The reducing efficiencies of the commonly used heat treatment methods and fermentation processes on aflatoxin M1 (AFM1) in Nigeria were investigated. Seventy samples of fresh cow milk from both conventional and traditional dairy cattle herds were collected and analyzed for the determination of AFM1 using Cobra-cell incorporated high performance liquid chromatography. Of these analyzed samples, 56 (80.0%) tested positive for AFM1 out of which 3 milk samples with high AFM1 concentrations were selectively pooled and subjected to varied conditions of heat treatments and fermentation processes using both indigenized and exotic strains of lactic acid bacteria ( Lactobacillus bulgaricus + Streptococcus thermophilus and L. rhamnosus and L. plantarum) as starter cultures respectively. Both processes used either singly or combined, demonstrated high degrees of reducing effects on AFM1 levels. Sterilization of the milk at 121˚C and 80˚C under the same condition of time (15 - 20) min showed significant reduction of up to 58.8% (p < 0.05) when compared with the fresh untreated cow milk of the same source. Application of heat treatments within the acceptable pasteurization conditions of 61˚C for 15 - 20 min showed no significant reduction (p > 0.05) in the level of AFM1 when compared with the initial mean AFM1 concentration of the untreated fresh milk. The situation was however different around the boiling temperature of 100˚C at which point the level of AFM1 reduction was found to be inconsistent. The indigenized combined strains showed some slight margins of AFM1 reduction in the proportions of (20.5, 30.8 and 43.9)% over and above that of the exotic strains (17.4, 30.0 and 41.1)% in 12 h, 48 h and 72 h of fermentation respectively. Generally, fermentation alone showed lower reduction of AFM1 in milk from 24.5% to 43.9% compared with the reducing activities of (35.4 to 58.8)% when heat-treated milk samples were subsequently subjected to varied fermentation conditions.
Aflatoxins M1 (AFM1) is the principal hydroxylated metabolite of aflatoxin B1 (AFB1). AFB1 is the major secondary metabolite produced by Aspergillus species specifically Aspergillus flavus. AFM1 was once classified as group 2B carcinogen to humans by the International Agency for Research on Cancer [
The USFDA puts the action levels for AFB1 and AFM1 as 5 ppb and 0.5 ppb respectively in dairy products; it implies that the potency of AFM1 is one tenth that of AFB1. In spite of this marked difference in the action levels, AFM1 still represents a potential carcinogen for humans [
Aflatoxin is a troublesome milk contaminant, in this paper; the authors have evaluated the heat treatment methods and fermentation processes to remove aflatoxin M1. Reports abound which support the heat stability of AFM1 in raw and processed milk products during pasteurization and processing [
The study was conducted in Kaduna State of Nigeria. Kaduna state is located at the centre of Northern Nigeria between latitude 5˚19'60"N and longitude 7˚45'0" E in the north central geographical zone of Nigeria [
A total of 70 samples of fresh raw milk were collected randomly from 3 conventional dairy farms and 2 traditional Fulani dairy herds within a period of 7 weeks. Milk collection began by cleaning the udder of the selected cows with clean disposable hand towels. Hand milking was adopted for the extraction of about 50 ml of milk from the mammary glands of the selected cows into a 50 ml capacity sample bottle under a firm restraint technique of the affected animal by the animal attendant. Two samples were collected weekly from each farm and at the end, about 7 sets of samples were collected.
Sample extracts were cleaned using “VICAM” method. Five gram of fresh milk sample was mixed with 1 g of salt (NaCl) and placed in a blender jar. To the sample was added 20 ml of methanol-water (80/20 v/v) and the mixture blended for 5 min. The mixture was filtered through fluted filtered paper in a clean vessel. From the filterate, 2 ml was collected and diluted to 5 ml with purified water and then filtered through a glass microfiber filter. From the filterate, 2 ml was collected and passed through IAC after which the column was washed successively with 5 ml of purified water. AFM1 was eluted with 1 ml of methanol and collected in a glass cuvette. The extract was finally dried under nitrogen stream and stored at −20˚C till HPLC analysis.
The Shimadzu Prominence UFLC Liquid chromatography system (Kyoto, Japan) was used for the HPLC determination. It consists of a Liquid Chromatography, LC-20AD which is fitted to a degasser, DGU 20A5R, auto sampler (injection) SIL 20A, communication bus module CBM 20A, column oven CTO 20A, photodiode array detector SPD M20A and fluorescent detector RF 20A XL, connected to a gigabyte computer with Intel Core DUO and Microsoft XP operating system. The analytes that fluorescence were detected at specific excitation and emission wavelengths also referred to as the compound’s fluorescence signature. Extracts from IAC were dissolved in 500 µl of HPLC grade acetonitrile. Samples were run at a flow-rate of 1 ml per minute (min−1) retention times. Aflatoxin analysis involved the coupling to the detector of a coring cell (CoBrA cell) (Dr Weber Consulting, Germany) as an electrochemical cell for the derivatisation of aflatoxins. The following mobile phases were used for the analysis of Aflatoxins-Methanol/Acetonitrile/Water (20/20/60, v/v/v) containing 119 mg of potassium bromide (KBr) and 350 ul of nitric acid (4M HNO3).
The Shimadzu Prominence UFLC Liquid chromatography system (Kyoto, Japan) was used for the determination of AFM1. The HPLC was fitted to photodiode array detector SPD M20A and fluorescent detector RF 20A XL with a detection limit of 0.0125 ng/l and recovery rate of 92% for milk. Aflatoxin analysis involved the coupling to the detector a coring cell (CoBrA cell) (Dr Weber Consulting, Germany) as an electrochemical cell for the derivatization of aflatoxins. Spiked concentrations of AFM1 were prepared in triplicates with concentrations of 75, 150 and 300 ng/l of AFM1 standards, mixed thoroughly and extracted. The calculated area under the curve for the standards is entered in a system of coordinates on a semi-logarithmic graph paper against the AFM1 concentration in ng/l. Then the concentration of AFM1 corresponding to the area of each sample was read and determined from the calibration curve using the formula, y = 2460.6x as shown in
The heat-treatment and fermentation experiments were presented as (2 × 4) and (2 × 3) factorial designs respectively. The AFM1 reduction experiment employed different heat treatment methods and fermentation processes using different
lactic acid bacterial (LAB) starter cultures. Phosphate buffer saline suspension of the AFM1 extract of the naturally contaminated milk sample was used as the positive control while the extract of the uncontaminated fresh milk suspended in PBS was used as the negative control. Sixteen (M1-M16) equal portions of 5 ml of AFM1 positive milk samples were used in the reduction experiments.
1) Reduction of AFM1 through Controlled heat treatment of milk
Three milk samples with considerable detectable levels of AFM1 were randomly selected amongst others for the experiment. The 3 samples were pooled to obtain a minimal sample size of 80 ml with AFM1 mean concentration of 0.24 ± 0.05 ng∙L−1. The pooled sample was divided into 16 portions of 5 ml each in test tubes by means of sterile pipettes. Then 6 portions (M1-M6) were used for this part of the experiment while (M7-M16) were utilized for other experiments as described below. The M1 (5.0 ml) represents the control (no heat treatment), M2 (5.0 ml) represents heat treatment at 61˚C for 15 min, M3 (5.0 ml) represents heat treatment at 61˚C for 20 min, M4 (5.0 ml) represents heat treatment at 80˚C for 20 min, M5 (5.0 ml) represents boiling of milk at 100˚C for 20 min, and M6 (5.0 ml) represents milk sterilization at 121˚C for 15 min. Samples M2-M6 were arranged individually in metal racks. Each sample except M1 and M6, was heated in a water bath tempered at each given temperature. But sample M6 was autoclaved while M1 was left as a positive control. All treated samples were immediately cooled before further analysis.
2) Reduction of AFM1 through unregulated heat treatment of milk
One portion, sample M7 (5.0 ml) of the pooled fresh milk was subjected to uncontrolled heat treatment. The sample in 5 ml quantity was placed in a metal rack inside a metal case cooking pot. The arrangement was made in such a way that the 5 ml sample level was just immersed in water inside the pot. The whole setting was traditionally set on fire for cooking for about 20 min. The sample was cooled immediately before processing for HPLC analysis.
1) Preparation of lactic acid bacterial innocula
The strains of LAB used in the study adopt modified methods of [
2) Reduction of AFM1 through fermentation of milk
The 1 ml suspension of each of the prepared innocula was directly inoculated, by the help of Eppendorf tube, into the test AFM1 contaminated milk samples.The last 9 portions (M8-M16) of 5 ml samples of fresh milk each were utilized for the fermentation experiments under 3 different probiotic culture systems using a standardized indigenized combined strains of Lactobacillus bulgaricus + Streptococcus thermophilus obtained from a local dairy institution (LBST) and single exotic strains of L. rhamnosus (ATCC 53103) and L. plantarum (ATCC 8014) as starter cultures. Samples M8-M16 were arranged in 3 groups. Each of the groups consists of 3 test tubes of 5 ml of fresh milk. The test samples in test-tubes were heated slowly to 85 ºC and maintained at that temperature for 2 minutes. This step was designed to kill undesirable contaminant microorganisms. It also denaturizes certain inhibitory enzymes that retard the subsequent fermentation processes. The milk was then allowed to cool in a cold water bath to 42˚C - 44˚C. The cooling process took about 15 minutes. The starter cultures as prepared were inoculated and mixed with a clean glass rod in accordance with the method described by [
3) Reduction by a combined heat treatment and fermentation of milk
In this section of the experiment, the test milk samples (M8 - M16) were initially subjected to pasteurization and sterilization temperatures as indicated in the early part of the methodology involving heat treatment of milk. The heat-treated milk samples were then allowed to cool in a cold water bath to about (42 - 44)˚C. The heat-treated milk samples were then resubjected to fermentation processes by inoculating the prepared probiotic starter cultures at the prepared inoculation doses as indicated in the previous method. The inoculated milk was then incubated at 42˚C and the levels of aflatoxin M1 were quantitatively determined using high performance chromatographic technique as described above.
Aflatoxin M1 (AFM1) remains a potential public health threat through the consumption of dairy products [
Heat treatment conditions | Untreated fresh milk | Heat-treated milk samples at different temperatures and time | Uncontrolled heat treatment | ||||
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Mean AFM1 conc. of initial samples (M1) | AFM1 conc. of M2 | AFM1 conc. of M3 | AFM1 conc. of M4 | AFM1 conc. of M5 | AFM1 conc. of M6 | AFM1 conc. of M7 | |
No heat treatment | 0.24 ± 5.2a | ||||||
61˚C for 15 min | - | 0.21a | |||||
61˚C for 20 min | - | - | 0.21a | ||||
80˚C for 20 min | - | - | - | 0.19a | |||
100˚C for 20 min | - | - | - | - | 0.20a | - | |
121˚C for 15 min | - | - | - | - | - | 0.13b | - |
Uncontrolled heat treatment for 20 min | - | - | - | - | - | - | 0.19a |
P < 0.05 exist between a and b implies consistent decrease in AFM1 concentration, implies inconsistent decrease in AFM1 concentration.
which reversible chemical reactions could occur. This finding may present grave public health concern as chemical rearrangements may result in the formation of even more deleterious intermediary chemical groups of serious health implications. The above findings are important in showing that heat treatment of milk, in any case, may not be the best means of removing AFM1 from dairy products.
Controlled fermentation processes using indigenized starter cultures of LAB have shown promising results in the reduction of AFM1 burden of fermented cow milk. In the current study, synergistic effects of the two indigenized LAB starter cultures comprising of Lactobacillus bulgaricus and Streptococcus thermophilus was compared with the exotic strains of L. rhamnosus and L. plantarum. It was observed that the AFM1 reducing efficiency of the indigenized strains of the starter culture was reasonably compared with any other starter cultures (
Heat treatment conditions | Post-heat-treatment fermentation (AFM1 concentrations) (µgL−1) | ||
---|---|---|---|
AFM1 conc. of sample (A) fermented using Lactobacillus rhamnosus (% reduction) | AFM1 conc. of samples (B) fermented using Lactobacillus plantarum (% reduction) | AFM1 conc. of samples (C) fermented using L. bulgaricus + Streptococcus thermophilus (% reduction) | |
61˚C for 15 min | 0.140 (41.6%) | 0.179 (25.4%) | 0.132 (45.0%) |
61˚C for 20 min | 0.140 (41.6%) | 0.179 (25.4%) | 0.132 (45.0%) |
80˚C for 20 min | 0.134 (44.2%) | 0.172 (28.3%) | 0.122 (49.2%) |
100˚C for 20 min | 0.133 (44.6%) | 0.170 (29.2%) | 0.123 (48.8%) |
121˚C for 15 min | 0.119 (50.5%) | 0.155 (35.4%) | 0.099 (58.8%) |
Uncontrolled heat treatment of milk for 20 min | 0.130 (45.8%) | 0.166 (30.8%) | 0.121 (49.6%) |
AFM1 concentration of the fresh, unprocessed milk is 0.24.
Galvano et al. (1998) [
Of interest in the study was the significant AFM1 reduction levels noticed when the pasteurized and sterilized milk samples were further subjected to fermentation processes. One way fermentation of contaminated milk only reduced the AFM1 level of contaminated milk from (24.5 to 43.9)%, but a more significant reduction from (35.4 to 58.8)% was however noticed when heat-treatment of milk was followed by fermentation as shown in
Findings from this study showed that the commonly used heat treatment methods and fermentation processes in Nigeria are capable of reducing the level of contamination due to AFM1 in milk and dairy products. Elevated heat treatment
temperatures and fermentation processes of cow milk demonstrated high reducing efficiencies on AFM1 burden of milk. However, reducing efficiency of heat treatment of milk exceeding a temperature of 80˚C may present risks associated with chemical breakdown and rearrangements; with the likelihood of forming more toxic substances. However, fermentation processes used in Nigeria using the indigenized and combined strains of L. bulgaricus and Streptococcus thermophilus showed a promising result and therefore alludes a more credible and reliable reducing efficiency of AFM1 in milk intended for human consumption. Subsequent fermentation of heat treated milk also suggests an assured way of significantly reducing the AFM1 burden of cow milk.
The authors wish to appreciate the efforts of all those who contributed technically to this work, particularly, the technical Staff of Animal Health Department, North West University, South Africa and the Department of Veterinary Public Health and Preventive Medicine, Ahmadu Bello University, Zaria, Nigeria. We also extend our appreciation to the TETFUND through her NEEDS ASSESSMENT Programme executed by the Management of University of Abuja. The individual effort of Prof. Hussein Makun of the Department of Biochemistry, Federal University of Technology (FUT), Minna, for creating links to execute the work overseas is highly appreciated.
There is no conflict of interest of any kind.
Omeiza, G.K., Mwanza, M., Enem, S.I., Godwin, E., Adeiza, M.A. and Okoli, C. (2018) Reducing Efficiencies of the Commonly Used Heat Treatment Methods and Fermentation Processes on Aflatoxin M1 in Naturally Contaminated Fresh Cow Milk. Open Journal of Veterinary Medicine, 8, 134-145. https://doi.org/10.4236/ojvm.2018.88013