Indonesian Journal of Life Sciences

LABORATORY REPORT

Ochratoxin A Analysis in Various Foodstuffs

Adella Aurel (20010025)1, Chery Tanadel (21010053)1, Farell Adithya

Simon (22010014), Joshia M. Djuharto (21010104)1, Stanislas Gosali
(21010171)1

1
Food Technology, Indonesia International Institute for Life Sciences,
Jakarta, Indonesia

ABSTRACT

Ochratoxin A (OTA) is one of mycotoxins produced by fungus or mold

that is mostly found in food and agricultural products. This toxic substance
can contaminate food and pose a threat to human health. It can cause renal
disease such as chronic interstitial nephropathy (CIN) and Balkan endemic
nephropathy (BEN). Several foodstuffs were tested for its OTA using ELISA
such as rice, roasted coffee beans, dried nutmeg, and corn. It was found
that coffee contains the highest level of OTA, while rice was found to have
the lowest level of OTA. The concentration of OTA is mainly caused by
Aspergillus and Penicillium which are two fungi that contribute the most
towards OTA production. Production of OTA depends on the fungal growth
and type of food. Aspergillus ochraceus were found to produce toxin the
most in coffee, while Penicillium verrucosum was found to produce
ochratoxin the most in rice. The OTA level should be below the regulations
by BPOM in order to avoid any risk of adverse health effects. Further
recommendation can be done by conducting the experiment more cautiously
such as accurate pipetting and balanced centrifugation.

Keywords: Ochratoxin A, Mycotoxins, Toxic, ELISA, Food

INTRODUCTION
Indonesian Journal of Life Sciences

Mycotoxin is a term which refers to the toxic substances that were

originally produced by fungus or mold (Munkvold et al., 2021). According to
Tao et al., (2018), one of the widespread mycotoxins which commonly found
in food and agricultural products is Ochratoxin A or so called OTA. Related to
this, Heussner and Bingle (2015) mentioned that Ochratoxin A is a type of
mycotoxin which mainly produced by several fungal species such as
Penicillium verrucosum, A. niger, A. carbonarius, Aspergillus ochareus.
Mycotoxins including OTA frequently contaminate food products either
directly or indirectly. Direct contamination of OTA could occur as OTA
contaminates the stored products or grain, while indirect contamination of
OTA occurs in food products as the ingredient which is used in the production
process is contaminated by OTA or feed animals that consumed is
contaminated by OTA prior to consumption by humans (Bianchini &
Bullerman, 2014).

Ochratoxin A exposure in food products does not only cause economic

losses, however, it could also become a threat to human health when
exposed towards humans (Wang et al., 2022). Related to this, it is also
mentioned by Bui-Klimke & Wu (2015) that Ochratoxin A exposure could
lead to some renal diseases including chronic interstitial nephropathy (CIN)
and also Balkan endemic nephropathy (BEN) and other renal diseases. Due
to the adverse health effects which could be caused by Ochratoxin A
exposure in the human body, it is important to evaluate the presence of OTA
in order to produce a safely consumable food product. According to Sun et
al., (2019), one of the methods that could be used to detect OTA in food
products is indirect competitive Enzyme-Linked Immunosorbent Assay or
also known as ELISA.

Competitive ELISA could detect the presence of OTA by measuring the

amount of its antigen present in the sample where they are competing with
the standard to bind in a limited number of antibody binding sites. In
competitive ELISA, the more target antigen present in the sample, the more
conjugated antibodies would be bound (Konstantinou, 2017). Thus,
consequently leads to a less amount of unbound antibody to be bound to
coated antigen. After all of the binding process occurred, the washing
process of the medium should be done in order to remove the unbound
antibodies. Then, enzyme substrate would be added and results in the
coloration which indicated a positive result and its concentration could be
measured by utilizing the absorbance of spectrophotometry (Aydin, 2015).
Indonesian Journal of Life Sciences

Furthermore, Competitive ELISA is a method that is frequently used to

determine the presence of antigen presence as Wang et al., (2017) stated
that it is one of the reliable methods to detect a specific compound as it is
sensitive and accurate.

Therefore, the main objective of this experiment is to evaluate the

level of Ochratoxin A contamination in various foodstuffs which originated
from Indonesia by utilizing the Competitive ELISA method.

MATERIALS AND METHODS

The experiment used rice, roasted coffee beans, dried nutmeg, and
corn as the selected foodstuffs samples. Additionally, distilled water was also
needed as the materials of this experiment. The equipment that were used
were safety gloves, scale, laboratory mill/mortar, centrifuge, horizontal
shaker, vortex, graduated cylinder 100 mL, micropipettes,
spectrophotometer (450 nm).

Wash Buffer Preparation

5 g of wash buffer salt were dissolved in 50 mL of distilled water. The

action was taken to obtain a 10-fold concentrated washing buffer, which is a
ready-to-use buffer.

ECO Extractor Buffer Preparation

50 mL of ECO extractor concentrate were dissolved into 500 mL

distilled water as a step to prepare the diluted ECO extractor.

Sample Preparation

The sample preparation started by 10 g of homogenized as well as

ground sample weight into a container. 50 mL of diluted ECO extractor was
then added to the sample and underwent a vortex for 10 seconds. The
sample was then shaken vigorously for 5 minutes and proceeded to be
centrifuge for 5 minutes at 3,500 g, room temperature. Lastly, 1 mL of
ready-to-use wash buffer was utilized to dilute 1 mL of the supernatant.

Test Procedure
Indonesian Journal of Life Sciences

All standards and samples were inserted into the microwell holder that
has been inserted with wells and run in duplications. The placement of each
sample was recorded to ensure no mistakes in the interpretation. 50 µL of
each standard and samples were pipetted in duplications into the according
wells. Afterwards, 50 µL of conjugate were added into each well and was
mixed gently by shaking the plate manually, followed by an incubation for 30
minutes at room temperature in the dark. The liquid of the wells were then
poured out and taped upside down vigorously on absorbent paper ensuring
removal of liquid from the wells and this washing step is run for 3 times. All
the wells were then filled with a 250 µL wash buffer and then the flow
through was poured out similarly to the previous step. Each well was then
added with 100 µL of substrate/chromogen and was shaken manually to
evenly homogenize the solution. After mixing gently, it was incubated for 15
minutes at room temperature in the dark. In order to finish the process, 100
µL of stop solution were added into each well and mixed gently again using
the plate and measured out the absorbance at 450 nm. The reading was
done 15 minutes after the addition of the stop solution.
Indonesian Journal of Life Sciences

RESULTS

Table 1 and Table 2 offer a thorough understanding of the Ochratoxin

A concentration findings in various food samples. Table 1 displays the
correlation between the absorbance values and the corresponding
concentrations and standard deviations for the coffee, rice, nutmeg, and
corn samples. These variations call for close examination. Table 2 shows the
wide range of Ochratoxin A concentrations in different regions by contrasting
these results with well-known references. The following sections will conduct
a thorough analysis, highlighting the implications for food safety and the
necessity of ongoing supply chain monitoring.

Table 1. Concentration Results of Ochratoxin in samples

Sample Absorbance Concentration Standard Deviation

Coffee 0.2409 2.422878788 0.0311411705

Rice 2.730975 -1.349962121 0.06721049955

Nutmeg 1.21015 0.9543181818 0.02849640328

Corn 2.83805 -1.51219697 0.09926760549

The Ochratoxin A concentration results from Table 1 show significant

differences between the examined samples. Coffee had the highest
concentration of all of them, 2.422878788 to be exact, with an absorbance
of 0.2409. On the other hand, corn had the lowest concentration, with an
absorbance of 2.83805, which is equivalent to a concentration of -1.5121.
These results demonstrate the range of Ochratoxin A contamination levels in
widely consumed food items, highlighting the significance of continuing
surveillance and regulatory considerations for food safety.

Table 2. Maximum and Minimum amount of Ochratoxin A in the Samples

Indonesian Journal of Life Sciences

Concentration
Sample Origin Reference
Minimum Maximum

Cambodia 0.19 µg/kg 1.12 µg/kg

Oeung et al. (2022)

Coffee Northern Thailand - 0.39 µg/kg Maman et al. (2021)

Singapore - 2.5 µg/kg Cressey et al. (2011)

China 0.3 μg/kg 3.2 μg/kg Lai et al. (2015)

Rice Iran 0.84 μg/kg 11.37 μg/kg Rahimi (2014)

Lebanon - 4.98 μg/kg Hassan et al. (2022)

Cubero-Leon et al.,
Germany (EU) - 15 µg/kg
(2017)
Nutmeg
Czech Republic 0.3 μg/kg 60.70 μg/kg Pickova et al. (2021)

Albania - 5 μg/kg Topi et al. (2015)

Corn Europe - 3 μg/kg Li et al. (2021)

Pakistan - 3 μg/kg Majeed et al. (2013)

A range of contamination levels can be seen by examining the

Ochratoxin A concentration data in different samples from Table 2.
Interestingly, Oeung et al. (2022) reported that coffee from Cambodia had
the lowest minimum concentration, at 0.19 µg/kg, while Pickova et al.
(2021) reported that nutmeg from the Czech Republic had the highest
minimum concentration, at 0.3 μg/kg. Conversely, the Czech Republic's
nutmeg was found to have the highest maximum concentration of ochratoxin
A, measuring 60.70 μg/kg. By comparison, Maman et al. (2021) found that
rice from Northern Thailand had the lowest maximum concentration, which
was 0.39 µg/kg.
Indonesian Journal of Life Sciences

Figure 1. Standard curve results of ochratoxin concentrations in different

absorbance levels.

The standard curve in Figure 1 shows a decreasing trendline of

concentration in the different absorbance levels of the standard. The
obtained equation from the curve was shown to be y = -0.66x + 1.84 with
an R2 value of 0.528.
Indonesian Journal of Life Sciences

DISCUSSION

Based on the data shown in Table 1 it can be concluded that the

highest concentration of ochratoxin A is found in coffee while the lowest
being in rice. Mold development, especially from Aspergillus and Penicillium
species, throughout the stages of bean cultivation, processing, or storage is
frequently the cause of ochratoxin A in coffee. High humidity, high
temperatures, poor ventilation during storage, and poor harvesting
techniques are all conducive to the growth of mold. In areas with more
moderate temperatures, such as many coffee-growing regions, these
elements may combine to provide an environment that is favorable for the
synthesis of ochratoxin A (Leitão, 2019).

On the other hand, corn in general is known to have low amounts of

ochratoxin A. Corn often maintains low amounts of Ochratoxin A due to
several factors related to its cultivation, processing, and storage. Corn is
generally grown in conditions less favorable for the growth of molds that
produce Ochratoxin A. Additionally, the harvesting practices for corn, when
done at the appropriate time and under proper conditions, contribute to
minimizing mold contamination (Topi et al., 2023). Furthermore, effective
drying methods are commonly employed during the processing of corn to
reduce moisture content, limiting the environment conducive to mold
growth. Adequate storage practices, such as maintaining low humidity and
proper ventilation, further help prevent the development of molds and the
subsequent production of Ochratoxin A (Salasib, 2020).

Aspergillus and Penicillium are the two main contributors of ochratoxin

A (OTA). There are many strains of both fungi and each has different
optimum growth conditions. For instance, Aspergillus carbonarius grows
optimally at 25-30C, Aw of 0.98-0.995, and pH level of 5-6.5 (Belli et al.,
2005). Meanwhile, Aspergillus niger is optimal at 24-37C, Aw of more than
0.95, and pH of 4-6.5 (Passamani et al., 2014). Furthermore, Aspergillus
ochraceus grows best at 25-30C, Aw of 0.99, and pH of 6, and Penicillium
verrucosum produces most ochratoxin at 25C with an Aw of 0.98
(Cairns-Fuller et al., 2005; Gil-Serna et al., 2014). Additionally, the
production of ochratoxin A is dependent on the type of food. For example,
the common fungi that produces ochratoxin in coffee is Aspergillus
ochraceus whereas in rice, Penicillium verrucosum is found to produce
ochratoxin the most.
Indonesian Journal of Life Sciences

The limit of Ochratoxin A level in Indonesia is regulated based on

Peraturan Kepala Badan Pengawas Obat dan Makanan Republik Indonesia
Nomor 8 Tahun 2018. The maximum level in rice and ground coffee is
5µg/kg body weight. They have different regulations for instant coffee,
which is 10 µg/kg body weight. Corn is categorized as cereals and the limit
for ready-to-eat cereal products is 3µg/kg body weight. The regulations have
a limit for aflatoxin levels in spices but do not have a specified limit for
Ochratoxin A in spices. None of the Ochratoxin levels shown in Table 1
exceed the Ochratoxin A regulation in Indonesia. However, since the
standard curve itself has an R-value of 0.528, it means that the data is not
the most reliable. The low reliability result may be due to the pipetting
errors, improper mixing of the samples, and deviation from the
recommended incubation time and temperatures.
Indonesian Journal of Life Sciences

CONCLUSION AND RECOMMENDATION

Ochratoxin A (OTA) is one of the mycotoxins which are mainly

produced by Penicillium verrucosum, A. niger, A. carbonarius, and
Aspergillus ochareus. This type of mycotoxin is commonly found in food and
agricultural products and its exposure could cause renal diseases. Among
coffee, rice, nutmeg, and corn which were analyzed in this experiment, it
can be seen that coffee has the highest level of OTA exposure, while corn
has the least level of OTA exposure. This exposure difference occurs due to
the growth environment and conditions of food samples which affect the
growth of fungi that produce OTA toxin. Furthermore, according to BPOM,
the maximum limit of OTA level varies for each type of food ingredient.

Further recommendations that could be implied to enhance the

reliability of this experiment are to prevent human error including inaccurate
weighing, inaccurate pipetting, and others. Moreover, during the experiment,
an imbalance centrifugation process occurred which could lead to an
inaccurate separation of samples and further create false results. Therefore,
a more proper and flawless procedure has to be implied in order to achieve
more reliable results.
Indonesian Journal of Life Sciences

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APPENDICES

Appendix 1. Absorbance results

Appendix 2. Plagiarism check