Detection of Aflatoxin Contamination of Dairy Production Chain in the Northwest Region of Rio Grande do Sul , Brazil

The present study evaluates levels of aflatoxin contamination involving small dairy farms from the in the Northwest region of Rio Grande do Sul, Brazil. The objectives of this trial is: quantify lactating cow diet and raw for bulk milk Aflatoxin B1 (AFB1) contamination; evaluate the use of a mycotoxin adsorbent on cows submitted to semi-confinement raising; and verify the presence of AFM1 contamination on UHT and powdered milk commercialized locally. For this, 17 farms were selected. During the experimental period, every lactating cow on each herd received 50 g/day of a commercial mycotoxin adsorbent for dairy cows (100% bentonite). From each farm, bulk milk, corn silage and concentrate samples were collected for aflatoxin quantification. Aflatoxin M1 was detected in all milk samples examined from each farm, but the levels were within the allowed by the legislation. Aflatoxin B1 was also detected in all samples of corn silage and concentrate. The mycotoxin adsorbent used reduced (p < 0.05) AFM1 contamination in milk. AFM1 milk contamination levels observed on commercialized milk also respected Brazilian requirements. It is possible to conclude that there is a high incidence of Aflatoxin B1 contamination on corn silage and dairy cows concentrates in family farms from South Brazil region. Based on the condition in which the present study was conducted, the use of a mycotoxin adsorbent reduced the levels of AFM in milk in production system. However, the locally produced milk and commercial milk were below the AFM levels accepted by the National Requirements for AFM1 contamination.


Introduction
Mycotoxins are secondary metabolites with low molecular weight produced by a wide range of filamentous fungi present in many animal feeds, including bulky and concentrated food (Bhatnagar et al., 2002;Gallo et al., 2015).Of these, those that have the greatest negative effect on dairy farming include aflatoxin, ochratoxin, vomitoxin, deoxynivalenol, zearalenone and fumonisin (Fink-Gremmels, 2008).Among all aflatoxins, the most important are classified as B1, B2, G1 and G2; they are secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus (Wilson & King, 1995).
The most abundant aflatoxin present in naturally contaminated foods is type B1 (AFB1) (Gallo et al., 2015).The toxic effects secondary to the consumption of the contaminated feed occur due to damage to the liver and cause reduction of growth rate, milk production, milk quality and reduction of resistance to infectious diseases (Pier, 1992;Abeni et al., 2014).Ruminants are more resistant to aflatoxin than monogastric animals, this is due to biotransformations that these animals can perform.When fed AFB1-contaminated foods, they are biotransformed metabolically in the hydroxylated form aflatoxin M1 (AFM1) by the enzyme cytochrome P and are excreted in the milk of lactating animals (Kuilman et al., 2000;Wu et al., 2009).
Thus, in addition to the economic damages related to animal production, these toxins are of great importance for public health.Its properties, toxic to humans, have a broad spectrum, with special attention to the carcinogenic effects (Ross et al., 1992;Chawanthayatham et al., 2017).AFM1 is allocated to the human carcinogenic group 1 by the International Agency for Research on Cancer (Bosch & Peers, 1991;IARC, 2012;Ostry et al., 2017).
In order to reduce the deleterious effects of aflatoxins, it has been proposed the use of additives such as adsorbents mixed with animal feed, which are able to decrease the absorption of mycotoxins.When added to the feed, such products are intended to bind to mycotoxins in order to transport them fully or partially out of the digestive tract, thereby preventing the absorption and intoxication of the animals (Kabak et al., 2006;Xiong et al., 2015;Nidhina et al., 2017).However, the use of inorganic adsorbents may reduce the nutrient density of the diet, as well as promote an excess of absortion capacity that may lead to a decrease in the availability of important microelements (Rabiee et al., 2010).
Given this scenario, this work aimed to analyze the chain that involves the occurrence of aflatoxins in the Alto Jacuí region, Northwest of Rio Grande do Sul (RS), Brazil.For this, studies were carried out to reach the following objectives: quantify the levels of AFB1 in the feed supplied to dairy cows and their reflection on the levels of AFM1 in raw milk produced in family farms of the RS region.A second objective of this study was also to evaluate the effect of the use of an adsorbent additive of mycotoxins in the milk produced in the facilities.The last objective was also to verify the presence of AFM1 in different brands of UHT and powdered milk commercialized in the region, comparing the results obtained with the current legislation.

Place of Experiment
To perform this study, 17 milk-producing farms were selected, located in six counties in the Northwest region of the State of RS, Brazil, these farms represented a representative sample of Alto Jacuí region, as they have representative characteristics of the region, as described by Lorenzoni, Araldi, and Mera (2012).The properties had up to 50 hectares dedicated to milk production, herds with up to 20 lactating cows, semi-confinement systems, two daily milking and family labor.These properties did not receive regular technical assistance and did not evaluate the raw material supplied to the animals.According to the authors, the average daily milk production was between 18 and 403 liters, per cow and per property, respectively.
In the farm selected for the study, the diet offered to the animals consisted of commercial protein concentrate or locally produced concentrate, corn silage, pastures grown in autumn-winter: black oats (Avena strigosa), white oats (Avena sativa) and ryegrass (Lolium multiflorum); and in the summer: sorghum (Sorghum bicolor) and millet (Pennisetum americanum).In all the evaluated properties, the concentrate was supplied individually in feed fences, in two daily meals.The milking was done in closed system with proper milking management and milk production occurred throughout the year.

Supply of Adsorbent
The mycotoxin adsorbent additive was supplied during the 10-day experimental period to all lactating cows.The animals received 50 g of a commercial adsorbent approved for use in lactating cows, composed of 100% bentonite (Toxfree MilkPower® Nutrifarma).The product was given orally, once a day (at the first meal), individually, mixed into the concentrate.

Sample Collection
In each farm silage samples were collected on days 0 and 5 of the experimental period in nine distinct points of the silo: three points of the upper third of the silo, three points of the middle third and three points of the lower third.The material was homogenized and the analysis performed in an aliquot of 100 grams, representative of the exposed face of the silo.The samples were stored in plastic containers and stored at -20 ºC until processed.
The collection of samples of concentrated ration was performed on days 0 and 5 of the experimental period.Samples were collected directly from the sheds where food was stored.The bags destined to the next feeding of the animals were selected, and 5 samples were collected at random points of the bags.All samples were frozen immediately until analysis.
Samples of raw milk were collected on days -4, -2, 0 and 5 of the experimental period, directly from the expansion tanks after milking the animals, cooling to 4 ºC and homogenization.Three samples of milk per property were collected.The samples were stored in 70 ml sterile tubes and immediately frizzed at -20 o C and then sent to the laboratory for analysis.

Analysis of Milk Marketed in the Region
Samples of milk marketed in regional establishments were purchased from boxes of 1 liter of UHT milk from 13 Figure 4 Northwe