Reduction of Aflatoxin M1

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Reduction of Aflatoxin M1

Порука  admin on Tue Feb 26, 2013 12:26 pm

Reduction of Aflatoxin M1 residue in milk utilizing chemisorption compounds and its effect on quality of milk
S Soha, MT Mazloumi, M Borji
Shaheed Beheshti University of Medical Science & Health Services, College of Nutrition Science and Food Technology & Research Center of Agricultural and Natural Resources of Markazy Province.


This study was conducted to determine the effect of chemisorption compounds on the level of Aflatoxin M1 in contaminated natural raw whole milk. Two Golpayegany cows in mid lactation were fed with a diet contaminated with Aflatoxin B1 for 15 days. Chemisorption compounds used in this experiment were bentonite and hydrated sodium calcium aluminosilicate (HSCAS). The samples of contaminated milk were tested at 3 concentrations (0,0.5% and 2%) of these compounds in room temperature (25ْْ C). Toxin levels, milk composition, milk pH and the residue of some elements (Al and Si) that may be present after treatment were analyzed. The type of chemisorption compounds, applied level and their interaction had a significant toxin decreasing effect on milk (P<0.01). Milk toxin decreased by more than 90% with bentonite B1. Milk protein, fat, lactose, non-fat solid and total solid values were affected (P<0.05), although values for the increase or decrease of milk components were negligible. As a result there were no residues of Al, but a low level of Si remained in the samples that were treated with chemisorption compounds. This study showed that utilization of chemisorption compounds has high efficiency for detoxification of Aflatoxin M1 from milk. This method can be further developed by research on the effects of these compounds on functional properties of milk.

Key words: adsorption, Aflatoxin M1, Milk, phyllosilicate clays


Mycotoxins are the secondary metabolites of fungi that may have toxic, carcinogenic, mutagenic and teratogenic effects. Aflatoxins are a group of highly toxic compounds of mycotoxins which are found in feeds and foods during growth, harvest and storage period.1The four major aflatoxins are B1, B2,G1 and G2 based on their fluorescence under UV light (blue or green) and relative chromatographic mobility during thin layer chromatography. Aflatoxin B1 is the most potent natural carcinogen known.2

Milk and milk products can also serve as an indirect source of aflatoxins. When cows consume aflatoxin B1 present in their feeds, they metabolically biotransform aflatoxin B1 into a hydroxylated form (4-hydroxy derivatives)1 in hepatic microsomal mixed function oxidase system and will produce aflatoxin M1.3,4 Some researchers5 have reported the transformation of aflatoxin B1 existing in feeds to aflatoxin M1 at around 1-3 % but this conversion has been also reported at 2.6-6.2 %.6,7,8

To reduce contamination with aflatoxins, specific regulations exist in many countries and practical programs are being developed, e.g. the codex committee on food additives and contaminants has developed a code of practice for reducing aflatoxin B1 in raw material (van Egmond et al, 1997; Codex, 1997).9,10 The best way to eliminate direct contamination of milk by the M aflatoxins is to prevent the initial contamination of feeds consumed by dairy cattle. However, despite the use of many prophylactic measures against fungal growth and elaboration of aflatoxins, contamination of feed and thus of milk is sometimes unavoidable and so a process to free milk of aflatoxin would be desirable (Applebaum & Marth, 1982b).11

Many methods of decreasing of aflatoxin M1 have been described, but most of the research indicate that heat processing (such as pasteurization, sterilization and roller drying) does not change the amount of aflatoxin M1 in milk.12,13,14 Degradation of aflatoxin M1 has also been attempted by combination treatments, such as ultra violet light,15 microwaves, gamma rays, and adsorption (van Egmond, 1999).16 Masimango et al.17 observed that aflatoxin B1 in liquid media was adsorbed on clays so strongly that only traces of the toxin remained in contaminated media. Because of similar structures between aflatoxin B1 and aflatoxin M1, Applebaum and Marth11 tried bentonite as a means of eliminating aflatoxin M1 from naturally contaminated raw whole milk.

Some in vitro tests18 have shown that various absorbing materials classifiable as aluminas, silicas and aluminosilicates are capable of binding aflatoxin in solution and hydrated sodium calcium aluminosilicates18-21 were particularly efficacious in binding aflatoxin. There are outstanding differences between absorbent compounds and toxins in their binding capacity and stability of the produced compounds. Furthermore, temperature and environmental pH can be regarded to have influences on the above factors.19

Unfortunately, in relation to effects of absorbent materials on milk components there is not much information available. Applebaum and Marth11 stated that the highest concentration of bentonite used, resulted in no greater than a 5% decrease in total protein. Studies have also demonstrated that levels of HSCAS as low as 0.5% resulted in adsorption and removal of aflatoxin M1 from naturally contaminated milk, with negligible change in nutritional quality as determined by proximate analysis (Ellis et al., 1990).22 In this survey, it was first attempted to evaluate the competency to absorb toxin M1 from naturally contaminated milks in the case of some chemisorption compounds; in the second instance, consumption effects of these materials on milk components were also studied.

Materials and methods

Studied cattle
The animals which were used were 2 Golpayegany multifarious cows (native breed) that were in mid lactation with a mean of 150 ±10 lactation days. The studied cows weighed about 350± 10 kg and fed a fixed amount of total mixed ration (TMR). Ration was calculated to meet the lactation requirements of low-milk producing cows (daily milk production of 5± 0.5 litres) according to ARC.23 The ration consisted of 60:40 forage to concentrate ration and contained barley grain, wheat bran, bakery waste, wheat straw and alfalfa hay. The cows were fed twice a day approximately at 7.00hrs. and 18.00hrs. Each cow was kept individually .Water and common salt was continuously available.

Aflatoxins were produced by fermentation of bakery waste by Aspergillus flavus, Aspergillus parasiticus and Aspergillus niger. The sterile substrate was inoculated with 2ml of each mold suspension. The substrate was cultured for 7 days at 28ºC and 40% relative humidity. To provide contaminated diet, different amounts of contaminated bakery waste was incorporated into the basal diet.

The amount of aflatoxin B1 was measured by thin layer chromatography method.24 When the aflatoxin B1 concentrations in total mixed feed exceeded the permissible contamination level, 20µg/kg(20ppb)( Ellis et al.,1990; Henry et al,.2001), it was used as feed.24,25

In order to measure cow adaptability, the contaminated ingredient was offered gradually in a 10d period. After receiving these diets for another 10 days, the aflatoxin M1 excretion into the milk was determined by ELISA accelerated method.25-30

In order to obtain the test sample, milking was done at 6.00hrs and at 17.00hrs on the 20th day after contaminated feeds were started; the collected milk was mixed and preserved at 7ºC to be used for determination of aflatoxin M1. When the level of aflatoxin M1 in milk exceeded the maximum level of world standard 50ng/kg (50ppt) (Codex, 2001), the specimen was considered as contaminated.31

Adsorbent material
In this study, three types of adsorbent substances, Bentonite B1, Bentonite X and hydrated sodium calcium alluminosilicate (HSCAS) (provided by ROIC Co), were used. For preparation of reaction mixtures, ten millilitres of naturally contaminated raw whole milk (NCRWM) was placed in 50ml Erlenmeyer flask, which was sealed with a foam plug. The effects of three types of adsorbents at three different concentrations (0, 0.5, and 2%), on levels of aflatoxin M1, milk pH, milk composition and residue of Al and Si in milk after application of adsorbent, were investigated. The adsorbent materials were directly added to the milk samples in each flask and were held at 25 ºC for 30 minutes. The toxin levels in each sample were then analyzed using ELISA method by its reader equipment (Awaraness 303) and related kits (R-Biopharm GmbH).

Other determined items in experiment
Lipid, protein, lactose, total solid and solid non-fat were determined through a Milko-Scan (Foss Electric group 133B). The pH of milk in blank tests and in those specimens to which the absorbent materials were added were determined by the use of a digital pH-meter (Methrom 7B). The remaining constituents of the adsorbent substances in milk was measured by atomic absorption spectrophotometer (spectr AA 20 plus varian) and Aluminum and Silicon elements were determined through using of existing lamps and standard solutions.

Statistical analyses
General linear models using SAS software were adapted to analyze the data as a 4×3 factorial.32 Type of adsorbent substances were represented as factor A, level of adsorbents as factor B, and their interaction effects as factor AB. For any treatment in toxin adsorption test, three replicates were used and in adsorbent substances effect test on milk constituents, four replicates were considered . Significance of differences was calculated in the analysis of variance, and comparison of means of treatments was made using Duncan’s multiple range procedure.33


Results indicated that two kinds of bentonite and HSCAS (in all levels) were capable of adsorbing aflatoxin M1 from naturally contaminated raw whole milk. The results of analysis of variance and comparison of mean treatments are given in tables 1 and 2, respectively. Data indicate a significant difference (p<0.05) in mean aflatoxin M1 adsorbed, amongst adsorbents. As shown in table 2, the maximum decrease in toxin levels occurred in milk treated with bentonite B1 which resulted in a decrease in aflatoxin M1 levels by about 90% when compared to the controls. The comparisons indicate that the effect of different adsorbents in decreasing aflatoxin M1 is higher in bentonite B1 followed by HSCAS and bentonite X in that order. Under the experimental conditions applied in this study, adsorbent capacities for aflatoxin M1 ranged from 49% to more than 90% for 0.5% of bentonite X and 2% of bentonite B1 respectively. Since there is no significant difference between minimum and maximum consumption levels of bentonite B1, it can be concluded that bentonite B1 at a minimum concentration (0.5%) plays a significant role in toxin adsorption. Contrary to the results seen in bentonite B1, amount of adsorbed aflatoxin M1 in bentonite BX and HSCAS was increased with the addition of more bentonite to milk.


Up to the present, several methods for decreasing or degrading aflatoxin M1 in milk involving chemical and physical treatment,25 as well as biological methods34 have been investigated. The chemicals that have been studied for their ability to degrade aflatoxin M1 are sulfites, bisulfate and hydrogen peroxide.11,14,35 Adsorption is a physical process that has been explored to decrease aflatoxin M1 in milk. Of all decontamination processes this is the most effective method (in aqueous solution).19 Applebaum and Marth11 added 2% bentonite to naturally contaminated raw whole milk and found that it had absorbed 89% of aflatoxin M1. A study on adsorption of aflatoxin B1 indicated that vermiculate, bentonite and other phyllosilicate clays absorbed the toxin from buffer solution.17 Due to the structural similarity between aflatoxin B1 and aflatoxin M1, possible adsorption of aflatoxin M1 in milk onto adsorbent materials were also explored (Applebaum & Marth, 1982b).11 Phillips et al.19 have shown that phyllosilicate clay (such as HSCAS) tightly binds aflatoxins in aqueous solutions, including milk. The β-dicarbonyl moiety in aflatoxin was found to be essential for tight binding by HSCAS; and molecular mechanism of aflatoxin binding may involve the chelation of metal ions in HSCAS with the β-dicarbonyl moiety in aflatoxin.19,21 Effects of bentonite and HSCAS (0.5 or 2%) on composition of NCRW (naturally contaminated raw whole milk) are presented in table 3.

A statistically significant (P<0.05) decrease of milk protein was observed when HSCAS (0.5% and 2%) and 2% of bentonite X were added to milk; but the maximum reported decrease in protein was only 2.7%. Bentonite B1, which has been shown to be the best adsorbent in the present study, only caused a decrease of 0.14-0.21% of milk protein content, which is regarded as negligible. Applebaum and Marth11 stated the maximum decrease in milk protein content through using bentonite is 5%. Although aflatoxin M1 seemed to be bound predominantly to casein, the association of aflatoxin M1 with hydrophobic casein13,36,37 means that it is expected that using adsorbent material to adsorb toxin would lead to adsorption of protein thereby causing a decrease in milk protein content.

The type of adsorbent material had a statistically significant effect (p<0.01) on concentration of milk fat but the level of adsorbent material used and their interactive effects (type × absorbent level) had no statistically significant effect on milk fat content (Table 3). But comparisons made between mean values did not show similar results, with no statistically significant difference between milk with added HSCAS (0.5 and 2%) and controls. There was a statistically significant difference between milk which had Bentonite B1 and bentonite X (0.5 and 2%) and milk of controls (P<0.05), but numerically the difference was not large and maximum reported decreased fat was 1.6% in bentonite B1 (the best adsorbent in this study) which is negligible.

The effect on lactose, solid non fat and total solid were similar, so that decrease in these nutrients with both type and level of adsorbent as well as their interactive effects was statistically significant.(p<0.01) (Table3). By comparing the mean values of lactose, solid non-fat and total solid contents in cows fed different adsorbents, no statistically significant difference were observed in these values between cows fed the adsorbents and those that were not; furthermore, changes in contents were noted for some adsorbent substances at different concentrations (table 4). It shows that there were significant increases (P<0.05). Two percent Bentonite X resulted in a 2.7 % increase in lactose, 2 % of Bentonite B1 caused 1.5 % increase in solid non-fat and 2 % Bentonite X caused 2.2 % increase in total solid compared to the control. Using of adsorbent materials will reduce protein and lipid contents and conversely increase the amount of lactose, so it can be expected that the milk's total solid and solid non-fat is increased, but this increase is not real. The use of Milko-Scan equipment for determining the milk's ingredients and also considering that some other factors such as additives, salts and so forth, may cause some alterations in sensitivity of the equipment (Foss Electric, 1989), it may be that this numerical increase is because of secondary variables, as the measurement of ashes remaining in milk pre and post-addition of chemisorption compounds shows (Table 5). Figure 1 shows effect of absorbent materials on milk's pH.

By comparing means of treatments, a relative increase in pH due to the application of adsorbent materials is seen compared to the control; although this difference is statistically significant for some materials (P<0.05), the increased rate is negligible and is 0.01-0.12 % unit of existing minimum and maximum changes. While the adsorbent materials are consisting of cations of alkaline-earth metals as well as basic aluminosilicate, an increased pH is expected. Philips et al19 stated that pH=7 in an aqueous solution creates appropriate conditions for stability of the toxin's components and adsorbent materials .The pH determined in the present study is close to 7, therefore, suitable conditions for toxin absorption are expected.

Despite the fact that the Gras list indicates that phyllosilicate clays such as hydrated sodium calcium aluminosilicate are used in food industries18 we investigated the possibility of the presence of aluminum and silicon elements in milk because of aluminosilicate base of such compounds and as no separation process for absorbent materials was made (Table 6). The results did not indicate any aluminum content; however, there were 0ppm, 3ppm and 6ppm silicon in Bentonite B1, Bentonite X and hydrated sodium calcium aluminosilicate adsorbent materials. However since the permissible amount of silicon in human food is up to 50 ppm and its dangerous limit leading to silicon poisoning is 70 ppm,38 presence of this element is safe and even can be regarded as a mineral material.


Results indicated that use of phyllosilicate clays can decrease aflatoxin M1 in milk and practically bring down the impermissible toxin limits to safe limits. The use of such materials will not have significant effects on milk constituents, and they will leave no remnants of toxic material or elements in milk.

Broj poruka : 758
Datum upisa : 06.10.2009

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