Accepted Manuscript

Effect of amino acids and frequency of reuse frying oils at different temperature
on acrylamide formation in palm olein and soy bean oils via modeling system

G. Daniali, S. Jinap, M. Sanny, C.P. Tan

PII: S0308-8146(17)31708-9
DOI: https://doi.org/10.1016/j.foodchem.2017.10.070
Reference: FOCH 21893

To appear in: Food Chemistry

Received Date: 13 June 2017

Revised Date: 2 October 2017
Accepted Date: 11 October 2017

Please cite this article as: Daniali, G., Jinap, S., Sanny, M., Tan, C.P., Effect of amino acids and frequency of reuse
frying oils at different temperature on acrylamide formation in palm olein and soy bean oils via modeling system,
Food Chemistry (2017), doi: https://doi.org/10.1016/j.foodchem.2017.10.070

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Effect of amino acids and frequency of reuse frying oils at different temperature on acrylamide
formation in palm olein and soy bean oils via modeling system

Daniali, G.a, Jinap S.a,b*, Sanny, M,a, Tan C.P.c

a
Department of Food Science, Faculty of Food Science and Technology, Universiti Putra
Malaysia, 43400 UPM, Serdang, Selangor, Malaysia, b Food Safety and Food Integrity, Institute
of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia,c
Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra
Malaysia, 43400 UPM, Serdang, Selangor, Malaysia

Corresponding author: *Email: jinap@upm.edu.my; sjinap@gmail.com

Abstract

This work investigated the underlying formation of acrylamide from amino acids in frying oils

during high temperatures and at different times via modeling systems. Eighteen amino acids

were used in order to determine which one was more effective on acrylamide production.

Significantly the highest amount of acrylamide was produced from asparagine (5987.5 µg/kg)

and the lowest from phenylalanine (9.25 µg/kg). A constant amount of asparagine and glutamine

in palm olein and soy bean oils was heated up in modelling system at different temperatures

(160,180 and 200 ºC) and times (1.5, 3, 4.5, 6, 7.5 min). The highest amount of acrylamide was

found at 200 ºC for 7.5 min (9317 and 8511µg/kg) and lowest at 160 ºC for 1.5 min (156 and

254 µg/kg) in both frying oils and both amino acids. Direct correlations have been found

between time (R2=0.884), temperature (R2=0.951) and amount of acrylamide formation, both at

p<0.05.

1
Keywords: acrylamide, asparagine, frying-oil, deep-frying, glutamine

2
 1. Introduction

Acrylamide is a chemical compound applied in various industries worldwide. In 2002,

acrylamide was found to form naturally in foods cooked at high temperatures (Tareke, Rydberg,

Karlsson, Eriksson & Törnqvist, 2002). Due to significant evidence of carcinogenicity in

experimental animals, acrylamide is considered to be a potential human carcinogen (IARC,

1994). Acrylamide has been detected in foods prepared by commercial processing and in home

cooking. High level variability of acrylamide was observed within particular categories of foods.

These fluctuations were probably attributed to the acrylamide precursors in raw materials,

difference in food composition and variations in process parameters, such as temperature and

time of baking or frying. A number of technologists have been developed to reduce acrylamide

concentration in thermally processed foods based on changing process parameters (time and

temperature of cooking). In this way food materials and pre-treatment unit operations that

modify process parameters can significantly diminish the acrylamide amount in heat processed

foods (Mariotti, Pedreschi, Carrasco & Granby, 2011; Pedreschi, 2011). During deep-frying in

various industries, oils are repeatedly used at high temperatures with an optimal value of 180 ºC,

in the presence of atmospheric oxygen and receive maximum oxidative and thermal abuse

(Andreu-Sevilla, et al. 2008; Frankel, 1998). However, early studies have shown that

unsaturated oils are healthier to use than hydrogenated oils, saturated or animal based fats

(Frankel, 1998; Medina-Navarro, Mercado-Pichardo, Hernández-Pérez & Hicks1,999), Recent

studies have shown that saturated fats are safer and generated the lowest amount of carcinogenic

compound than unsaturated oils (Daniali, Jinap, Hajeb, Sanny &Tan, 2016; Katragadda, 2010).

Studies on model systems of amino acids and sugars have indicated that acrylamide can be

generated from asparagine or from amino acids that can produce acrylic acid either directly such

3
as β-alanine, aspartic acid or indirectly such as cysteine and serine. The main pathway

specifically involves asparagine and produces acrylamide directly after a sugar-assisted

decarboxylation with 1, 2-elimination steps; and the second pathway involves the initial

formation of acrylic acid from different sources and its subsequent interaction with ammonia to

produce acrylamide. Furthermore, certain amino acids such as serine and cysteine were found to

generate pyruvic acid that can be converted into acrylic acid and generate acrylamide when

reacted with ammonia (Yaylayan, Wnorowski & Perez, 2003; Sohn & Ho, 1995).

The structural characteristics of several amino acids and polyamines suggested that following a

Strecker degradation (decarboxylation plus oxidative deamination), or an oxidative deamination

respectively, the formation of 3- substituted propanals occurred. It was already known, on a

qualitative basis, methionine degradation resulted in the formation of methional and acrolein (

Lieberman, Kunishi, Mapson & Wardale, 1965; Tada, Kobayashi & Kogayashi, 1971) and that

some sympaticomimetic amines could be oxidatively deaminated in vitro with the ascorbic acid-

dehydroascorbic acid system (Beyer. 1943).

The formation of acrolein from several new sources under common and widely used physico-

chemical conditions is quantitatively reported. Methionine, homoserine, homocysteine, and

cystathionine in decreasing order, generated significant amounts of acrolein in aerobic

interactions at neutral pH and 100°C with compounds capable of producing the Strecker

degradation of α-amino acids. Such decarboxylation and oxidative deamination of the above

amino acids would result in 3-substituted propanals, structure which according to previous

studies readily decomposes with the formation of acrolein. Interactions of spermine and related

polyamines with these agents, H, O, or with photoactivated acrylic acid, which is an oxidation

product of acrolein and glycerol, produces a significant amount of acrylamide with ammonia

4
(Yasuhara, Tanaka, Hengel & Shibamoto, 2003). To our knowledge no study has been carried

out on the effects of total amino acids on acrylamide formation during frying, with more

emphasis on time and temperature.

The objectives of the study were to identify contribution of amino acids in acrylamide formation

during frying and to determine the influence of time, temperature, asparagine and glutamine on

the formation of acrylamide in cooking oils using modelling system

2. Materials and Methods

2.1. Materials

Soy bean and palm olein oils were purchased from a local market (Serdang, Malaysia). Isotopic

[13C3] acrylamide (isotopic purity, 99%) used as an internal standard was obtained from

Cambridge Isotope Laboratories (Andover, MA, USA) at the concentration of 1 mg/ml; it was

diluted to 50 ppb to form a working solution, which was used in the sample, standard and spiking

acrylamide preparation. Acrylamide (standard for GC, assay ≥ 99.8%) was obtained from

Sigma–Aldrich (Hong Kong, China) in powder form, whereas Oasis MCX 3 cc (60 mg) liquid

phase and Oasis HLB 3 cc (60 mg) solid phase extraction cartridges were purchased from Waters

Corporation (Waters, Milford, MA, USA). Asparagine and glutamin were purchased from

Sigma Alderich (Hong Kong, China).

2.2. Instrumentation

Liquid chromatography–mass spectrometry (LC–MS-MS) analysis was performed on a TSQ

Quantum Ultra (Thermo Scientific, San Jose, CA, USA) triple quadrupole mass spectrometer
5
connected to an Accela High Speed LC quaternary high pressure pump and an Accela

autosampler (Thermo Finnigan, San Jose, CA, USA). An atmospheric pressure chemical

ionization (APCI) source, produced and introduced ions into the mass spectrometer equipped

with X-calibur software (Thermo Scientific, San Jose, CA, USA), was used for separation,

detection and quantification. The analytical column was a porous graphitic carbon Hypercarb

column (2.1 mm × 50 mm ID; 5 µm) (Thermo Electron, Bellafonte, PA, USA) maintained at 45

°C. The mobile phase was 100 % water with the flow rate maintained at 150 µl/min. The

injection volume was 10 μl. Acrylamide was analyzed using the APCI in positive ion mode.

Selective reaction monitored mode (SRM) was acquired with the characteristic fragmentation

transitions m/z 72 > 55 ([M+H-NH3]+) for acrylamide and m/z 75 > 58 for [13C3] acrylamide.

The APCI-optimized parameters were: discharge current, 4 µA; capillary temperature, 250 °C;

sheath gas pressure, 10 arb; and auxiliary gas pressure, 12 arb. Tube lens offset voltages were

optimized for acrylamide using the automated optimization procedure in syringe infusion mode

provided by the manufacturer. The argon collision gas pressure was adjusted to 2.9e-5 psi (1.5

mTorr) for MS/MS.

2.3. Experimental design

Eighteen amino acids were examined to generate acrylamide during frying. A model system was

designed to study the influence of time, temperature, asparagine and glutamine on the formation

of acrylamide in cooking oils. Frying temperature is 180°C and frying time is 3min so the model

system was created from the lower (160ºC) frying (180ºC) to higher(200°C) temperatures. The

effect of three different temperatures (160,180 and 200ºC), five different frying durations (1.5, 3,

4.5, 6 and 7.5 min) and two types of vegetable oils (soybean and palm olein oils) were

determined. The information obtained (the best amount of asparagine, 0.2 mg according to

6
previous study (Daniali, et al. 2016) was added to all samples. Ten grams of each cooking oil

were mixed with 0.2 mg asparagine and 0.2 mg glutamine individually. Subsequently, the

samples were transferred to a glass petri dish and heated up at160, 180 and 200 °C. After

cooling, the acrylamide concentration was determined. The experiment was replicated thrice.

2.4. Sample preparation

Modified methods of previous studies have been applied to extract acrylamide from samples

(Daniali, et al., 2016). The samples were heated and stirred with a magnetic stirrer and allowed

to cool to room temperature. A 0.5 g sample of the heated mixture of oil and amino acids were

transferred to a 50 ml centrifuge tube and the volume was adjusted to 10 mL with 50 ppb 13C3-

labelled acrylamide as an internal standard. Samples were mixed using a reciprovertical shaker

(RS-1, Jeio Tech Co., Gyeonggi-do, Korea) for 30 min at medium speed (ca. 285 pulses/min).

The homogenate was centrifuged using a refrigerated centrifuge (3–18 K, Sigma, Gillingham

Dorset, UK) at 10,000 rpm (10956 RCF×g) for 30 min at 4 ºC. A syringe filter (0.45 µm) was

used to filter an approximately 4 ml aliquot beneath the oil layer. Oasis HLB and Oasis MCX

cartridges were conditioned and equilibrated with 3 ml of methanol followed by 3 ml of water

before being loaded with the filtrate. The filtrate (2 ml) was passed through the Oasis HLB

cartridge (Waters, Milford, MA, USA) gravitationally and discarded. The acrylamide containing

fraction from the Oasis HLB cartridge was eluted with 2 ml of water; the eluent was collected

and then loaded onto the Oasis MCX cartridge (Waters, Milford, MA, USA). The collected

eluent was transferred to an amber vial for LC–MS/MS analysis.

3. Statistical Analysis

7
Analysis of variance (ANOVA) was used to analyze the significance of differences between

acrylamide concentrations in different cooking oils. The ANOVA analysis was performed by

using Minitab (Release 14 for Windows, Pennsylvania, USA). Significance was determined at

the 95% confidence level (p < 0.05), and Tukey’s test was used for further statistical analysis.

3. Results and Discussion

Fig.1 shows the chromatogram of acrylamide. According to Table 1 on the generation of

acrylamide from amino acids during frying, the rate of acrylamide formation were L-Phenyl

alanine <L-Lysine <L-Valine <L-Isoleucine <L-Serine <L-Tyrosine <L-Alanine <L-Histidine

<L-Cysteine <L-Lucine <L- Norvaline <L-Glycine <L- Proline <L-Aspartic acid <L-Thronine

<L-Argenine <L-Glutamine <L-Aspargine. These results indicated that all the amino acids used

in this study were able to produce acrylamide during frying. L-Phenyl alanine and L-Lysine

generated the lowest amount of acrylamide 9.25 and 16.25 µg/kg respectively. The highest

amounts of acrylamide were found in L-Asparagine 5987.50 µg/kg and L-Glutamine 1636.50

µg/kg. L-Aspargine and L-Glutamine were polar and uncharged but L-Lysine and L- Phenyl

alanine were neutral non polar and L- Lysine was charged, acrolein preferentially reacted with

lysine .The reaction of acrolein with a lysine derivative was carried out and acrolein was

bounded to oxidized acrylic acid. This could be the reason for the lowest amount of acrylamide.

Furthermore carbonyl compounds formed in lipid oxidation (acrylic acid) would react with

amino acids, especially asparagine, and produce acrylamide (Yasuhara, et al. 2003). Acrolein

readily formed from glycerol produced from lipid glycerides. Once the acrolein was formed, it

oxidized to form acrylic acid, which subsequently reacted with ammonia from an amino acid to

8
yield acrylamide under high temperatures. Ammonia was formed from an amino acid upon

Strecker degradation with the presence of a carbonyl compound (Schonberg, Moubacher &

Mostafa, 1948). Based on studies of acrolein, acrolein failed to be produced from some amino

acids. These negative results were expected, since unlike methionine, a Strecker degradation of

glutamic acid, lysine, could not give rise to 3-substituted propanals or alternate acrolein-

producing moieties (Schonberg & Moubacher, 1952; Alarcon, 1976). Asparagine and glutamine,

with the same amide moiety at the end of their molecule, were heated and had the same reaction

but asparagines produced more acrylamide which was in agreement with other researchers’

findings. When asparagine and glutamine were heated separately at 180 °C for 30 min, 0.99 µg/g

and 0.17 µg/g of acrylamide were formed respectively (Shibamoto & Bjeldanes, 2009) .

Some amino acids such as aspartic acid and glutamine can generate acrylic acid directly during

their thermal decomposition (Stadler, et al. 2003). Such amino acids require the presence of

ammonia to convert acrylic acid into acrylamide. One of the main sources of ammonia in food is

the free amino acids (Sohn, et al., 1995). Researchers have identified asparagine, glutamine,

cysteine and aspartic acid as the most efficient ammonia generating amino acids under thermal

treatment (Sohn, et al., 1995). Acrolein generation during interaction of diverse amino acids had

been known since decades ago (Schonberg, et al. 1952; Alarcon, 1976), and glycerol, acrolein

and acrylic acid had been found to be producers of acrylamide (Yasuhara, et al. 2003; Alarcon,

1976).

Based on these results, L-Asparagine and L-Glutamine were chosen for further study and a

modeling system was designed to find out the effect of temperature and time on acrylamide

generation during frying. The results showed that the total amounts of acrylamide present in the

cooking oils were positively correlated with the frying temperatures and times. Tables 2 and 3

9
represent the acrylamide generation rate for cooking oils as affected by the heating temperature

and time. Total concentrations of acrylamide increased with longer frying time and temperature,

at 160ºC for asparagine from 343 µg/kg at 1min and 30 sec to 542, 598, 721; with 906 µg/kg at

3, 4.5, 6, 7.5 min in palm olein and for glutamine at the same temperature and same time were

254, 301, 336, 502 and 610 µg/kg acrylamide. Furthermore, at 180ºC and the same set of time,

the presence of asparagine resulted in the production of1802, 2134, 2593, 3010, 3700 µg/kg

acrylamide whereas glutamine produced 1056, 1240, 1345, 1355 and 1400 µg/kg acrylamide.

The same trend of results were also obtained for 200ºC, 180ºC and 160ºC; 3574, 4225, 6588,

7315, 9317 µg/kg acrylamide; also 2096, 3120, 5373, 6030 and 7010 µg/kg acrylamide, for

aspargine and glutamine respectively. Similar patterns were found for soy bean oil at 160ºC in

the presence of asparagine from 383 µg/kg at 1min and 30 sec to 507, 512, 690 and 784 µg/kg at

3, 4.5, 6, 7.5 min in palm olein; and for glutamine in same temperature and same time were 156,

275, 384, 446 and 556 µg/kg acrylamide. Furthermore, at 180ºC and the same set of time were

1608, 1798, 2299, 2831, 3290 µg/kg acrylamide in the presence of asparagine also 976, 1113,

1235, 1317 and 1516 µg/kg acrylamide at same time set in the presence of glutamine

respectively. Also the results at 200ºC were supported by the ones at 160ºC and 180ºC as well;

2096, 2720, 3837, 5105, 8511 µg/kg acrylamide, with 1574, 2256, 3588, 4331 and 5431 µg/kg

acrylamide, for aspargine and glutamine respectively. There were significant differences (p

<0.05) between times and amounts of acrylamide formation and also the same patterns were

found between heating temperatures and amounts of acrylamide formation in both the cooking

oils and amino acids.

The correlation between the amount of acrylamide formation and rate of temperature (R2 =0.951;

p < 0.05) was higher than time (R2 =0.884; p < 0.05) in all samples as shown in Table 4. It had

10
been described that dependence of volatile emissions with temperature was more evident for

alkanals and hydrocarbons (Stevens & Maier, 2008). The data published so far had indicated

that a temperature >120°C was required for acrylamide formation. Acrolein effects can be

formed as a by-product of certain metabolic pathways such as lipid metabolism, glycolysis, the

amino acid turnover or the oxidative deamination of polyamines (Stevens & Maier, 2008;

Shibamoto, Stadler & Lineback, 2009; Esterbauer, Schaur & Zollner, 1991; Mottram, Wedzicha

& Dodson, 2002). The results of this study had been in agreement with the results of other

researchers regarding the volatiles compound and formation of acrylamide: when edible oil had

been heated up to temperatures above 180 °C (roasting/deep-frying) formation of acrylamide

increased as the temperature increased ( Kim, Kim, Lee, Yoo & Lee, 2011),

Nevertheless it can be presumed that thermally treated foods, depending on the type of thermal

treatment, may contain significant amounts of acrylic acid. The formation of acrylic acid during

the heating of oil depends on the fatty acid composition, the heating time and the temperature

(WHO, 2002). . Acrylic acid contents had been reported in the lower trace range up to 20 μg/kg.

In contrast used deep-frying fats showed markedly increased amounts (Ewert, Granvogl &

Schieberle, 2011: Ghilarducci & Tjeerdema, 1995). Depending on the conditions (e.g. oil grade,

temperature, time) 5 to 250 mg acrylic acid/kg of used oil may be released (Li & Ho, 2005;

Yaylayan & Harty‐Majors, Ismail, 1999; Yaylayan & Keyhani, 2000; Umano & Shibamoto,

1987; Fullana, Carbonell‐Barrachina & Sidhu, 2004). Acrylic acid has also been detected in

processed foods, in products containing heated animal fats and plant oils as well as in the volatile

components of certain foods such as fish, bread, poultry and beef (Li, Kimura, Endo, Maruyama

& Fujimoto, 2005).

5. Conclusion

11
All amino acids could produce acrylamide during the process of deep frying. The dependence of

acrylamide formation on temperature had been found to be more evident. The obvious

conclusion therefore was to use the lowest possible temperature and time for reaching the desired

quality and safety of the end products. This had been supported by the past results of other

researchers and their research efforts regarding the total concentrations of volatile compounds

which would increase with higher frying temperatures (Katragadda, 2010; Yaylayan, et al. 2003;

Sohn, et al. 1995).

Conflicts of interest

We have no conflicts of interest to disclose.

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Figure 1. Chromatogram of acrylamide detected by LC-MS/MS

16
Table 1. Data for the generation of acrylamide µg/kg from amino acids during frying
Amino acids Acrylamide contents (µg/kg) Formula
L-Aspartic acid 739.50±21

L-Histidine 227.75±10

L-Phenyl alanine 9.25±1

L-Isoleucine 138.15±11

L-Lysine 16.25±2

L-Alanine 180.25±11

L-Glutamine 1636.50±60

L-Tyrosine 168.75±12

L-Serine 142.75±9

L-Valine 132.50±8

L-Thronine 1098.00±90

L-Asparagine 5987.50±102

L-Cysteine 263.00±15

L-Lucine 461.00±10

Glycine 555.75±17

L-Argenine 1560.50±20

L-Proline 612.25±7

L-Norvaline 546.75±4

17
Table 2. Acrylamide content ( µg/ml) for different frying times and temperatures of palm olein

Amino acids Acrylamide content ( µg/ml) for different frying times (min) at 160 °C

1.5 3 4.5 6 7.5 Control

Asn 343± 43dA 542±45cA 598±31cA 721±45bA 906±31aA -

Gln 254±51dB 301±80 cB 336±51cB 502 ±34bB 610±16aB -

Acrylamide content ( µg/ml) for different frying times(min) at 180 °C

1.5 3 4.5 6 7.5 Control

Asn 1802±89eA 2134± 81dA 2593±128cA 3010± 89bA 3700±81aA -

Gln 1056±68 dB 1240±51cB 1345±69bB 1355±98bB 1400±44aB -

Acrylamide content ( µg/ml) for different frying times(min) at 200 °C

1.5 3 4.5 6 7.5 Control

Asn 3574± 128eB 4225± 174 dA 6588±530cA 7315± 1034bA 9317 ± 739aA -

Gln 2096± 60eB 3120±157 dB 5373± 318cB 6030 ±435 bB 7010±582 aB -

*Similar capital letter in each column shows insignificant difference (p > 0.05).
*Similar letter in each row shows insignificant difference (p > 0.05).

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Table 3.Acrylamide content ( µg/ml) for different frying times and temperatures in soy bean oil

Amino acids Acrylamide content ( µg/ml) for different frying times(min) at 160 °C soy bean oil

1.5 3 4.5 6 7.5 Control

Asn 383±47 dA 507±80 cA 512 ±54 cA 690± 43bA 784±31 aA -

Gln 156±31.3eB 275 ±45.5dB 384 ±45.5 cB 446 ±51bB 550 ±16aB -

Acrylamide content ( µg/ml) for different frying times(min) at 180 °C

1.5 3 4.5 6 7.5 Control

Asn 1608 ± 24eA 1798 ±89dA 2299±82cA 2831± 93bA 3290±14aA -

Gln 976±95dB 1113± 14cB 1235±84cB 1317±58bB 1516±13aB -

Acrylamide content ( µg/ml) for different frying times(min) at 200 °C

1.5 3 4.5 6 7.5 Control

Asn 2096 ± 128eA 2720± 174 dA 3837±530bA 5103± 1034cA 8511 ± 739aA -

Gln 1574 ± 760eB 2256±157 dB 3588± 318cB 4331±435 bB 5431±582 aB -

*Similar capital letter in each column shows insignificant difference (p > 0.05).
*Similar letter in each row shows insignificant difference (p > 0.05).

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Table 4. Correlation between acrylamide formation from amino acids during times and temperatures

Oils Amino acids Time Temperature

R2 P value R2 P value
Soy bean Asn 0.884 0.05 0.951 0.01
Glu 0.865 0.05 0.851 0.01
Palm olein Asn 0.851 0.05 0.941 0.01
Glu 0.845 0.05 0.875 0.01

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Highlights

 All amino acids can be used as a nitrogen source of acrylamide producer

 The highest amount of acrylamide was produced from asparagine
 Direct correlation was found between time, temperature and amount of acrylamide
 Correlation between amount of acrylamide and temperature was higher than time

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