ANTONIO, Jazmine C.

BS Biology 4-2 MB
Prof. Ace Bryan S. Cabal Food Microbiology

1. Select a particular pathogen (bacteria, virus, fungi or protozoan) that may be food-borne).
Discuss its biology, epidemiology, signs and symptoms, diagnosis, prevention, and control
measures and how does it cause disease to the exposed individual.
AFLATOXIN, A GLOBAL THREAT TO HUMAN HEALTH
Introduction
In 1960 a pestilence on Turkeys in England decimated thousands of birds which were latter
identified to be caused by consuming feeds, laced with fungi- contaminated Brazilian nuts. The
fungi, Aspergillus flavus, produces a potent compound AFLATOXIN B1 which attacks the liver of
the infected fowls and causes multitude of damage not only to the liver but also other internal
organs (Kumar A. et al, 2021).
In 2003, in Kenya, residents of a rural community consuming maize from locally grown
crops became ill with a disease which severely damaged their liver, causing 120 deaths among
the 350 population who contracted the infection. Again, investigation on the possible causes of
the epidemic narrowed down to the consumption of fungi-contaminated maize, a staple diet of
the Kenyan village. The fungi, Aspergillus flavus, proliferates on the humid and hot conditions of
storage of the maize kernels stored in a damp area.
These two instances of epidemic scale deaths wrought by consuming fungi-
contaminated food supply, from the initial stage of plant growth through the harvesting,
transport, and storage stages, emphasized the vital importance of maintaining the safety of the
food supply against microbial contamination. As the world’s population is dependent on
agricultural products, either local or imported, for their food needs the importance of
monitoring the level of microbial contamination is paramount.

Growth Characteristics of Aspergillus spp.

Aspergillus flavus and Aspergillus parasiticus, the two fungi species which are mainly
responsible for Aflatoxicosis outbreaks, grow best at a temperature range of 28 degrees
Centigrade to 35 degrees Centigrade. A relative humidity of 85% or higher, absence of sunlight,
presence of amino acids, bivalent metals such as Ca, Mg, Zn, sugars such as glucose, ribose,
sucrose, xylose, and glycerol are excellent substrates for fungal growth. The presence of fats
and oils in the substrate promotes the proliferation of Aspergillus fungi. The optimum pH range
of Aspergillus species is 3-7 while the presence of oxygen favors fungal growth but carbon
dioxide retards growth (Kumar et al., 2017)
Toxigenic Products of A. flavus and A. parasiticus
Aspergillus flavus and Aspergillus parasiticus synthesize six main toxigenic compounds
AFB1, AFB2, AFG1. AFG2, AFM1 and AFM2. Aspergillus flavus synthesizes only AFB1 and AFB2,
compounds which emit blue fluorescence when exposed to UV irradiation while A. parasiticus
produces mainly AFG1 and AFG2 which emit green fluorescence under UV irradiation. A.
parasiticus also produces AFB1 and AFB2 in smaller quantities. AFM1 and AFM2 are oxidation
products of AFB! and AFB2 contamination of animal products such as milk and eggs. Of the six
highly toxigenic compounds AFB1 is the most potent and produces a wide range of harmful
effects on the human body.

Origin of Fungal Contamination

A. flavus and A. parasiticus are soil-living organisms. Contamination of crop plants
occurs during the pre-harvesting, harvesting, transport and storage stages when the crop
harvest comes in contact with soil and fungal spores floating in air. A wide range of crop plants
serve as host for the Aspergillus fungi such as cereals, (maize, rice, sorghum, millet, wheat), oil-
producing seeds (peanut, soybean sunflower cotton), spices (chilies, black pepper, turmeric,
coriander, ginger), nuts (almond, walnut, Brazilian nut, coconut) yam and milk products. These
wide range of crops intended for both human and animal consumption afforded the Aspergillus
fungi a diverse source of food supply for growth and proliferation.

Methods of Detection and Quantification of Aflatoxin

Older methods of measuring levels of AFLATOXINS such as High Performance Liquid
Chromatography (HPLC), Thin Layers chromatography (TLC) and liquid Chromatography- Mass
Spectroscopy (LC-MS) are now being replaced by simpler, cheaper, faster and more sensitive
detection and measurement methods such as ELISA (Enzyme-linked Immunosorbent Assay),
Polymerase Chain Reaction (PCR), Fluorescence Near Infra-Red Spectroscopy (FS/NIRS), Hyper
Spectral Imaging (HIS). Newer methods of detection and quantification are also more sensitive
which can detect Aflatoxin contamination down to 0.03- 0,05 ng/ml. This detection limit applies
to Time-Resolved, Fluorescence Immune Chromatography Assay (TRFICA) and anti-idiotype
nanobody- Phage Display mediated Immune – Polymerase Chain Reaction (PD-IPCR) The latest
method of detection using Biosensors permits rapid detection of Aflatoxin contamination under
field conditions and are fast, portable, much cheaper with minimal sample pretreatment.
These new methods of detection and quantification meet the requirement for rapid
identification and quantification because human lives as well as feed- based livestock’s’ safety
are at stake. Availability of fast, reliable, and economical methods of detecting Aflatoxins will
save lives as well as reduce economic losses.
The US Food and Drug Administration set an upper limit of 20ppb for Aflatoxin contamination
on food crops intended for human and animal consumption while the European Union has set a
more stringent upper limit of 2-4 ppb for imported agricultural crops. These upper limits are
well within the capability of both the older methods as well as newer methods of detection and
quantification of Aflatoxin contamination.
Current Practice to Minimize Aflatoxin Contamination of Agricultural Crops and Animal
Byproducts
Current practices to minimize or mitigate Aflatoxin contamination of food crops are
divided into three methods- Physical, Chemical and Biological Methods (Kumar et al., 2021).

 Physical Methods
Roasting at 150 degrees Centigrade for at least 15 minutes, heating using steam
under pressure, exposure to sunlight, UV, Gamma, and Infrared radiation were found to
be effective and reduce Aspergillus fungi growth to a significant degree. Heating
methods, however, may degrade the nutritional content and palatability of foods
intended for human consumption.
 Chemical Methods
Exposure to C02, Ozone, Ammonia reduces the level of aflatoxin compounds.
Chemicals such as sodium bisulfite, calcium hydroxide, formaldehyde, sodium
hypochlorite, sodium borate, citric acid, food preservatives such as benzoic acid, boric
acid, crystal violet, sodium acetate, propionic acid inhibit the growth of aflatoxin fungi.
 Biological Methods
Various microorganisms such as Flavobacterium aurantiacum B-184 and
antagonistic strains of Pseudomonas, Trichoderma, Ralstonia, Lactobacilli, Burkholderia,
Bacillus spp. were found to reduce growth of Aspergillus fungi when introduced to pre-
harvest crops. A combination of Biological and Chemical methods would be a better
option, but it has not yet been tested.
Besides these methods of minimizing Aspergillus contamination, the avoidance of
conditions favoring fungal growth must be observed during storage of harvested crops.
Aspergillus fungi grows best at around 30℃ temperature and high relative humidity of 85% and
above. Reducing the storage temperature to 5-℃ and Relative Humidity below 30% will retard
fungal growth. However, these storage conditions are rarely met because of financial
constraints. At best, periodic microbial tests to detect presence of Aspergillus fungi will provide
timely warning to institute remedial measures immediately.

Medical Diagnosis for Aflaxitosis

Aflaxitosis is a serious medical condition that warrants hospitalization. It can be
classified into 2 groups- Acute Aflaxitosis due to the ingestion at one instance of large quantities
of Aflatoxin- containing food and chronic Aflaxitosis due to the consumption of smaller
quantities of Aflatoxin –containing food over a period (Dhakal, et al, 2023).
Acute Aflaxitosis is manifested by fever, nausea, vomiting, abdominal pain, bleeding,
digestion problems, edema, malabsorption, mental changes, convulsion, coma and ultimately
death. Chronic Aflaxitosis results in impaired growth and stunted growth of children, immune
system suppression, weight loss, abdominal mass, anorexia, vomiting, nausea, bleeding, and
psychosis.
Both acute and chronic Aflaxitosis ultimately lead to hepatocellular carcinoma.
Hospitalization to mitigate symptoms of Aflaxitosis will reduce the incidence of fatality. Since
there is no known antidote for Aflaxitosis removal of contaminated food and optimal dietary
regimen and supplementation with antioxidant vitamins will aid recovery. Remedial measures
to mitigate severe symptoms are best done under a hospital setting.
Once hepatocellular carcinoma is diagnosed then treatment options include the
following: surgical resection, thermal ablation, radiotherapy, systemic chemotherapy + radio
embolization, trans arterial embolization. The survival of the victim ultimately depends upon
the person’s health status, nutritional status, immunity, and age. Children are more at risk for
morbidity and death than adults.

Etiology of Aflaxitosis
Of the 6 major aflatoxin AFB1 is the most potent. Once it enters the human body it
preferentially deposits in the liver where it is converted to AFB! 8, 9 – epoxide by the
cytochrome p-450 system in the liver. The AFB1 8,9- epoxide then interacts with DNA causing
damage by lipid peroxidation or by oxidation reactions and ultimately to hepatocellular
carcinoma. The epoxide causes mutation on the third base of codon 248 of the p53 system
which guards the cell from any gene mutation. Apart from liver damage Aflatoxin also
negatively impairs the functioning of the kidney, heart, testes, and brain.
2. How will you detect this pathogen in food? Design a schema on how you go about the
detection? Explain your diagram.
Traditional methods of detection such as initial culturing of the food sample and
microscopic examination latter on are too slow and can only serve as confirmatory validation of
the identity of the Aspergilllus fungi. Faster methods of detection such as HPLC, TLC and LC-MS
are costly, tedious and require skilled personnel. The latest detection techniques such as ELISA,
PCR and High Spectral Imaging are faster, more sensitive, and cheaper than the previously
mentioned methods. Field testing for Aspergillus contamination is desirable to detect as early
as possible Aspergillus contamination and the latest method of Biosensor detection holds great
promise for this requirement. A combination of methods will ensure that the Aspergillus fungi is
correctly identified. As there are Aspergillus species which are non-aflaxitogenic correct
identification under field conditions is important. Below is a flowchart for the accurate and fast
identification of A. flavus and A. parasiticus.
Figure 1. Flowchart for the accurate and fast identification of A. flavus and A. parasiticus
 Upon receipt of an agricultural crop- either from the field or from storage, part of the
sample is tested for AFLATOXIN contamination using a portable Biosensor. If The Biosensor
indicates presence of AFLAXITOGENIC Fungi further confirmatory test using ELISA or PCR is
made before testing for Aflatoxin presence. If Aflatoxin is present, it is measured using Time-
Resolved Fluorescence Immuno-Chromatographic Assay (TRFICA) or anti – idiotypic nanobody-
Phage Display-mediated Immune –Polymerase Reaction (PD-IPCR) which are sensitive down to
0.03ng/ml. If the results of the quantitative measurements showed Aflatoxin level to be less
than 20ppb then the food crop is stored at low temperature of 5-10degree centigrade and
Relative Humidity of less than 30% prior to farther processing. If Aflatoxin level is above 20 ppb
the agricultural cop is subjected to either chemical or biological mitigation to reduce Aflatoxin
levels below 20ppb.
In the meantime, part of the sample is cultured in a microbiological laboratory then
tested via ordinary methods of identification such as microscopic examination to confirm
identity of the fungi. If the fungi are identified as either A. flavus or A. parasiticus then
measurement of Aflatoxin level will determine whether it is to be subjected to further Aflatoxin
reduction methods.

Conclusion
The detection, measurement, and mitigation of the effects of Aflatoxin contamination of
the world’s food supply is of vital importance to the world’s human population as well as to
livestock animals dependent on man for their food. This is particularly true for third world
countries such as the Philippines where a combination of high temperature, high humidity, and
poor storage conditions of food crops such as rice and corn foster the rapid growth of
Aspergillus fungi. It is estimated that about 30% of the world’s agricultural crops are
contaminated with Aflatoxigenic fungi and this causes significant damage to human health as
well as economic losses to agricultural production. A particular example is rice production
which is the staple food of half of the world’s population. It was discovered that rice samples
contained Aflatoxin levels above the 20-ppb limit of the USFDA and well above the 2-4 ppb limit
of the European Union. This high Aflatoxin contamination poses a great risk to the health of
most of the world’s population, particularly children, and urgent measures should be instituted
by many nations to address the situation.

References
Dhakal, A., Hashmi, M. F., & Sbar, E. (2023). Aflatoxin Toxicity. In StatPearls. StatPearls
Publishing. http://www.ncbi.nlm.nih.gov/books/NBK557781/
Kumar, A., Pathak, H., Bhadauria, S., & Sudan, J. (2021). Aflatoxin contamination in food crops:
causes, detection, and management: a review. Food Production, Processing and
Nutrition, 3(1), 17. https://doi.org/10.1186/s43014-021-00064-y
Kumar, P., Mahato, D. K., Kamle, M., Mohanta, T. K., & Kang, S. G. (2017). Aflatoxins: A Global
Concern for Food Safety, Human Health and Their Management. Frontiers in
Microbiology, 07. https://doi.org/10.3389/fmicb.2016.02170