed to the experimental conditions employed, a model explaining the AA formation mechanism still needs to be investigated further. On the other hand, to the best of our knowledge, the data used for fitting the proposed kinetic models were typically obtained from chemical models with a defined ratio of reducing sugars to asparagine but seldom from real foods. Thus, herein, a series of runs were conducted using a real-food frying system under well-defined laboratory conditions (see Materials and Methods) for investigating the kinetic behaviour of AA formation in French fries by correlating the experimental data with the kinetic model of TSCR. As shown in Fig. 3a, the total AA concentration–time profiles, i.e., MTAA–θ curve, increased with time, followed by levelling off. However, if the hypothesis for the mechanism of the TSCR model was obeyed, the features of 17

the MTAA–θ curve should theoretically exhibit intermediate behaviour (Levenspiel, 1999), where a peak value for MTAA can be observed during frying, followed by a sharp drop or a gradual decline depending on the frying temperature (Corradini & Peleg, 2006). The TSCR model does not appear to be suitable for explaining the results from the MTAA–θ curve. On the basis of the arguments about the EGA results and the kinetic features of MTAA–θ profiles outlined above, the subsequent thermal decomposition of AA in

French fries is not significant in our real-food testing studies. The proposed kinetic model of AA formation could be simplified by assuming that the kinetic behaviour of AA formation mainly relied on the manipulation of two major factors — oil temperature and frying time — caused by constraining the effects of other factors. This conclusion can be substantiated as follows: a) variations in chemical composition linked to AA-forming potential, such as that of asparagine, fructose, glucose, and sucrose, in the selected test Kennebec potatoes were measured in a narrow range (Vivanti et al., 2006), and b) the moisture content of sliced potatoes reduced to a stable value of 16.8%. As a result, a simple lumped kinetic model modified from Eq. (5) for net AA formation was proposed, and its rate expression can be expressed as follows: rNet − AA = rAA = k f f1 (reactants) f 2 (T , θ )

(6)

Here f1 (reactants) and f 2 (T , θ ) represent the reactant-dependent function and a lumping function of fried oil temperature and frying time for AA formation, respectively, under actual frying with certain constraints on the properties of used potatoes. Furthermore, the rate equation of the net formation of AA ( rNet − AA ) can also be expressed as follows: rNet −TAA =

d [ M TAA ] dθ

18

(7)

The increase in the exponential decay model as listed in Eq. (8) provided a good fit to the experimental data from the MTAA–θ curves (Fig. 3a).

θ τ

(8)

M TAA = M e (1 − exp( − ))

Here, Me represents the asymptotic value of the MTAA–θ curve at the steady state, and

τ represents the characteristic time that quantifies the AA formation rate. By substituting Eq. (8) into Eq. (7), we obtain

rNet −TAA =

d [ M TAA ] θ = τ −1 M e exp(− ) ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! "#$ dθ τ

−1 k As compared to Eq. (6), τ is equivalent to the rate constant f , Me indicates the

characteristic value of the reactant-dependent function

f1 (reactants) , and the

exponential term represents the lumping function f 2 (T , θ ) . Table 1 lists the kinetic parameters of

kf

and Me obtained by fitting the experimental data shown in Fig. 3a

to Eq. (8). Similarly, the rate constant

kf

can be expressed in the Arrhenius form:

k f = k ∗f exp(−

EF ) RT

(10)

k ∗f is the pre-exponential factor, and EF is the activation energy for AA formation. As shown in Fig. 3b, AA formation obeyed Arrhenius kinetics, which allowed for the determination of activation energy using a non-linear regression model. The k ∗f and

EF values were estimated to be 5.26 × 105 ± 1.37 × 105 min−1 and 51.3 ± 1.3 kJ/mol, respectively. Interestingly, in a real food model, a similar result for the estimated EF value was observed from a potato frying process under operating conditions (T = 120–170 °C and water content = 17.6%) similar to those employed in this study (T = 135–190 °C and water content = 16.8%), which used a pseudo first-order kinetic model of AA formation to obtain an activation energy of 62 kJ/mol (Amrein et al., 2006). 19

Table 2 provides a review of the available published data related to the activation energy of AA formation (EF) and/or AA decomposition (ED) during the last decade. Notably, a large variation was observed in the EF values (35–208 kJ/mol) produced from different experiments based on laboratory model systems. Likewise, there was an enormous difference in the EF values between 21.8 and 116 kJ/mol in real food tests. Generally, although the estimated activation energies were considerably empirical, it is difficult to justify discrepancies among measured EF and ED values because of the various testing approaches, different operating conditions, and complex mechanisms of AA formation. However, in contrast to laboratory model systems, the TSCR model that seemed most effective for fitting the data obtained from laboratory model systems was not suitable for describing the kinetic phenomena of AA formation in actual food processing because thermal decomposition was not observed in the frying temperature range of 135–190 °C. As observed in Table 2, this may result from the type of heating medium used, which is an important factor affecting the degree of thermal decomposition of AA. The EGA results may probably explain the difference of the applied heating medium, in that the formation of AA in real-food occurred in the fried-oil phase, in which a rather high temperature was required (310

°C), which was beyond the operating range of fried temperature (135–190 °C), to initiate the thermal decomposition of AA, while AA formation in laboratory model systems generally occurred either in the aqueous or N2 gas phase, leading to a low temperature (80 °C) for initiating the pyrolysis of AA. Theoretically, the pyrolysis of organic compounds involves bond breaking followed by the formation of free radicals (Jiang et al., 2010). Moreover, frying-oil phases exhibit high viscosity; such high viscosity possibly induces the solvent-cage effect, which may decrease the generation efficiency of free radicals and in turn decrease the pyrolysis of AA (Bunyard et al., 2001). Thus, as expected, the results show that the activation energy required to 20

trigger the thermal decomposition of AA in the fried oil phase (ED = 181.8 ± 10.5 kJ/mol) is higher than that of AA in the aqueous or N2 gas phase (ED = 91.8 ± 0.1 kJ/mol). Fig. 3c shows the ratio profile of the degradation rate (kd) derived from pyrolysis test of AA powder in fried oil to the AA formation rate (kf) during the heating process. Interestingly, it can be seen that AA destruction will occur at T > 300 °C, suggesting that there was no significance in AA elimination step at the operating temperature range for frying (135௅190 °C).

4. Conclusions

The actual chemical mechanism for the formation of heat-induced AA in foods is still quite unclear presumably because it is extremely complex and scientifically difficult to obtain mechanistic details of AA formation in heated foods. Nevertheless, several simplified kinetic models for AA formation have been well adopted for the interpretation of experimental results. However, most of the experiments on the kinetic studies of AA formation or elimination have been conducted using laboratory model systems. Even in actual food tests, kinetic analysis has been typically conducted only utilising the AA measured in foods, not considering the possible release of AA in both fried oil and vapour phases from heated foods. This study has demonstrated that the AA formed during a real process of preparing French fries is eventually distributed in three phases: solid (French fries), liquid (fried oil), and air (AA vapour). Previous studies have reported that the concentration profile of AA formation rapidly increased to a peak value at the start, followed by a sharp decline; however, herein, the concentration profile based on the total amounts of AA (MTAA) included in three phases gradually increased and reached a stable value. Consecutive reactions, i.e., thermal decomposition, did not occur within the operating temperature ranging from 135 to 190 °C after AA formation. Thus, the commonly used TSCR 21

model is inappropriate for the characterisation of kinetic behaviour of total amounts of AA formation. According to the results obtained from EGA, the solvent (frying oil) cage effect is hypothesised to hinder the pyrolysis of AA at the operating temperature range for frying (135–190 °C). In summary, this study is of considerable importance and interest both to the scientific and fast food communities, as it provides a detailed insight into the relationship between the intrinsic kinetics of AA formation during the actual frying of French fries and the distribution of the formed AA into both fried oil and gas phases from French fries; it also identifies the types of people at major risk from exposure to AA via inhalation and precautionary measures of appropriately changing reusable frying oil.

Acknowledgement

The study was funded by the Ministry of Science and Technology of Taiwan (MOST 104-2221-E-039-007).

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27

Figure captions Fig. 1. Typical profiles of frying oil temperatures during the frying of French fries (a)

and comparison between frying oil temperature and core temperature of potato slice for French fries during frying at 135 and 190 °C (b). Fig. 2. Concentration profiles showing the distribution of the AA formed from frying potato in three phases of French fries (a), frying oil (b), and vapour (c) at different oil

temperatures during frying. The corresponding profiles of the mass fraction of the formed AA, showing the distribution of AA in French fries (d), frying oil (e), and vapour (f) are shown. Error bars indicate the standard error of the mean for triplicate measurements. Fig. 3. Profile of the total AA concentration, MTAA, formed under different frying oil

temperatures during frying (a); The Arrhenius plot for the formation of MTAA in French fries during frying (b); profile of the ratio of degradation rate, kd, to formation rate, kf, of AA during the heating process (c). Error bars indicate the standard error of the mean for triplicate measurements. Fig. 4. Evolution profiles during the thermal decomposition of dry AA powder in

nitrogen gas and AA powder mixed with frying oil (a); Profile of conversion fraction of AA during the pyrolysis of dry AA powder in nitrogen gas and AA powder mixed with frying oil (b); Arrhenius plot for the SIM signals of AA during pyrolysis at temperatures ranging from 80 to 140 °C for dry AA powder in nitrogen gas and 310 to 500 °C for AA powder mixed with frying oil (c). Error bars indicate the standard error of the mean for triplicate measurements.

28

Temperature of fried oil, T (oC)

(a) 200 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125

190 175 160 135

0

2

4

6

8

10

± 2 oC ± 2 oC ± 2 oC ± 1 oC

12

14

Frying time, θ (min)

(b) 223 212 201 Temperature, T (oC)

190 179 168 157 146 135 Oil temperature during at 135oC Core temperature of potato slice during at 135oC Oil temperature during at 190oC Core temperature of potato slice during at 190oC

124 113 102 91 0

1

2

3

4

5

6

7

8

9

10 11 12 13 14

Frying time, θ (min)

Fig. 1. Typical profiles of fried oil temperatures during the frying of French fries (a) and comparison between fried oil temperature and core temperature of potato slice for

French fries during frying at 135 and 190 °C (b).

29

(d)

(a)

1.0 o

Mf-190 C

o

Mass fraction of AA in French fries, φf

AA conc. in French fries, Mf (ng-AA/g-Potato)

450 o

Mf-175 C

375

Mf-160oC o

Mf-135 C

300

225

150

75

0

190 C o 175 C o 160 C o 135 C

0.8

0.6

0.4

0.2

0.0 0

2

4

6

8

10

12

14

0

2

4

Frying time, θ (min)

(b)

10

12

14

10

12

14

10

12

14

1.0 o

Mo-190 C Mo-175oC

1750

Mass fraction of AA in oil phase, φo

AA conc. in oil phase, Mo (ng-AA/g-Potato)

8

(e) 2100

o

Mo-160 C o

Mo-135 C

1400

1050

700

350

0

0.8

0.6

0.4 190oC 175oC o 160 C o 135 C

0.2

0.0 0

2

4

6

8

10

12

14

0

2

4

Frying time, θ (min)

6

8

Frying time, θ (min)

(c)

(f) 1.0

2100 o

o

Mv-190 C

Mass fraction of AA in vapor phase, φv

AA conc. in vapor phase, Mv (ng-AA/g-Potato)

6

Frying time, θ (min)

o

Mv-175 C

1750

Mv-160oC o

Mv-135 C

1400

1050

700

350

0

190 C o 175 C 160oC o 135 C

0.8

0.6

0.4

0.2

0.0 0

2

4

6

8

10

12

14

0

Frying time, θ (min)

2

4

6

8

Frying time, θ (min)

Fig. 2. Concentration profiles showing the distribution of the AA formed from frying potato in three phases of French fries (a), fried oil (b), and vapor (c) under different fried oil temperatures during frying. The

corresponding profiles of the mass fraction of the formed AA, showing the distribution of AA in French fries (d), fried oil (e), and vapor (f). Error bars indicate the standard error of the mean for triplicate measurements. 30

Total AA concentration, MTAA (ng-AA/g-Potato)

(a)

2700 2400 2100 1800 1500 1200 900

MTAA-190oC MTAA-175oC MTAA-160oC MTAA-135oC

600 300

Fitted model, Eq. 8

0 0

2

4

6

8

10

12

14

16

Frying time, θ (min)

(b)

1.0

-1

kf (min )

0.8

0.6

0.4

0.2

0.0 0.0020

Exp. data 95% Confidence Interval Nonlinear regression, Eq.10 (r2=0.985) 0.0021

0.0022

0.0023

0.0024

0.0025

360

440

0.0026

1/T (oK)-1

(c)

1.6 1.4 1.2

kd /kf

1.0 0.8 0.6 0.4 0.2 0.0 80

120

160

200

240

280

320

400

480

520

Heating temperature,T (oC)

Fig. 3. Profile of the total AA concentration, MTAA, formed under different frying oil

temperatures during frying (a); The Arrhenius plot for the formation of MTAA in French fries during frying (b); Profile of the ratio of degradation rate, kd, to formation rate, kf, of AA during the heating process (c). Error bars indicate the standard error of the mean for triplicate measurements. 31

Normalized SIM singnal intensity, λ (%)

(a)

Dry AA powder in nitrogen AA powder mixed with fried oil

100

80

60

40

20

0 60

120

180

240

300

360

420

480

540

600

660

o

Heating temperature, T ( C)

(b) Dry AA powder in nitrogen AA powder mixed with fried oil

Fractional conversion, α

1.0

0.8

0.6

0.4

0.2

0.0 0

100

200

300

400

500

600

700

o

Heating temperature,T ( C)

(c)

2

ln[(d(α)/dT)/(1-α)]

0

-2

-4

-6

-8

-10 0.0012

Dry AA powder in nitrogen AA powder mixed with fried oil 0.0015

0.0018

0.0021

0.0024

0.0027

0.0030

o

1/T ( K)

Fig. 4. Evolution profiles during the thermal decomposition of dry AA powder in

nitrogen gas and AA powder mixed with fried oil (a); Profile of conversion fraction of AA during the pyrolysis of dry AA powder in nitrogen gas and AA powder mixed with fried oil (b); Arrhenius plot for the SIM signals of AA during pyrolysis at temperatures ranging from 80 to 140 °C for dry AA powder in nitrogen gas and 310 to 500 °C for AA powder mixed with fried oil (c). Error bars indicate the standard error of the mean for triplicate measurements. 32

Table captions Table 1. Kinetic Parameter Estimates for the Formation of MTAA in French Fries during Frying Table 2. Comparison of Activation Energy of AA Formation and/or Elimination under Different Testing Methods, Operating Conditions, and Kinetic Models

Table 1. Kinetic Parameter Estimates for the Formation of MTAA in French Fries during Frying

Frying temperature

a

Kinetic parameters of MTAA formation

T (°C)

kf (min−1)a

Me (ng/g/min)a

r2

135

0.099 ± 0.025

1214 ± 50

0.99

160

0.376 ± 0.020

1335 ± 33

0.86

175

0.482 ± 0.043

1534 ± 29

0.84

190

0.817 ± 0.047

2270 ± 37

0.93

Kinetic parameters are expressed as the meanௗ±ௗstandard error of triplicate measurements.

Table 2. Comparison of Activation Energy of AA Formation and/or Elimination under Different Testing Methods, Operating Conditions, and Kinetic Models

Testing

Heating Operating conditions

approach LMSa

ED

(kJ/mol)

(kJ/mol)

94.4 ± 11

85.1 ± 14

Kinetic model medium

0.2 M equimolar

EF

Water solution

TSCRc

Reference

glucose–asparagine; LMS

LMS

pH = 6.8; T = 120–200 °C 0.1 M equimolar glucose–asparagine; pH = 5.5; T = 120–200 °C 0.1 M equimolar

Knol et al. (2005)

Water solution

TSCR

35 ± 27

g

Knol et al. (2010)

Water solution

TSCR

130.3–208.1d

84.2–158.5

De Vleeschouwer et al.

glucose–asparagine;

(2006)

pH = 4−6; T = 140–200 °C LMS

LMS

5 µmol equimolar fructose–asparagine; T = 120–200 °C 10% equimolar glucose–asparagine with water activities of 0.88–0.99; T = 120–200 °C

Limited water

TSCR

52.1

72.9

Gökmen & ùenyuva (2006)

Dry N2 gas

TSCR

147−171e

60−180

De Vleeschouwer et al. (2008)

AFPb

Potato chips fried under

Fried oil

traditional frying; AFP

AFP

T = 150–190 °C Potato powder under the influence of moisture content (5.0%−60.7%); T = 120–170 °C Potato slices with a water content of 16.8%;

Logistic

21.8

kinetic model Fried oil

Pseudo first-order

Granda & Moreira (2005)

62−116f

Amrein et al. (2006)

51.3 ± 1.3

This study (2015)

kinetic model Fried oil

Lumped kinetic model

T = 135–190 °C a

LMS represents laboratory model system. bAFP represents an actual food process. cTSCR represents two-step consecutive reaction. dThe lower

the pH, the higher the EF and ED. eThere was no significant effect on EF at water activities of 0.88–0.99. fThe lower the moisture content, the higher the EF. gThere was no significance in AA elimination step.

(1)Acrylamide formed during a real process of preparing French fries is distributed in three phases: French fries, frying oil, and air. (2)Thermal decomposition of AA during French fry preparation is not significant. (3)Considering the total acrylamide amount present in these phases, a lumped kinetic model was proposed. (4)The findings of a high acrylamide level in air imply that occupational exposure to acrylamide via inhalation may be a hazard for fast-food workers.

37