**1. Introduction**

[21] BRASIL. Ministério da Agricultura. Portaria n. 07, de 09 de novembro de 1988. Esta‐ belece os padrões mínimos das diversas matérias primas empregadas na alimentação animal. Diário Oficial da União, Poder Executivo, Brasília, DF, 09 de novembro de

[22] Arrus, K., Blank, G., Clear, R., Holley, R.A. Aflatoxin production by Aspergillus fla‐

[23] Lourenço, L. F. H., Santos, D. C., Ribeiro, S.C.A., Almeida H., Araujo, E.A.F .Study of adsorption isoth.erm and microbiological quality of fish meal type "piracuí" of Acari-Bodo (Liposarcus pardalis, Castelnau, 1855). Procedia - Food Science 2011, 1, 455-462.

[24] Santos, J. R. C. & Freitas, J. A. Characteristics and quality of a piracuí fish derived

[25] Cerdeira, R.G.P. Fish consumption and others food itens by the riverine population of the Lago grande de Monte Alegre, PA-Brazil, Acta Amazonica 1997, 27(3):213-228.

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fish. MSc. INPA, 165 p. 2009.

1988.

206 Aflatoxins - Recent Advances and Future Prospects

Aflatoxins are a group of structurally related mycotoxins produced by certain species of the genus *Aspergillus*, particularly *A. flavus*, *A. parasiticus* and *A. nomius*, which can grow on a variety of food and feed commodities [1]. Aflatoxin production is influenced by several fac‐ tors: for example, temperature and humidity [2]. It has been shown that aflatoxin B1 (AFB1) is the most potent hepatocarcinogen of this group of mycotoxins. Aflatoxin M1 (AFM1) is a hydroxylated metabolite of AFB1 produced by the hepatic microsomal cytochrome P450, and is secreted in the milk of mammals that have consumed AFB1-contaminated foods. AFM1 is also a hepatocarcinogen and is classified in Group 1 as carcinogenic to humans by the International Agency for Research on Cancer [3]. In terms of food safety and public health concerns, exposure to AFM1 through milk products is considered to be a serious problem.

According to worldwide regulations for mycotoxins in food and feed compiled by the Food and Agriculture Organization of the United Nations, 60 countries have already established regulatory limits for AFM1 in raw milk and milk products. The report also indicates that the limits vary from ND (not detectable) to 15 µg/L [4]. The values of 0.05 µg/L and 0.5 µg/L are the two most prevalent regulatory limits for AFM1 in milk products, enforced in 34 and 22 countries, respectively. The maximum permitted level for AFM1 established by the Europe‐ an Community is 0.025 µg/kg for infant formulae and follow-on formulae, including infant milk and follow-on milk, while the limit for raw milk and heat-treated milk is 0.05 µg/kg [5]. The U.S. regulatory standard for AFM1 is 0.5 µg/L [4]. There are still several countries, in‐ cluding Thailand, that have not yet established regulatory limits for AFM1 in dairy products.

© 2013 Ruangwises et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Ruangwises et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The law that regulates the quality of milk products in Thailand is the Notification of the Ministry of Public Health No. 265, which regulates only cow milk products. However, the law does not specify the regulatory standards for AFM1 but states that "…*milk products may be contaminated with aflatoxins at a level that is not harmful to human health*" [6]. The only guide‐ line that regulates the quality of raw goat milk is the Thai Agricultural Standard TAS 6006-2008 of the National Bureau of Agricultural Commodity and Food Standards, Ministry of Agriculture and Cooperatives [7]. Like Notification No. 265 for cow milk products, the TAS 6006-2008 guideline does not specify the recommended limit for AFM1 in goat milk.

the years 2009–2011. Both types of milk samples were frozen at –20 °C until analysis (within one month from the collection date for raw milk, or 2 months from the manufacturing date for pasteurized milk). A total of 90 milk samples were collected and analyzed in this study.

The extraction procedure was performed using the manufacturer's recommendations, as previously described by Ruangwises et al. [13]. Briefly, 50 ml of raw milk or pasteurized milk sample was pipetted into a 50-ml plastic centrifuge tube. Milk samples were defatted

was then transferred into a 50-ml plastic syringe with a Luer tip which was attached to an immunoaffinity column. The skimmed milk was allowed to flow into the column by gravity at a flow rate of approximately 1 ml/min. After the skimmed milk had run through, 20 ml of HPLC water was used to wash the column. AFM1 was eluted from the column with 1.25 ml of acetonitrile:methanol (3:2) and 1.25 ml of HPLC water. The eluate (a total volume of 2.5 ml) was filtered through a nylon syringe filter for HPLC with pore size 0.45 µm (Whatman, UK). AFM1 in the final solution was determined using HPLC. Each milk sample was extract‐

A complete liquid chromatographic system (ProStar; Varian, Palo Alto CA, USA) consisted of a HPLC pump (model 240), an auto injector (model 410), a column oven (model 510), and a fluorescence detector (model 363). The HPLC conditions for analysis of AFM1 were as fol‐ lows: column, Spherisorb ODS-2 (Waters, Milford MA, USA); column temperature, 40 °C; mobile phase, water:methanol:acetonitrile (57:23:20); flow rate, 1 ml/min; and detector, fluo‐

The Q2B procedure of the U.S. Food and Drug Administration [14] was used for determina‐ tion of the limit of quantification (LOQ) for AFM1. Milk samples (50 ml) were fortified with standard AFM1 at four concentrations of 0.025, 0.050, 0.125 and 0.250 µg/L, while blank sam‐ ples were not fortified with standard AFM1. Concentrations of AFM1 in AFM1-fortified milk samples and blank samples were quantified as described above in Section 2.3 using

TM immunoaffinity columns. All samples were analyzed for AFM1 in duplicate.

Individual linear regression lines were obtained from least-square regression analyses of the residual peak areas versus the four concentrations of fortified AFM1 (0.025, 0.050, 0.125 and 0.250 µg/ml). The residual peak areas were peak areas of AFM1-fortified samples minus the peak area of blank sample. A total of 12 regression lines (six regression lines each for intra‐ day and interday analyses) were obtained by least-square linear regression. The LOQ of the method was calculated using the equation LOQ = 10 σ/S, where σ is the standard deviation

of *y*-intercepts and S is the average slope of the 12 linear regression analyses [14].

rescence spectrophotometer (excitation 360 nm; emission 440 nm).

C. Fat was separated; the resulting skimmed milk

Occurrence of Aflatoxin M1 in Raw and Pasteurized Goat Milk in Thailand

http://dx.doi.org/10.5772/52723

209

**2.3. Extraction and determination of aflatoxin M1**

by centrifugation at 3,500 *g* for 20 min at 4o

ed and analyzed for AFM1 in duplicate.

**2.5. Determination of limit of quantification**

**2.4. Instrument**

AflaM1

In Thailand, the number of dairy goats is approximately 5% that of dairy cows [8–10]. Goat milk is consumed by only a small percentage of the country's population, particularly Thai people who have an allergy to cow milk. Goat milk has been shown to form finer and softer curds than cow milk following acidification under conditions similar to those in the stom‐ ach, thus making it more readily digested [11]. It has been reported that micellar caseins of human and goat milk were 96% hydrolyzed by pepsin and trypsin in *in vitro* studies, while the hydrolytic rate of cow milk was 76–90% [12]. With the knowledge that goat milk is more easily digested, some Thai adults prefer goat milk products. As a result, the number of dairy goats in Thailand has been gradually increasing in recent years. In 2009, the number of dairy goats in Thailand was 20,830; the numbers increased to 22,630 and 33,363 in 2010 and 2011, respectively [8–10].

Thailand is administratively divided into four regions: central, north, northeast and south. The central region was selected for this study, since this region has the highest number of dairy goats and the highest rate of goat milk production, accounting for approximately 60% of the national total [8–10]. There are no internationally published reports regarding the quality and levels of AFM1 in goat milk produced in Thailand.

The purpose of this study was to investigate whether the concentrations of AFM1 in raw and pasteurized goat milk produced in Thailand are within the acceptable level for consumption.
