Rapid Detection and Quantification of Acrylamide Formation and Its

Precursor Components from Recovered Cooking Oil and Fried Products

based on Different Processing Parameters Using Near-Infrared Spectroscopy

David Tjandra Nugraha

1. Introduction
Acrylamide is one of the organic chemical substance, which is commonly found within
food products related with high processing temperature. Acrylamide itself is an odorless, white
crystal substance that is highly soluble in water. Acrylamide was reported to be one of the
suspected cause of carcinogenic components in human body (Daniali, Jinap, Hajeb, Sanny, & Tan,
2016). In its pure unbounded form, acrylamide has a boiling point and melting point of 125oC and
87.5oC respectively (Pamies & Vilanova, 2014). The monomer form of acrylamide is especially
hazardous and recognized as a neurotoxin. One of the main sources of acrylamide can be found in
fried foods. Acrylamide itself appears in food that has sufficient amount of amino acid and reduced
sugar, in a heated environment. There were findings regarding the pathway of acrylamide
formation. The mechanisms proposed were the decarboxylation and dehydration of amino acids,
degradation of starch, fat, free amino acid (asparagine, alanine, methionine, and glutamine), and
directly formed from asparagine due to its similarity in formation with acrylamide (thermal
breakdown). The first mechanism is reported to be more significant, as a previous attempt of
heating asparagine component did not establish noticeable amount of acrylamide. Although
components that are commonly found in the cooking oil as a by-product of high temperature
processing had been reported as a precursor of acrylamide formation, namely acrolein and acrylic
acid (Dybing & Sanner, 2003; Katragadda, Fullana, Sidhu, & Carbonell-Barrachina, 2010; Pamies
& Vilanova, 2014).
Another point that might be important, there is a possibility of exudation to the heat
transfer media. As cooking oil is the most common heat transfer media in food processing and
food industry, there is a high possibility that the necessary precursors of acrylamide formation
stayed within the cooking oil and concentrate the compounds in the following batch of processing.
Some of the components from the food might be discharged to the cooking oil, and started to cause
the appropriate reactions for acrylamide forming. In this case, the observation of commercial
cooking oil and recovered cooking oil can give us a better idea about the related by-products from
food processing. While the Food and Agriculture Organization (FAO) recommends a maximum
acrylamide consumption within 0.3-0.9 µg/kg of a person’s weight/day, recent findings in the
acrylamide content of fried foods are quite alarming, reporting acrylamide content of 1200 µg/kg
in potato chips, 450 µg/kg in fried fries, and 100-200 µg/kg in cereals and bakery products (Semla,
Goc, Martiniaková, Omelka, & Formicki, 2017). Although there are standardized analytical
methods for acrylamide determination, rapid and efficient quantification methods with good
reproducibility can be developed for more efficient in-line food production, and food safety
measures.
Near-infrared spectroscopy (NIRS) has been recorded to successfully detect acrylamide
compounds from various food products such as fried fries and potato chips with the limit of
detection around 50 µg/kg from a matrix having a range of acrylamide concentration from 50-
8000 µg/kg (Brandl et al., 2002). Lower detection can be achieved using more specific
wavelengths measurement and specified NIRS technique. Utilizing aquaphotomics method that is
taking the advantage of highly sensitive water’s hydrogen bond under the first overtone range of
O-H bonds (1300-1600 nm), hidden spectral pattern can be disclosed, revealing more specific
information with higher sensitivity. This approach will consider and study the changes from water
molecules as a collective matter and energy mirror, and theoretically will be testing the limits of
detection of smaller concentration of compounds (Tsenkova, Muncan, Pollner, & Kovacs, 2018).
Application of chemometrics and multivariate methods such as principal component analysis
(PCA), linear discriminant analysis (LDA), and partial least squares regression (PLSR) are
commonly used in interpreting the spectral data to give a better image of the experiment setup.
The mentioned methods would be supplemented by conventional analytical methods using
chromatographic quantification and verify the robustness of the model from the developed rapid
technique. The results of this study would bring practical implications of rapid and non-destructive
analytical methods in regards of food safety and food processing.
2. Objectives
The proposed research would aim to produce and publish findings related to:
1. Kinetic of acrylamide formation and its precursor availability from different food
processing.
2. Parameters related to acrylamide formation and its precursor components from
different food products.
 3. Rapid quantification of acrylamide and its precursor componetns using near infrared
spectroscopy.
4. Classification and identification of acrylamide containing matrixes for rapid quality
check.
3. Literature Review
3.1. Acrylamide and Formation Pathways
Acrylamide was a major ingredient that was used for coagulating drinking water and waste
water treatment, in the form of its polymer (polyacrylamide). Acrylamide is an unsaturated amide
components that is very reactive with nucleophilic compounds. Acrylamide is a reactive organic
molecule that can undergo free radical reactions. It is found in a white crystal solid at room
temperature with a formula of C3H5NO, and molecular weight of 71.08 Dalton. The structure of
the compound can be seen in Figure 1 (Eriksson, 2005).

Figure 1. Acrylamide chemical structure.

The pathways of acrylamide formation has been reported in previous researches, and one
of the most common compound in most of the pathway is asparagine amino acid. The reaction
scheme for acrylamide formation through asparagine can be seen in Figure 2 (Yasuhara, Tanaka,
Hengel, & Shibamoto, 2003).

Figure 2. Acrylamide pathway from asparagine and carbonyl compound under high temperature.
 There was also a proposal related with Maillard browning precursor might be related with
the acrylamide formation. This theory rose because of the similarity in the precursor components
of both pathway, reducing sugars and amino acids. This theory suggests that the flavor and
browning phenomena from the Maillard reaction, produces acrylamide in parallel of the former
components. A research considering these two main pathways had investigated the acrylamide
formation in browned food products. As mentioned before, acrylamide from asparagine thermal
degradation alone is not significant, resulting in a level of 0.99 µg/g asparagine. The case is not
the same when asparagine was sprayed with acrolein gas at 180oC, where significant level of
acrylamide was detected at 114 µg/g asparagine. Comparing the result of this small experiment,
glutamine was used in place of asparagine, and acrolein gas was sprayed, resulting in a low
acrylamide concentration of 0.18 µg/g glutamine. Alarming concentration of acrylamide was
detected in an experimental setup where ammonia gas and acrolein was heated, resulting in
acrylamide concentration of 753 µg/g ammonia (Yasuhara et al., 2003). Acrolein itself is important
in this regard, as it is the form of a simple unsaturated aldehyde caused by glycerol in the
breakdown of fat products. Acrolein is a clear, colorless or light yellow liquid that is volatile. It
can be easily detected by human olfactory system even at the concentration as low as 0.25 ppm in
air. The structure of acrolein and its oxidized form can be seen in Figure 3 (FDA, 2016; Totani,
Yawata, Takada, & Moriya, 2007).

Figure 3. Acrolein compounds and its derivate.

Acrylic acid as the oxidation product of acrolein, plays a pivotal role in the formation of
acrylamide, as acrylic acid and ammonia under 180oC of temperature produces acrylamide with
astounding 190000 µg/g ammonia, revealing that both ammonia and acrolein has a distinct role in
acrylamide formation, especially in foods that contains a lot of lipid, and in similar environment
(Yasuhara et al., 2003).
3.2. Near Infrared Spectroscopy (NIRS)
Infrared based spectroscopy; commonly known as Near Infrared Reflectance Spectroscopy
(NIRS), is a secondary method that is used to evaluate food products. The characteristics of this
method is rapid, and non-destructive (Osborne, 2000). In these recent years, NIR is often used to
evaluate various food. This method is utilized as a quantitative analysis rather than qualitative
because of its ability to do a rapid test, without hefty sample preparations, and abundant parameters
can be collected at once (O’Sullivan et. al., 1999). NIR instrument is based on the reflection of
infrared radiation hovering near the sample, and the detector measures it. Spectra range of NIR is
780-2.500 nm (13.000-4.000 cm-1) and provides more complex structural information related to
the vibration types of the molecules, and the bonds (Osborne, 2000).
3.3. Statistical Methods
PCA is a method to reduce the dimension of a data set consisting large number of related
variables. The variation of data set is maintained as much as possible by transforming the data set
into new set of variables, the principal components (PCs); uncorrelated data. PCA technique is
considered an orthogonal transformation due to its axes rotation from the original axes (Wold et
al., 1987). PCA was often used to remove the redundant data or noise that have good impacts on
the classification problem. With this feature, PCA was commonly used as an extraction method of
information. More important data or important data that can be used to discriminate two or more
classes can be retained and projected on the principal component. Visualization of principal
components projection can be seen in the figure below (Figure 4.). The solid lines in the Cartesians
graph represent the eigenvectors, while blue and green lines are the projection of the data
components on the first and second principal components (Tharwat, 2016).

Figure 4. Visualized example of PCA technique

The data sample are projected into the two axes with the most variance affecting the data
set. The values in the graphs can also be determined as component scores.
 Similar to PCA, the aim of linear discriminant analysis (LDA) technique is to project the
original data matrix into lower dimensional space. The projection is achieved by calculating the
separability between the classes (between-class variance), calculating the distance between mean
and samples of each class (within-class variance), and construct lower dimensional matrix to
maximizes between-class variance and minimize within-class variance.

Figure 5. Visualized LDA Technique

The solid lines again, represent the eigenvectors in which the data set are being projected.
The separation in this case is done to two of the most dominant principal components (PCs), while
in this visualization; the eigenvector 1 will show greater separation than projection on eigenvector
2. The within-class variance is much smaller, thus projecting on eigenvector 1 will minimize the
within-class variance (Tharwat, 2016).
PLSR is a combination technique from PCA and multiple regression. PLSR is often used
to predict a set of dependent variables from very large independent variables. PLSR predicts Y
from X and describe the common structure. Usually, this feat can be done with an ordinary multiple
regression, but when the number of independent variables are too large, X will likely be singular
and multicollinearity will occurs, making regression no longer feasible. The aim of PLSR in this
case is to find a set of components (latent vectors) that explain as much possible of the covariance
between X and Y. PLSR statistical values can also be used as validation technique in the overall
experiment. The scores from the experiment can be quantified and calculated the error and
correlation coefficient. This can give us better understanding between predicted and experiment
values, in this case, bias is also calculated (Workman, Mobley, Kowalski, & Bro, 1996).
4. Research Design
The research would be divided into segments that would promote better evaluation of the
results, making a proper foundation before moving to the following steps of the experiment. The
proposed segments are:
4.1. First Phase
The first experimental phase would be to collect several commercial cooking oils in the
vicinity of the study. The cooking oil would be analyzed using analytical methods, for both
acrolein and acrylamide components. The analytical methods proposed for this early determination
will utilize chromatographic method for detailed and accurate quantification. The cooking oil
sample will be then adjusted to its processing temperature, going as low as 120oC (the melting
point of acrylamide), and as high as around 220oC, commonly found in food processing industry.
The temperature can be increased in 20oC intervals, and the components will be quantified again
analytically. The components would be scanned also with an NIRS instrument, to analyze the
spectral data and spectral changes after the processing. Chemometrics approach would be used to
classify and study the tendency of the cooking oil from molecular perspective, through the angle
of spectroscopy. The results from the previous quantification can also be used to further create a
model using PLSR based on the spectral data, and create a calibration set that can be tested in
different parameters later on.
4.2. Second Phase
The second phase of the experiment would involve the processing of simple food products
using the said cooking oils. The food products would be in different form factors (such as whole
potato, potato slices, potato fries), and the proximate analysis will be conducted firstly to know the
amount of possible precursors for acrylamide component. The food product will be submerged in
the cooking oils according to the previous temperature for a determined amount of time, and both
the cooking oil and food products will be measured analytically for acrylamide component. Both
the recovered cooking oil and food products will also be scanned using NIRS instrument to analyze
the spectral data, and possibly, creating a model, where rapid quantification is possible.
5. PhD Timeline
Following the Japanese standard for PhD education in 6 semesters (3 education years), the
following schedule is proposed and planned for the fulfillment of this project. Assuming that the
educational year for the graduate school will begin in October the following year.
 2021 2022 2023 2024
No. Research Stage
1 2 3 4 5 6
1. Research proposal
2. PhD confirmation
3. Introduction and institutional background for
the first phase
4. Data collection
5. Data analysis
6. First phase writing
7. Introduction and background for the second
phase
8. Data collection
9. Data analysis
10. Second phase writing
11. Finalization of PhD thesis

6. References

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