The Aflatoxin Contamination On Selected Food Supplement Using Enzyme-Linked Immunosorbent Assay

Paulita Grace Villanueva Agudelo (pgagudelo2014@gmail.com)
Graduate School Department, Centro Escolar University
March, 2014
 
PERSONAL DATA
Paulita Grace Villanueva Agudelo residing at 1217-A Casanas St. Sampaloc Manila. I'm 30 years old and was born on September 28, 1984. I'm a graduate of BS Pharmacy at Centro Escolar University in the Philippines and also taking up Masteral of Science in Pharmacy in the same school. Currently employed as a branch pharmacist at South Star Drug Incorporated which is one of the leading drugstore in the Philippines.
 

Abstract

Abstract

Aflatoxins are produced as secondary metabolites by the fungus Aspergillus flavus and Aspergillus parasiticus. Aflatoxins are carcinogenic mutagenic, teratogenic, and immunosuppressive to most animal species and humans. Humans are exposed to aflatoxins via risky foods such as agricultural crops and dairy products. The Food and Drug Administration sets an action level for aflatoxins at 20 ppb for all foods including animal feeds and 0.5 ppb for milk and dairy products. The Joint FAO/WHO Expert Committee on Foods Additive recommended maximum tolerable daily intake of 4 ppb of total aflatoxins in cereals, wheat, and products for direct human consumptions and 0.05 ppb on milk, and other dairy products. This study aimed to determine the aflatoxin contamination on selected food supplements such as wheat, cereals, and milk which are commercially available in the market, and to compare which of the products that contain aflatoxin is in conformance with the FDA, and FAO/WHO acceptable level.

For this purpose, a total of 27 samples consisting of 9 brands of food supplements in 3 different lot numbers, with 2 representations in each samples were obtained from different drug outlets in Metro Manila. The food supplements were labeled as wheat 1, wheat 2, and wheat 3 for the wheat food supplements. For the cereal food supplements were labeled as cereal 1, cereal 2 and cereal 3. And as for the milk food supplements were labeled as milk 1, milk 2 and milk 3. Enzyme-Linked Immunosorbent Assay AgraQuant Total Aflatoxin Test Kit (4-40ppb) was used to determine the aflatoxin contamination on the food supplements. Statistical treatment using mean and standard deviation was done to show the difference on the total aflatoxin concentration on the selected food supplements.

Based on the ELISA results and statistical treatment, most of the food supplements used in this study were found to have aflatoxin contamination. Wheat 1 product but still in conformance with the FDA acceptable level, however as for the FAO/WHO recommended maximum tolerable limit only wheat 2 products does not conformed.

Keywords: aflatoxin, aflatoxin contamination, ELISA, food supplements, FDA, and FAO/WHO acceptable level


1. Introduction

Food supplements were defined as a product that contains vitamins, minerals, herbs or other botanicals, amino acids, enzymes, and other ingredients intended to supplement the diet, it is also known as Dietary supplements. The Food and Drug Administration has a special labeling requirement for food supplements and treats them as foods, not drugs. Unlike drugs, food supplements are not intended to treat, diagnose, prevent, or cure diseases (FDA Food Supplements, 2008)

The use of food supplements is increasing not only in many industrialized countries but also in the Philippines, many of whom believe that being natural products, food supplements are harmless. Using the term natural on a label will fool many into believing that food supplements will do no harm. Some supplements do provides benefits to certain consumers, but there are also side effects that can be experienced because food supplements are frequently contaminated with toxigenic fungi generated from the soil, or from the plants themselves, during harvesting or storage. Esplanada, (2010)

According to Weaver and Truckess (2010) aflatoxins are naturally occurring fungal toxins causing serious food quality and safety issues worldwide. Aflatoxins are produced as secondary metabolites by the fungus Aspergillus flavus and Aspergillus parasiticus they can affect a wide range of important agricultural products. Among the 18 different types of aflatoxins identified the major members are aflatoxin B1, B2, G1, and G2 which is usually found in foods and feeds such as corn, peanuts, wheat, cereals, rice, and spices. And the hydroxylated aflatoxin derivatives, aflatoxin M1 and M2 are presented in dairy products such as milk and cheese from animals that had been feed with toxic meal. Fungal growth and and aflatoxin contamination are the consequence of interactions among the fungus, the host, and the environment. Water stress, high temperature stress, and insect damage of the host plant are major determining factors in mold infestation and toxin production.

Food and Drug Administration sets an action level for aflatoxins at 20 ppb for all foods including animal feeds. Studies conducted demonstrated that levels of aflatoxins below 20 ppb could be fed without presenting a danger to the health of the consumers and animals. The Joint FAO/WHO Expert Committee on Foods Additive implemented in March 2001 a recommended maximum tolerable daily intake of 4 ppb of total aflatoxins in cereals, wheat, edible nuts, and ground nuts for direct human consumptions and 0.05 ppb on milk and dairy products in some parts of the world such as European Union, Latin America, and Africa.

Aflatoxin is a chemically stable compound that is odorless, colorless, and tasteless. Due to the low concentrations involved and the uneven distribution aflatoxins are difficult to detect. Several methods of aflatoxin determination are developed, including the Enzyme-Linked Immunosorbent Assay. ELISA has become a common rapid method for detection of aflatoxins in food and animal feeds this method provides a quicker and simpler assay and is used where speed and simplicity are at premium.

1.1 Objectives

The research study hoped to determine the aflatoxin contamination in selected food supplements using the Enzyme-Linked immunosorbent Assay. The following are the main objectives:

1. To determine the aflatoxin contamination in selected food supplements.

2. To determine the concentration of aflatoxin present in each sample.

3. To compare which of the product that contains aflatoxin is in conformance
to FDA and FAO/WHO acceptable level.

2. Methods and Procedures

2.1 Collection of Samples

A total of 27 samples were used consisting 9 different brands of food supplements in three different lot numbers, with two representations in each samples. The samples were analyzed in duplicate. The food supplements were selected based on the criteria considered by the researcher which includes; must have an active ingredients that may possibly contain aflatoxin, all samples must be a registered products listed in the FDA data, lot numbers of the products, date of manufacture and expiration of the products, the place of manufacture whether locally produced or foreign, availability of the products, popularity of the food supplements, and it must be made for the consumption of different age group.

The selected food supplements were obtained in different drug outlets in Metro Manila from April 2011 to November 2013. The samples were labeled as; Wheat 1(312 AKDV, 312 ANAV, and 312 APAV), Wheat 2 (M048SIOE, M007SIOH, and M027SIOJ), Wheat 3 (93100, 93015, and 93013), Cereal 1 (11701135VE, 11931135RA, and 12741135VF), Cereal 2 (12170597A1K1, 12440597A1B1, and 12250189A1J1), Cereal 3 (12003, 10033, and 12533), Milk 1 (B101412A, P211825B, and P211652C), Milk 2 (120801893A, 12401893B, and 122501893A) and Milk 3 (6B02, 6B18, and 6A19). The sample food supplements were properly stored until the analysis.

2.2 Sample Preparation

Sample preparation procedures were performed by adding 25 ml of 70% methanol to 5 g of the samples. Afterwards, the solution was vigorously shaken in 3 minutes, and allows the samples to settle. The extract was filtered through a Whatman #1 filter paper and filtrate was collected; 50 L of samples per well was used in the assay.

2.3 Test Procedure of Total Aflatoxin

An appropriate number of dilution well were placed for each standard total aflatoxin solutions and sample solutions. Using 8 channel pipette, about 100 L of Conjugate solution was dispensed into each dilution well. An equivalent amount of 50 L of each standard total aflatoxin solutions and sample solutionswas added using a single channel pipette into the appropriate dilution well containing 100 L of Conjugate solution and it was mixed carefully by pipetting up and down 3 times.

About 100 L of the standard total aflatoxin solution and sample solutions from each dilution well was immediately transferred into the corresponding Antibody coated microwell and it was incubated at room temperature for 15 minutes. After incubation period the standard total aflatoxin solutions and the sample solutions were washed by filling each microwell with distilled water, and dumped the water from the microwell strips for five times. The microwell strips was tapped with dry towels to expel as much residual water as possible, and it was dried using a dry cloth.

About 100 L of Substrate solution was dispensed into each microwell and it was observed that the color of the solutions was turned into blue; afterwards it was incubated at room temperature for another 5 minutes. After incubation period the solutions were treated with an equivalent amount of 100 L Stop solution, the color of the solutions changed from blue to yellow. An ELISA plate reader using 450 nm filter with 630 nm differential filter was used to read the strip and measure the absorbance level.

2.4 Statistical Treatment

Mean and standard deviation was done to show the difference on the total aflatoxin concentration on the selected food supplements. Data processing and statistical analysis was conducted at the Centro Escolar University Center Evaluation and Data Processing Department

3. Results and Discussion

3.1. To determine the aflatoxin contamination on selected food supplements.

The presence of aflatoxin was determined with the use of AgraQuant Aflatoxin (4-40ppb) Quantitative ELISA Kit which is a direct competitive ELISA. Based on the results, only wheat 2 and wheat 3 samples were found to have aflatoxin contamination on wheat products. For the cereal products aflatoxin contamination was detected on cereal 1, cereal 3, and lot numbers 12170597A1K1 and 12250189A1J1 on cereal 2 samples.Levels of total aflatoxin were also found on lot numbers 120801893A and 124401893B on milk 2 samples, and lot number 6A19 on milk 3 samples.

3.2 To determine the concentration of total aflatoxin present in each sample.

Absorbance level of the samples and the standard solutions were measured using ELISA microwell reader. Absorbance value of the standard and samples were used to compute for the concentration of the total aflatoxins. The concentration of the total aflatoxin of the samples was calculated using the Romer Log/Logit Excel Spreadsheet which when the measured absorbance value of the samples was encoded in the spreadsheet the concentration is automatically computed and a graphically interpreted showing a straight line which verify the validity of the results. Statistical treatment using mean and standard deviation was used to determine the difference on the concentration of the total aflatoxin on the selected food supplements.


Table 1
Total Aflatoxin Concentration on Wheat Products

Product Lot Number Absorbance Concentration Mean Std. Dev.
Wheat 1 312 AKDV 1.510 0
312 ANAV 1.271 0 0 0
312 APAV 1.565 0

Wheat 2 M048SIOE 0.675 5.47
M007SIOH 0.705 5.08 5.09 0.35781
M027SIOJ 0.732 4.75

Wheat 3 93100 1.119 1.19
93015 1.202 0.52 1.0733 0.4627
93013 1.079 1.51

Data concerning the presence of total aflatoxin on wheat products are summarized in Table 1, the results showed that only Wheat 1 samples indicates the absence of aflatoxin contamination.Wheat 2 products used in this study were found out to be contaminated with aflatoxin based on the computed mean. The results proves that the amount of total aflatoxin concentration on these products show almost identical concentration with slight deviation from each other as indicate on the computed standard deviation.Among the wheat 2 products lot number M048SIOE has the highest total aflatoxin contamination, followed by lot number M007SIOH. And lot number M027SIOJ is the least contaminated wheat 2 product.

The computed mean proves that wheat 3 products were positive with total aflatoxin contamination, and it was verified on the computed standard deviation that the level of
aflatoxin concentration on these products was slightly different from each sample. Wheat 3 product on lot number 93013 has the highest level of total aflatoxin. Followed by lot number 93100 and the least contaminated among wheat 3 product is on lot number 93015.


Table 2
Total Aflatoxin Concentration on Cereal Products


Product Lot Number Absorbance Concentration Mean Std. Dev.
Cereal 1 11701135VE 1.159 0.88
11931135RA 1.206 0.49 0.853 0.4787
12741135VF 1.119 1.19

Cereal 2 12170597A1K1 0.957 2.50
12440597A1B1 1.350 0 1.306 1.1381
12250189A1J1 1.090 1.42

Cereal 3 12003 1.119 1.19
10033 1.202 0.52 1.0733 0.4627
12533 1.079 1.51


Aflatoxin contamination is common on cereal products, 3 brands of cereal food supplements were used in this study to find out if these products are free from aflatoxin contamination. Table 2 showed the presence of aflatoxin in the examined cereal samples. Based on the computed mean indicates the presence of aflatoxin contamination on the selected Cereal 1products. The results confirmed that lot number 12741135VF presented the highest contamination. Followed by lot number 11701135VE and the lowest level of aflatoxin contamination were detected in lot number 11931135RA. The concentration on the amount of total aflatoxin on the selected Cereal 1 products was quite different from each other based on the computed standard deviation.Cereal 2 products were found to be positive to aflatoxin contamination.
These products vary on the amount of total aflatoxin based on the computed mean and a great difference on the computed standard deviation. The highest aflatoxin contamination was detected on lot number 12170597A1K1. Lot number 12250189A1J1 was the second contaminated products. And only lot number 12440597A1B1 exhibits no contamination among the Cereal 2 products.The result also proves that Cereal 3 products contain aflatoxin contamination based on the computed mean. The concentration on the total aflatoxin level of these products slightly differs from each other as indicated on the computed standard deviation. Based on the computed total aflatoxin concentration, it was found out that lot number 12533 has the highest aflatoxin contamination, followed by lot number 10033. And the least contaminated sample is on lot number 12003.


Table 2
Total Aflatoxin Concentration on Milk Products


Product Lot Number Absorbance Concentration Mean Std. Dev.
Milk 1 B101412A 1.466 0
P211825B 1.421 0 0.00 0.000
P211652C 1.258 0

Milk 2 120801893A 1.203 0.51
124401893B 1.240 0.12 0.2100 0.24887
122501893A 1.297 0

Milk 3 6B02 1.556 0
6B18 1.400 0 0.20 0.316
6A19 1.190 0.62

According to the study of Filazi, that aflatoxin metabolite is present in milk of cows fed with a diet contaminated with aflatoxin. The quantity of total aflatoxin contamination on Milk products was summarized in Table 3.
Based on the results Milk 1 samples were found to be free from aflatoxin contamination. Aflatoxin contamination found on Milk 2 products on lot number 120801893A which has to be the highest contaminated sample, and on lot number 124401893B as indicated on the computed mean. The amount of total aflatoxin concentration on these products was quiet different from each other base on the computed standard deviation. A trace of aflatoxin contamination was detected on milk 3 lot number 6A19 based on the computed mean. While on lot numbers 6B02 and 6B18 shows no contamination, this explains the variation on the amount of the total aflatoxin concentration among the milk 3 products.

3.3To compare the total aflatoxin concentration in each sample with the FDA and FAO/WHO acceptable level.

According to the FDA action levels and regulatory guidelines in food safety the allowable requirements of total aflatoxins are 20 ppb for all human foods and animal feeds and 0.5 ppb for dairy products especially in milk. The Joint FAO/WHO Experts Committees on Food Additives recommend a maximum tolerable daily intake of 4 ppb on wheat, cereals, nuts, and foods for direct human consumption and 0.05 ppb on milk and other dairy products.



Table 4
Comparison of the Total Aflatoxin Concentration of Wheat Products
with the FDA and FAO/WHO Acceptable Limit


Product Lot Number Conc. (ppb) FDA 20 ppb limit FAO/WHO 4 ppb limit

Wheat 1 312 AKDV 0 Below the Limit Below the Limit
312 ANAV 0 Below the Limit Below the Limit
312 APAV 0 Below the Limit Below the Limit

Wheat 2 M048SIOE 5.47 Below the Limit Above the Limit
M007SIOH 5.08 Below the Limit Above the Limit
M027SIOJ 4.75 Below the Limit Above the Limit

Wheat 3 93100 1.19 Below the Limit Below the Limit
93015 0.52 Below the Limit Below the Limit
93013 1.51 Below the Limit Below Limit

The total aflatoxin of the wheat products were compared with the acceptable limit sets by the FDA and FAO/WHO standard which is shown on Table 4.
Comparison with the FDA acceptable level on total aflatoxin which is 20 ppb, based on the obtained total aflatoxin concentration all of the wheat samples conformed to the FDA requirements. These wheat food supplements can be taken by consumers without presenting danger to their health because the obtained total aflatoxin concentration is below the permissible level. However, these wheat products can be taken with caution since it was detected to be contaminated with aflatoxin.
As for the FAO/WHO recommended maximum level of aflatoxin which is 4 ppb, only wheat 2 products does not conformed because it was found out to have a total aflatoxin concentration higher than the acceptable limit. The wheat 2 food supplements were packed in a sachet and it is ready for 1 serving and it can be taken three times a day 30 minutes before every meal, and if the consumers followed the recommended dosage there is a possible high aflatoxin level that can be ingested by the consumers and it may caused harmful effect on their health and based on the conducted studies on total aflatoxins human consumption on contaminated foods can produce acute as well as long-term health problems.


Table 5
Comparison of the Total Aflatoxin Concentration of Cereal Products
with the FDA and FAO/WHO Acceptable Limit


Product Lot Number Conc. (ppb) FDA 20 ppb limit FAO/WHO 4 ppb limit

Cereal 1 11701135VE 0.88 Below the Limit Below the Limit
11931135RA 0.49 Below the Limit Below the Limit
12741135VF 1.19 Below the Limit Below the Limit

Cereal 2 12170597A1K1 2.50 Below the Limit Below the Limit
12440597A1B1 0 Below the Limit Below the Limit
12250189A1J1 1.42 Below the limit Below the Limit

Cereal 3 12003 0.28 Below the Limit Below the Limit
10033 0.75 Below the Limit Below the Limit
12533 1.40 Below the Limit Below the Limit


Data concerning on the comparison between the total aflatoxin concentration of cereal products with the FDA and FAO/WHO acceptable level for aflatoxin is summarized in Table 5. The concentration on the total aflatoxin on Cereal products is accepted on the requirements sets by the FDA, since the obtained total aflatoxin level is lower than the 20 ppb acceptable level. And based on the guidelines of the FAO/WHO these cereal food supplements conformed on the maximum tolerance limit since the obtained level of aflatoxin is below 4 ppb. These cereal food supplements are safe to the health of the consumers. However, these products can be taken with caution since it exhibits the presence of aflatoxin contamination.


Table 6
Comparison of the Total Aflatoxin Concentration of Milk Products
with the FDA and FAO/WHO Acceptable Limit


Product Lot Number Conc. (ppb) FDA 0.5 ppb limit FAO/WHO 0.05 ppb limit

Milk 1 B101412A 0 Below the Limit Below the Limit
P211825B 0 Below the Limit Below the Limit
P211652C 0 Below the Limit Below the Limit

Milk 2 120801893A 0.51 Above the Limit Above the Limit
124401893B 0.12 Above the Limit Above the Limit
122501893A 0 Below the Limit Below the Limit

Milk 3 6B02 0 Below the Limit Below the Limit
6B18 0 Below the Limit Below the Limit
6B19 0.62 Above the Limit Above the Limit

Table 6 shows the comparison of the total aflatoxin concentration on the selected milk products with the acceptable level for aflatoxin sets by the FDA and FAO/WHO.
Comparison with the FDA acceptable level for milk products which is 0.5 ppb, only Milk 2 lot number120801893A and Milk 3 lot number 6A19 does not conformed because the amount of total aflatoxin concentration detected in this product was more than the acceptable level set by the FDA. As for the FAO/WHO requirements on the maximum tolerance limit for total aflatoxin on milk products which is 0.05 ppb, Milk 2 lot numbers 120801893A and 124401893B, and on Milk 3 lot number 6A19 were not accepted since the computed total aflatoxin concentration is above the acceptable limit. Consumption of milk products contaminated with aflatoxin may possibly cause harmful effect on the health of consumers, this may further suppress the immunity of the consumers and it can lead them to be susceptible to other diseases.

4. Conclusion
The researcher therefore concludes that most of the food supplements used in this study found to have aflatoxin contamination but it still in conformance with the FDA and FAO/WHO acceptable level.

5. References

Esplanada, J. 2010. Public Warned vs. Food Supplements. Philippine Daily Inquirer, p.C1.

FDA. 2008. FDA Food Supplements. USA: FDA.
http://www.fda.gov/food/dietarysupplements/

FAO/WHO. 2004. Aflatoxin Contamination in Foods and Feeds in
The Philippines FAO/WHO Regional Conference on Food

Safety for Asia and Pacific. Serembran, Malaysia: FAO/WHO.
http://who.int/mediacentre/events/2004/foodsafety.asiapacific/en/index/html.

Lawley, R. 2007. Aflatoxins. Toxin Rev. United States of America 3
(1):143-145.
Manetta, A.C. 2011. Aflatoxin-Detection, Measurement, and Control. University of Teramo Italy: In Tech Publishing.

Weaver, C.M., and M. Truckess. 2010. Determination of Aflatoxins
in Botanical Roots by a Modification of AOAC Official Method SM 991.31 Single Laboratory Validation. J AOAC International 93 (1): 184-190.

6. Acknowledgment

The researcher wishes to express her sincerest appreciation and gratitude to those who have extended their support, time, assistance, concern, encouragement to her, through the entire period of the research work until the time of its completion.
C.E.U Graduate School headed by Dr. Tereza R. Perez, Dr. Maricar W. Ching and staff for their assistance, encouragement, and moral support;
Dr. Learni Bautista, her adviser for the kindness, patience, guidance, suggestions, and encouragement and for her endless motivation and moral support;
Dr. Erna Yabut and staff of C.E.U. Center for Data Analysis for their advices and treatment of the statistical data for interpretation;
Mr. Rogelio Cruz and Ms. Avelina Ronquillo for their assistance, and valuable suggestions for the improvement of this study.
Ms. Janice Escueta, Ms Rina Roxas and Mr. Joel Lorenzo of Ariela Marketing for their accommodation, assistance, and patience during the instrumental analysis of this research;
Her friends and classmates in C.E.U Graduate School for their moral support help, and suggestions during the completion process of this research;
Her relatives for the prayers and financial assistance extended to the researcher;
Her parents, Mr. Fidel Agudelo and Mrs. Salome Agudelo, her brother Mr. Yuri Agudelo, her sister in-law Mrs. Cresmie Agudelo, and her niece Maria Ysobel Agudelo for their unending love, guidance, encouragement, moral, spiritual, and financial support;
Above all, the Lord Almighty, for His guidance and blessings that He gave for the fulfillment of this research.