Aflatoxin M1 in dairy products: causes and strategies to overcome it

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Optimization and validation of two methods to determine the levels of AFM1 in milk and cheese samples using immunoaffinity columns for extraction and HPLC-FLD for quantification

Publication . Vaz, Andreia; Gomes, Francileni Pompeu; Alves, A.; Rodrigues, Paula; Venâncio, Armando

Consumption of dairy products has expanded rapidly over the past decade and constitutes an important source of dietary protein. 1 Aflatoxin M1 (AFM1) is a potent carcinogen metabolite that can be present in milk from dairy cows that consume feed contaminated with Aflatoxin B1. Even though it is less toxic than its parent compound, AFM1 is hepatotoxic and carcinogenic, and is stable during milk pasteurization, storage and preparation of various dairy products. 2,3 Due to the toxicity of this molecule, its detection and quantification is extremely important. The objective of this work was to optimize and validate two methods, according to Commission Regulation (EC) nº 401/2006 of 23 February, to determine the levels of AFM1 in milk and in cheese, using immunoaffinity columns (IAC) for extraction and HPLC with fluorescence detection for quantification.4 The method for milk samples was adapted from VICAM – the supplier of the IAC, and for cheese samples was from r-biopharm and VICAM.5,6 For both methodologies, three levels of spiking in triplicate on two different days were performed. The calibration curve was linear from 0.047 to 4.7 μg L⁻¹ and the detection and quantification limits for milk and cheese were 0.001 μg L⁻1 and 0.003 μg L⁻¹, and 0.006 and 0.02 μg kg⁻¹, respectively. For milk samples, average recoveries determined at spiking levels of 0.020, 0.050 and 0.10 μg L⁻¹ were in the range of 62 % – 87 %, with intra-day precision (RSDr) in the range of 3.4 % – 9.5 %, and inter-day precision (RSDr) in the range of 5.4 % – 6.2 %. For cheese samples, average recoveries determined at spiking levels of 0.050, 0.10 and 0.25 μg L⁻¹ were in the range of 47 % – 74 %, with intra-day precision (RSDr) in the range of 3.8 % – 7.0 %, and inter-day precision (RSDr) in the range of 3.8 % – 5.8 %. Results of the validation process indicate that, except for the recovery in cheese samples, both methods are agree with the provisions of Commission Regulation (EC) nº 401/2006. Despite the recovery for cheese, both methods are precise for the quantification of AFM1 in milk and cheese.

2019Conference object

Open access

Detection methods for aflatoxin M1 in dairy products

Publication . Vaz, Andreia; Silva, Ana C. Cabral; Rodrigues, Paula; Venâncio, Armando

Mycotoxins are toxic compounds produced mainly by fungi of the genera Aspergillus, Fusarium and Penicillium. In the food chain, the original mycotoxin may be transformed in other toxic compounds, reaching the consumer. A good example is the occurrence of aflatoxin M1 (AFM1) in dairy products, which is due to the presence of aflatoxin B1 (AFB1) in the animal feed. Thus, milkbased foods, such as cheese and yogurts, may be contaminated with this toxin, which, although less toxic than AFB1, also exhibits hepatotoxic and carcinogenic effects and is relatively stable during pasteurization, storage and processing. For this reason, the establishment of allowed maximum limits in dairy products and the development of methodologies for its detection and quantification are of extreme importance. There are several methods for the detection of AFM1 in dairy products. Usually, the analytical procedures go through the following stages: sampling, extraction, clean‐up, determination and quantification. For the extraction stage, the use of organic solvents (as acetonitrile and methanol) is still the most common, but recent advances include the use of the Quick, Easy, Cheap, Effective, Rugged, and Safe method (QuEChERS) and proteolytic enzymes, which have been demonstrated to be good alternatives. For the clean‐up stage, the high selectivity of immunoaffinity columns is still a good option, but alternative and cheaper techniques are becoming more competitive. Regarding quantification of the toxin, screening strategies include the use of the enzyme‐linked immunosorbent assay (ELISA) to select presumptive positive samples from a wider range of samples, and more reliable methods—high performance liquid chromatography with fluorescence detection or mass spectroscopy—for the separation, identification and quantification of the toxin.

2020Journal article

Open access

Aflatoxin M1 in Europe between 1990-2018

Publication . Vaz, Andreia; Mourão, Filipa; Costa, Patrícia; Rodrigues, Paula; Venâncio, Armando

Aflatoxin M1 (AFM1) is a carcinogen metabolite that can be present in milk from lactating animals that consume aflatoxin B1 (AFB1) contaminated feed. AFM1, in addition to being hepatotoxic and causing carcinogenic effects, is relatively stable during milk pasteurization, storage and processing of various dairy products, which makes it a potential food contaminant. Consumption of dairy products has expanded rapidly over the past decade and, given the toxicity of this compound, its presence in milk and milk products poses a high risk to the health of the consumer. The occurrence of AFM1 in milk has been reported in many studies and considering the impact of AFM1 on human health and the economy, it is extremely important to study and understand the occurrence of this toxin in different countries of Europe over the years (from 1990-2018). The objective of this work was to study the trend of the occurrence of AFM1 in dairy products, including milk, cheese, butter and yogurt. To achieve this objective, an extensive literature search was made, in the databases Science Direct, Web of Sciences, Scopus and B-on, on quantitative AFM1 data, to evaluate the possible correlation between AFM1 concentration and sampling year, different types of milk products, applied heat treatment, and animal species' influences. For the study of heat treatment and animal species, only milk samples were considered. Data from 106 papers (representing 65,901 samples) were extracted and analyzed using IBM SPSS Statistics 27.0 statistical software. Data collected did not present homogeneity of variance nor followed a normal distribution. The discussion is based Welch and Brown-Forsythe's methods, followed by post hoc Games-Howell to evaluate differences betweens groups. Regarding the AFM1 distribution over the years, three decades were analyzed, 1990’s (1990-1999), 2000’s (2000-2009), and 2010’s (2010-2018), and no significant differences were observed between the two last decades. On the other hand, it was observed a significant difference between the two last decades and the 1990´s decade, presenting the last decades the highest AFM1 levels. In what regards dairy products, milk and yogurt were statistically similar and were the groups with the lowest levels of AFM1. Contrarily, butter and cheese samples were statistically different, with butter samples presenting the highest AFM1 concentration, probably due to a mass concentration effect. Concerning animal species, significant differences were noticed between all species analyzed. Namely, cow milk samples were the samples presenting the highest AFM1 levels, while buffalo milk samples had the lowest AFM1 levels. Regarding heat treatment, no significant differences were observed between raw, pasteurized and UHT milk samples. This study revealed that the levels of AFM1 in the last two decades are higher than the 1990´s decade, and the concentration in butter and cheese samples is higher than in milk samples. Therefore, considering the importance of dairy products, special measures should be taken to protect feed from contamination with AFB1, since the final levels of AFM1 will depend on the initial levels at the primary production.

2021Conference object

Open access