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Biochemistry & Analytical Biochemistry
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Unreported Aflatoxins and Hydroxylate Metabolites in Artisanal Oaxaca Cheese from Veracruz, Mexico

Vargas-Ortiz M1,2, Carvajal-Moreno M1*, Hernández-Camarillo E1,3, Ruiz-Velasco S4 and Rojo-Callejas F5

1Laboratorio C-119 De Micotoxinas, Departamento de Botánica, Instituto de Biología, Ciudad Universitaria, Universidad Nacional Autónoma de México (UNAM), CP 04510 CdMx, México.

2CONACYT-CIAD (Centro de Investigación en Alimentación y Desarrollo), Coordinación Culiacán. Carretera El Dorado Km 5.5, Col. Campo El Diez, Culiacán Sinaloa 80110, México.

3Instituto Tecnológico de Veracruz, Tecnológico Nacional de México, SEP, Calzada Miguel Angel de Quevedo 2779, Col. Formando Hogar, CP 91897 H. Veracruz, Ver., México.

4Departamento de Probabilidad y Estadística, Instituto de Investigaciones en Investigaciones y en Sistemas, Ciudad Universitaria, UNAM, CP 04510 CdMx, México.

4Departamento de Química Analítica, Facultad de Química, Ciudad Universitaria, Universidad Nacional Autónoma de México (UNAM), CP 04510 CdMx, México.

*Corresponding Author:
Magda Carvajal-Moreno
Laboratorio C-119 de Micotoxinas
Departamento de Botánica
Instituto de Biología, Ciudad Universitaria
Universidad Nacional Autónoma de México (UNAM)
Coyoacán. CP 04510 CdMx, México
Tel: +(5255) 56 22 91 38
Fax: +(5255) 5550 1760
E-mail: [email protected]

Received Date: March 09, 2017; Accepted Date: June 12, 2017; Published Date: June 15, 2017

Citation: Vargas-Ortiz M, Carvajal-Moreno M, Hernández-Camarillo E, Ruiz-Velasco S, Rojo-Callejas F (2017) Unreported Aflatoxins and Hydroxylate Metabolites in Artisanal Oaxaca Cheese from Veracruz, Mexico. Biochem Anal Biochem 6: 322. doi: 10.4172/2161-1009.1000322

Copyright: © 2017 Vargas-Ortiz M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Aflatoxins (AFs) are toxic secondary metabolites of the fungi Aspergillus flavus, A. parasiticus and A. nomius. The fungi produce these AFs in cereals, oilseeds and spices. AFs have damaging effects on all organisms, including humans, and their symptoms can be classified as acute (vomiting, hemorrhage and death) or chronic (immunodepression, Reye syndrome, Kwashiorkor, teratogenesis, hepatitis, cirrhosis, and various cancers). The common AFs (AFB1, AFB2, AFG1, AFG2) are metabolized in the liver or by microbes that produce hydroxylates (AFM1, AFM2, AFP1) and aflatoxicol (AFL), which makes them soluble in water. This means that AFs can be excreted in fluids such as milk or urine, and AFs are not destroyed in the process of making cheese. Other AFs can also be excreted in milk, but they have not been reported until now. The purpose of this study was to identify and quantify the AFs present in 30 samples of artisanal Oaxaca-type cheese sold in the City of Veracruz. The average concentrations of AFs detected in the 30 samples of artisanal cheese were AFB1 (11.2 ng g-1) in 77% (23/30); AFL (19.1 ng g-1) in 70% (21/30); AFG2 (0.2 ng g-1) in 63% (19/30); AFM1 (3.0 ng g-1) in 53% (16/30); AFP1 (0.1 ng g-1) in 50% (15/30); AFM2 (0.2 ng g-1) in 20% (6/30); AFG1 (0.03 ng g-1) in 13% (4/30); and a trace amount of AFB2 (

Keywords

Aflatoxins; Fresh cheese; Carcinogens; Food contamination

Introduction

Cheese is an economically important commodity worldwide. In Mexico, 76,696 tons of Oaxaca-type cheese were produced in 2005, with a value of 2,700 million Mexican pesos [1,2]. Most of the information about aflatoxins (AFs) in cheese is related to industrial production and sale through formal commercialized channels. However, most of the Oaxaca-type cheese consumed in Mexico is handmade artisanally, and there have been no reports about AFs in cheese and the quantities sold in Mexico.

The State of Veracruz is the sixth largest producer of milk in Mexico [3], and 53% of the total milk produced in the state is used without pasteurization to produce artisanal cheeses [4], which are sold in large cities such as the Port of Veracruz. One of the main artisanal cheeses produced in this region is Oaxaca-type cheese, which is made in the same way throughout the country. The process begins with warming milk to temperatures between 18°C and 25°C; the milk is then heated to 38°C, and rennet is added (9-12 mL for 100 L of milk). The milk is then acidified with acetic acid at pH 5.5, and upon curdling the curds are cut into 2-cm squares. The curds are left to rest for 25 min and are later shredded by hand and left to acidify for 20 min. The whey is drained, and the curds without whey are melted when mixed with hot water (60°C). The product is stretched by hand to form threads of 3 cm to 6 cm wide and then cooled with water (18°C). The cheese is left to drain, and salt is added (11 g to 50 g salt per 1 kg of threads). Finally, ball hanks are formed with the cheese threads [5].

Aflatoxins (AFs) are toxic secondary metabolites that chemically correspond to bis-dihydro-furanecoumarins and have well-known physicochemical properties [6]. AFs are mainly produced by the fungi Aspergillus flavus, A. parasiticus and A. nomius [7]. The common AFs found in cereals, which are present in balanced cow feed, are aflatoxin B1(AFB1) and aflatoxin B2(AFB2), which have blue fluorescence, and aflatoxin G1(AFG1) and aflatoxin G2(AFG2), which have green fluorescence [8].

The acute symptoms of AFs include vomiting, miscarriage, hemorrhage, diarrhea and death, and chronic symptoms include immunosuppression, fetal malformation, hepatitis B and C, cirrhosis, and carcinoma of the liver [9,10], cervix [11], colorectal system [12], breast [12] and pancreas [12]. AFs are considered potent carcinogens, and the International Organization for the Research on Cancer (IARC) classified them as Grade I in humans [13].

AFs can be present in balanced cattle feed [14] in countries with tropical weather, where high humidity and warm temperatures in storage warehouses facilitate fungal growth, as well as in agricultural and unregulated local markets [15]. Cheese is an important source of nutrients for humans, and it is frequently contaminated with AFs [16]. AFs are present in the cereals and oilseeds that are used as ingredients in feed and silage for cattle [17]. They are common in countries where storage, harvest and climate conditions are suitable for fungal growth and in countries without food regulation legislation [17,18].

When dairy cattle consume fodder contaminated with AFB1 or AFB2, these toxins are rapidly absorbed. The animal’s liver and the microbial metabolism mitigates the damaging effects of the toxins by introducing a hydroxyl (OH-) into the AF molecule, forming hydroxylate metabolites that allow the toxins to be dissolved in water and expelled from the body via urine or milk. In this way, AFB1, AFB2, AFG1 and AFG2 from balanced feed of ruminants or from fodder are biotransformed into AFM1, AFM2, AFP1 and aflatoxicol (AFL) [19], and these less toxic but still carcinogenic hydroxylate metabolites are secreted in cow’s milk (Figure 1).

biochemistry-and-analytical-biochemistry-structure

Figure 1: Chemical structure of aflatoxins.

Aflatoxins in cheese

AFs bind to proteins such as milk casein via a hydrophobic interaction [20]. Therefore, AFM1 is present in cheese when contaminated milk is used. AFM1 distributes in a 40% to 60% ratio between curd and whey, depending on the cheese-making method [21]. AFM1 can withstand temperatures up to 320°C before decomposing and is resistant to thermal treatments, such as pasteurization, ultrapasteurization and acidification, that are used during the processing of cheeses [22,23]. Although AFM1 is less toxic than AFB1, it is still carcinogenic, and it is frequently reported in dairy products. Nonetheless, other AFs certainly contribute to the risk associated with AFs due to the high consumption of dairy products. Therefore, many countries have established regulations for the maximum tolerable levels of AFs in milk and dairy products [24]. AFM1, AFM2, AFB1 and AFL have been reported in Mexican milk [25,26]. Oaxaca–type cheese has economic, cultural and alimentary importance; therefore, the detection of AFs in artisanal cheeses commercialized in Veracruz is necessary. The purpose of this research was to find other AFs (AFB1, AFB2, AFG1, AFG2) and the hydroxylate metabolites AFM1 and AFM2 for comparison as well as AFP1 and AFL, which have not been reported, and to discuss their importance as carcinogens.

Materials and Methods

Sampling

The study consisted of 30 samples of 750 g of Oaxaca-type artisanal handmade cheese purchased in groceries and markets in the City of Veracruz. The cheese samples originated from 5 townships (Acajete, Medellin, Tlalixcoyan, Soledad and Veracruz) of the State of Veracruz, Mexico (Figure 2). These townships practice dual-purpose cattle raising for both meat production and small artisanal cheese making with no sanitary legislation. A Matlab algorithm was used to randomly select the places from which the samples were purchased in the City of Veracruz. The cheeses were refrigerated immediately after sampling and were subjected to a drying process for a period of less than 12 hours. Samples of Oaxaca-type cheeses were purchased in March 2016, which is in the dry season when the cows are fed nutritionally balanced feed; during the rest of the year, cows typically eat grass.

biochemistry-and-analytical-biochemistry-samples

Figure 2: Origin of samples from the State of Veracruz, bought in the City of Veracruz.

Each cheese sample was manually unthreaded, the cheese samples were placed in a tray drier, and the dry samples were stored frozen until AF extraction and chemical analysis were performed.

Chemical extraction method for Aflatoxins

The R-Biopharm [27] method has been recommended for use with Total aflatoxin Easi-Extract Immunoaffinity Columns (IAC) (R-Biopharm Rhône Ltd., Glasgow, Scotland, UK). This method was performed according to the following protocol.

First, 15 g samples of dry, ground Oaxaca-type cheese were blended (Waring ETL laboratory blender 7010S model WF 2211214, Torrington, CT, USA) for 2 minutes at high speed with a mixture of 100 mL of MeOH/water (80:20 v/v) and 2 g NaCl to clarify the extract. The mixture was centrifuged at 4500 rpm for 15 min, and an amount of supernatant equivalent to 1 g of sample was dissolved in phosphatebuffered saline (PBS) at pH 7.4 at a proportion of 1:4(v/v) and homogenized for 1 minute in a vortex. Before adding the samples, each IAC was equilibrated with 20 mL of PBS at pH 7.4 applied at a flux of 5 mL/min. The buffered sample was passed through the IAC, and AFs were eluted using 1.5 mL of HPLC-grade MeOH followed by 1.5 mL of distilled water with reflux. The eluate was dried at 40°C in an oven (F135A Novatech Model, México City, Mexico) and then derivatized.

Derivatization

Derivatization is a process to increase the AF fluorescence [28,29] of AF standards to make calibration curves and to quantify the AFs in cheese samples Figure 3. The derivatization reaction with trifluoroacetic acid is the transformation of AFB1 and AFG1 that are less fluorescents, in their hemiacetals B2a abd AFG2a that are highly fluorescent. AFB2 and AFG2 are not affected by this reaction due to their saturated structure [28].

biochemistry-and-analytical-biochemistry-transforms

Figure 3: The derivatization reaction with trifluoroacetic acid that transforms AFB1 and AFG1, lessfluorescent, in their hemiacetals B2a and AFG2a that are highly fluorescent. AFB2 and AFG2 are not affected due to their saturated structure.

Eight dry AF standards (AFB1, AFB2, AFG1, AFG2, AFM1, AFM2, AFP1 and AFL from Sigma-Aldrich; St. Louis, MO, USA) that were used to determine the AFs’ linearity and percentage of recovery validation were dissolved in 200 μL of HPLC-grade acetonitrile (ACN), and 800 μL of derivatization solution was added. The derivation solution was prepared with 5 mL of trifluoroacetic acid (Sigma-Aldrich, St. Louis, MO, USA), 2.5 mL glacial acetic acid (Merck, Naucalpan, Estado de Mexico, Mexico) and 17.5 mL deionized distilled water and then vortexed (Vortex G-560, Bohemia, NY, USA) for 30 seconds. The vials containing the dry eluates were heated in a vapor bath at 65°C for 10 min. The derivatized samples were cooled to room temperature, and triplicate 60-μL samples were analyzed by HPLC with fluorescence (HPLC-FL).

Validation of the extraction method

The validation of the analytical method and the analysis of the 30 Oaxacatype cheese samples were performed using known parameters [30].

Linearity of the system (Calibration curves)

Solutions with different concentrations of AFs were prepared from a stock concentration of 1000 ng AFM. The 0.25 mg AFM standards were diluted with benzene:acetonitrile (98:2 v/v), following a previously reported methodology [31], so that the pure AFs did not decompose.

a. The spectrophotometer (Genesys 10 UV Thermo Electron Corporation. Madison, WI, USA) was calibrated before the experiments to measure the absorbance of the AFM standard solutions at 357 nm.

b. Different formulas [31] were applied to calculate 1000 ng stock solutions of each AF concentration:

c. Twelve concentrations (0.01, 0.05, 0.1, 0.5, 1, 2, 4, 8, 16, 32, 64 and 128 ng) of the 8 different AFs were independently created from the 1000 ng stock solution. These standard dilutions were then used to plot the analytic signal (area below the curve of each chromatographic peak) against the AF concentrations. The curve equation and statistical parameters were obtained. The slope value (b1), ordinate to origin (bo), determination coefficient (R2), confidence interval for the slope to origin (IC(β)), variation coefficient percentage (% CV), standard deviation (SD), and the LOD and LOQ were calculated using Excel 2003.

LOD and LOQ

The LOD of the equipment was established in relation to the noise in the chromatogram. The LOD is equal to the AF concentration that gives a signal three times greater than the noise. The LOQ equals the AF concentration that is 10 times greater than the noise [32].

Recovery percentages

The recovery percentage is a measure of the accuracy of the method and expresses the proximity between the theoretical and experimental values. The arithmetic average, standard deviation, percentage of variation coefficient and confidence interval were calculated. To obtain accurate measurements, the AFs of the samples of dried, ground Oaxaca-type cheese, in 1 g aliquots, diluted in PBS (1:4 v/v), were individually spiked with three different concentrations (5, 20 and 40 μg kg-1) of the eight individual AF standards (AFB1, AFB2, AFG1, AFG2, AFM1, AFM2, AFP1) and AFL; one aliquot without spiked AF was used as the control, which gave the basal contamination level. The samples were individually processed using the R-Biopharm extraction method [27]. AFs were purified and concentrated using an IAC, derivatized, and quantified by HPLC-FL, and the percentage of recovery for each AF was obtained. After the derivatization mixture was cooled to room temperature, triplicates of 60 μL of each sample were injected into the HPLC-FL method.

HPLC-FL quantitation

The HPLC-FL chromatographic system was an Agilent Series 1200 HPLC (Agilent Technologies, Inc., Santa Clara CA, USA) and consisted of an isocratic pump (Model G1310A); a fluorescence detector (Model G1310A Series DE62957044, Agilent Technologies, Inc., USA), which was set to an excitation wavelength of 360 nm and to two emission wavelength maxima of 425 nm for AFB1, AFB2, AFM1, AFM2, and AFL and 450 nm for AFG1, AFG2 and AFP1; and an autosampler (G1329A Series DE64761666). The chromatographic column was a VDS Optilab VDSpher 100 C18–E 5 μm 250 mm × 4.6 mm that was maintained at room temperature (22°C) with a mobile phase of water:ACN:methanol (65:15:20 v/v/v) that was degassed for 30 min under vacuum and added at a flux of 1.0 mL/min. The injection volume was 60 μL.

Statistical analysis

The statistical analysis was performed using Minitab version 16. Variance analyses with the Tukey test at 95% were performed in triplicate, considering each cheese as an experimental unit. The graphs showing the data from the Tukey test and the standard deviations were produced using Kaleida Graph version 3.5. Kruskal-Wallis statistical analysis was used to find significance and differences of AFs. The Wilcoxon Rank Sums test was performed to find differences for each pair of samples and Bonferroni corrections of the samples.

Results and Discussion

Validation parameters

The Limit of Detection (LOD) in ng g-1 and recovery percentage (Rec%) for AFs were AFB1 (LOD 0.01, Rec 97%), AFB2 (LOD 0.02, Rec 95%), AFG1 (LOD 0.05, Rec 93%), AFG2 (LOD 0.05, Rec 96%), AFM1 (LOD 0.01, Rec 95%), AFM2 (LOD 0.05, Rec 97%), AFP1 (LOD 0.05, Rec 95%) and AFL (LOD 0.01, Rec 98%). These data, the retention times and R2 are given in Table 1.

Aflatoxin LOD
(ng g-1)
Linearity (curves) Recovery percentage
Retention time
 (minutes)
R2
AFB1 0.01 7.085-8.849 0.9986 97%
AFB2 0.02 17.452-20.228 0.9988 95%
AFG1 0.05 5.722-5.876 0.9626 93%
AFG2 0.05 11.215-14.513 0.9946 96%
AFM1 0.01 8.514-8.769 0.9834 95%
AFM2 0.05 20.208-22.447 0.9946 97%
AFP1 0.05 15.563-19.318 0.9960 95%
AFL 0.01 3.032-5.569 0.9978 98%

Table 1: Validation parameters of the study of aflatoxins and hydroxylates.

Quantification of all AF

The averages of the AFs detected in the 30 samples of artisanal cheese were as follows: AFB1 (11.2 ng g-1) in 77% (23/30); AFL (19.1 ng g-1) in 70% (21/30); AFG2 (0.2 ng g-1) in 63% (19/30); AFM1 (3.0 ng g-1) in 53% (16/30); AFP1 (0.1 ng g-1) in 50% (15/30); AFM2 (0.2 ng g-1) in 20% (6/30); AFG1 (0.03 ng g-1) in 13% (4/30); and AFB2 in only 3% (1/30) traces (<LOD), as shown in Table 2 and Figures 4-6. In the chromatograms the same letters mean that the samples were statistically the same, and were analyzed in a variance analysis with Tukey Test (P<0.05).

biochemistry-and-analytical-biochemistry-fluorescence

Figure 4: Chromatograms of non-reported Aflatoxins found in Oaxaca-type cheese in the City of Veracruz. LU=Concentration in Luminiscence Units. The fluorescence data of AFs: a) AFB1, AFB2, AFM1, AFM2 Exc=360, Em=425 nm, and b) AFG1, AFG2 and AFP1 at, Exc=360 nm and Em=450 nm. A) AFB1 in sample 24 replication (rep) 1; B) AFL in sample 14 rep 1; C) AFL and AFB1 sample 6 rep 2 and D) AFG1 and AFG2 sample 6 rep 2; E) AFM1 and AFB2 sample 10 rep 2 and F) AFP1 sample 10 rep 2; G) AFL, AFB1, AFP1 and AFB2 sample 10 rep 2; and H) AFB1, AFM1 and AFM2 (<OD) sample 17 rep 1.

biochemistry-and-analytical-biochemistry-aflatoxins

Figure 5: Unreported aflatoxins (A) AFB1, (B) AFG1 and (C) AFG2. The same letters represent samples statistically the same with Tukey test (P<0.05). No AFB2 was detected.

biochemistry-and-analytical-biochemistry-aaa

Figure 6: Reported AFM1 (A) and AFM2 (B), and non-reported aflatoxin hydroxylates AFP1 (C) and Aflatoxicol (D) in artisanal cheese of Veracruz. The same letters represent samples statistically the same with Tukey test (P<0.05).

AFB1 and AFL were the most abundant AFs. The AFB1 was not a product of the cow metabolism but rather indicated that maize starch had been added during the manufacturing of the cheese. This addition is legal [33] and accepted for fresh cheeses, but it is not good practice from the point of view of AF contamination. The amount of AFB1 was very high, and it is the most carcinogenic AF. AFB1 transforms itself into AFL, which is the interconverting hydroxylate form [34]. These two toxic metabolites are more frequent in Oaxaca-type cheese than are AFM1 and AFM2. Other AFs, such as AFP1 and AFG1, were present in trace amounts [19], and AFG2 appeared more frequently. AFL can be formed through the enzymatic or synthetic reduction of AFB1, and it has high toxicity and carcinogenicity [34]. Although the toxicity of AFL is 18 times lower than that of AFB1, both molecular structures have similar potency to form an exo-epoxide analogue that can bind to DNA [34]. AFL interconverts with AFB1, has electrochemical properties like those of AFB1, and these compounds have been experimentally demonstrated to have high carcinogenicity and toxicity.

AFM1 contamination was in third place in Oaxaca-type cheese samples (Table 2), consistent with the results obtained for other kinds of cheese, such as cream cheese [35], white pickled cheese [36], sheep curd [37], Grana Padano cheese [38], parmesan [39], Turkish kashar cheese [40], and Serbian hard cheese [41]. AFM2 contamination has been less frequently reported. There have been several studies [42] on carryover from cows fed AFB1-contaminated rations to AFM1 in milk. The degree of toxicity and carcinogenicity of AFs is in the following order: B1>G1>B2>G2.

Aflatoxins ng g-1
Origin in Veracruz Sample AFB1 AFB2 AFG1 AFG2 AFM1 AFM2 AFP1 AFL AFt
Tlalixcoyan 1 0.1 0 0 0 0.03 3.43 0 71.0    74.5
Jamapa 2 0 0 0 0.5 0 0 0.32 10.6 12.6
Jamapa 3 0.1 0 0 0.1 0 0 <LOD 25.3 25.5
La Mixtequilla 4 0.03 0 0 0 0.01 0 0 13.9 13.9
Soledad de Doblado 5 0.1 0 <LOD 0 0.7 0 <LOD 2.9 3.8
Unknown  6 0.1 0 <LOD 0.3 0 0 0 9.9 10.3
Unknown 7 0 0 0.1 3.1 0 0 0 0.02 3.2
Unknown  8 0 0 0 0 0.04 0 0 2.1 2.5
Unknown  9 0.6 0 0 0.6 0.03 0.6 <LOD 24.7 26.6
La Antigua 10 9.4 0.02 0 0.1 8.5 0 1.25 104.2 123.5
Unknown 11 0.04 0 0 0.5 0 0 0 12.4 13.0
La Mixtequilla 12 0 0 0 0 0.9 0 <LOD 47.9 48.9
Soledad de Doblado 13 0 0 0 0.2 2.5 <LOD <LOD 45.2 47.9
Tlalixcoyan 14 2.8 0 0 0.1 0.1 <LOD 0 45.2 48.1
Tlalixcoyan 15 0.02 0 0 0.1 30.6 0 0 0 30.6
Unknown  16 0 0 0 0 0.1 0 <LOD 51.0 51.1
Malibranmarket 17 37.1 0 0 0 3.2 <LOD 0 0 40.4
Unknown 18 37.9 0 0 0.1 0 0 <LOD 0 38.0
Tierra Blanca 19 38.7 0 0 0.6 0 0 <LOD 0 39.2
Boca el Río 20 36.7 0 0 0 0 0 <LOD 0 36.8
Unknown 21 49.2 0 0 0 0 0 0 0 49.2
Soledad de Doblado 22 37.9 0 0 0 0 2.62 <LOD 1.8 42.4
Unknown 23 0 0 0 0.2 41.8 0 0 0 42.0
Soledad de Doblado 24 48.0 0 0 0.3 0.01 0 0 0 48.3
Malibrán market 25 0.2 0 0 0.1 0.7 0 0 26.9 27.8
Unknown 26 0.3 0 0.7 0.1 0 0 0 20.0 21.1
Unknown 27 0.4 0 0 0.1 0 0 <LOD 24.7 25.2
Unknown 28 0.3 0 0 0 0 0 0 16.2 16.5
La Joya, Jalapa 29 3.0 0 0 0.2 0 0 <LOD 17.4 20.6
Unknown 30 32.6 0 0 0.2 0.04 0 <LOD 0 32.8
Sum *   335.3 0.02 0.8 7.3 89.6 6.7 3.5 573 1016
Average   11.2 <LOD 0.03 0.2 3.0 0.2 0.1 19.1 33.9

Table 2: Averages of triplicate counts of reported and unreported aflatoxins in artisanal Oaxaca-type cheeses of Veracruz.

We performed a Kruskal-Wallis analysis to find differences in the concentrations of AFB1, AFB2, AFG1, AFG2, AFP1 and AFL among the 30 samples, as shown in Table 3. There were statistically significant differences among the samples for AFB1 and AFP1. For AFG2, the differences were not statistically significant at 5%, but if a different significance value is considered (10%, for example), the differences among the samples may be significant. Table 4 presents the Wilcoxon Rank Sums test to find the differences for each pair of samples for AFB1. We found that 21 of the 25 samples had concentrations that were not significantly different from zero, but when the Bonferroni correction was applied, these samples differed from the remaining four samples, 18 to 21.

Aflatoxin Chi-square (29 df) p-value
AFB1 44.897 0.0302
AFB2 29.000 0.4651
AFG1 26.180 0.6159
AFG2 41.971 0.0565
AFP1 42.646 0.0491

Table 3: Kruskal-Wallis statistical analysis to find significance and differences of aflatoxins.

Sample Mean Median Standard deviation Standard error Equalmedians Bonferronicorrection
4 0.0267 0 0.0462 0.0267 a a
11 0.0400 0 0.0693 0.0400 a a
1 0.0633 0 0.1097 0.0633 a a
16 0.0633 0 0.1097 0.0633 a a
6 0.0667 0 0.1155 0.0667 a a
8 0.0867 0 0.1501 0.0867 a a
3 0.0967 0 0.1674 0.0967 a a
26 0.1133 0 0.1963 0.1133 a a
5 0.1267 0.13 0.1250 0.0722 a a
25 0.1700 0.17 0.1700 0.0982 a a
28 0.2700 0.37 0.2364 0.1365 a a
27 0.3000 0.3 0.3000 0.1732 a a
9 0.3467 0.36 0.3402 0.1964 a a
14 2.7933 0 4.8382 2.7933 a a
29 3.0067 0.04 5.1731 2.9867 a a
10 9.3800 0.12 16.1428 9.3201 a a
17 14.2433 0 24.6702 14.2433 a,b a
15 15.4467 0.05 26.7111 15.4217 a,b,c a
30 22.9200 23.53 22.6212 13.0603 a,b,c a
24 24.1833 0 41.8868 24.1833 a,b,c a
22 32.1767 45.81 27.9737 16.1507 a,b,c a
20 36.7400 44.06 13.6778 7.8969    b,c b
18 37.8667 34.34 8.1166 4.6861    b,c b
19 38.6467 38.3 0.9488 0.5478    b,c b
21 49.1500 45.89 7.4182 4.2829    b,c b

Table 4: Wilcoxon Rank sums test to find difference for every pair of samples.

For AFP1, we found that 13 of the 15 samples had concentrations that were not significantly different from zero. When the Bonferroni correction was applied, these samples differed from the remaining two (samples 16 and 10). It is important to note that most samples had at least one replicate different from zero. Samples 2 and 5 had two replicates different from zero, and samples 10 and 16 had three replicates different from zero. Table 5 shows the statistics for AFP1 in the 15 samples that had values different from zero. Table 6 shows the Wilcoxon Rank Sums of Values of 20 samples with AFL concentrations different from zero. The Wilcoxon Rank Sums test for all the pairs indicated that there is some inconsistency in the results of the tests. This might be due to some of the samples having a very large variation coefficient. Employing Bonferroni correction, we found that all samples with observations different than zero were equal and significantly different than zero.

Sample Mean Median Standard deviation Standard error Equalmedians Bonferronicorrection
30 0.0033 0 0.0058 30 a, c a
9 0.0067 0 0.0116 9 a, c a
22 0.0067 0 0.0116 22 a, c a
5 0.0100 0.01 0.0100 5 a, c a
13 0.0100 0 0.0173 13 a, c a
19 0.0100 0 0.0173 19 a, c a
27 0.0100 0 0.0173 27 a, c a
12 0.0133 0 0.0231 12 a, c a
18 0.0200 0 0.0346 18 a, c a
20 0.0367 0 0.0635 20 a, c a
29 0.0400 0 0.0693 29 a, c a
2 0.1600 0 0.2771 2 a, b, c a
7 22.9433 1.20 38.7044 7 a,b, c a
16 0.0250 0.02 0.0229 16 a,b b
10 1.2500 0.72 1.4686 10 b b

Table 5: Statistics for AFP1 in the 15 samples that have values different than zero.

Sample Mean Median Standard deviation Standard error
7 0.0212 0.007 0.0250 0.0144
22 1.8033 0 3.1235 1.8033
8 2.0567 1.95 0.5676 0.3277
5 2.9143 1.46 2.9173 1.6843
6 5.7000 4.18 6.5928 3.8063
2 10.6233 11.14 1.1829 0.6829
4 12.5100 12.30 10.8265 6.2507
26 13.6633 16.37 12.5312 7.2349
25 14.9100 17.13 13.9333 8.0444
28 16.1967 21.15 10.1509 5.8607
29 17.4167 19.94 16.3021 9.4120
3 20.9867 4.97 32.142 18.5572
27 24.6667 31.80 12.4507 7.1884
9 24.7367 24.20 8.5976 4.9638
1 30.7633 28.58 31.9111 18.4239
13 45.1533 64.52 39.2355 22.6526
14 45.1933 61.44 39.6504 22.8921
12 47.9300 70.70 40.4737 23.3675
16 50.9700 34.41 29.7631 17.1837
10 104.2370 142.74 67.6794 39.0747

Table 6: Wilcoxon Rank Sums of Values of 20 samples with AFL concentrations different from zero.

AFL is produced in several fungi: Aspergillus flavus, A. parasiticus, A. niger, Eurotium herbariorum, Rhizopus spp. and other non aflatoxicogenic A. flavus [43]. Reduction of the 1-keto group of AFB1 produces AFL [44]. AFL is equally carcinogenic as AFB1, so its formation is not a significant detoxification mechanism [45,46]. AFL has approximately 70% the mutagenicity of AFB1 [47], and it has two forms, A(Ro) and B, both of which are produced from the biological reduction of AFB1 and mainly by Tetrahymena pyriformis, Dactylium dendroides and Rhizopus spp. AFL A is 18 times less toxic than AFB1 in the duckling biliary hyperplasia assay, and the biological activity of AFL B is unknown [48,49]. AFL is the major metabolite of AFB1 in many plants and animals, and it has been detected in milk [26,50], fermented dairy products [51], cereals and nuts [52], eggs [53], blood [54,55], human brain [56], the sera and liver of humans with kwashiorkor and marasmic kwashiorkor in Ghana and Nigeria [57-60], human urine [61], urine of heroin addicts [58], a breast-fed infant with neonatal hepatitis [62], the muscle of broiler chickens fed with contaminated diets [63], and poultry fed chronic low doses of mycotoxins, with the liver having the highest levels [64]. AFB1, AFM1 and AFL accumulate in the tissues and urine of calves [65,66]. AFL–DNA adducts that were produced in vivo were identical to those produced by AFB1 and had similar molecular dosimetry responses and toxicity to the target organ [67]. Regarding DNA adduction and hepatocarcinogenicity in rainbow trout, the tumorigenic potencies were AFB1=1.00, AFL=0.936, AFM1=0.086. AFL is a more potent toxin than AFM1, which can reconvert with AFM1, becoming AFL M1 [68]. AFL-induced hepatocellular carcinomas in rats and fish have a lower tumor incidence than those induced by AFB1 [69]. There is an interconversion of AFB1 and AFL, mediated by intracellular enzymes in rat blood [70]; guinea pigs [71]; sharks, which reconvert 30% of AFL to AFB1 [72-74]; and cultured human epidermal cells [75]. AFL converts into AFB1, which is the most carcinogenic and toxic of all AFs [76]. AFL is oxidized readily back to AFB1, so it can serve as a ‘reservoir’ for AFB1 in vivo, thereby prolonging the effective lifetime in the body [77]. If pH has a role in the interconversion AFB1–AFL, it could act in the normal human digestion of milk, where pepsin lowers the pH. The isomerization of AFL to AFB1 was observed in culture media with a low culture pH [76].

The genotoxicity of AFM1 has been demonstrated by in vitro and in vivo experiments. The carcinogenic potency of AFM1 is 2% to 10% weaker than that of AFB1 [78]. Therefore, the Food Safety Commission of Japan in 2013 [78] concluded that the AFB1 that is present in animal feed is extremely unlikely to affect the health of humans who have consumed contaminated milk or other livestock products. However, AF and the hydroxylate metabolites are also genotoxic carcinogens and are more likely to be found in livestock products, so AFB1 contamination in feed and AFM1 contamination in milk need to be reduced as much as possible. In particular, attention should be paid to the fact that the intake of milk per 1 kg of body weight is higher in infants than in other age groups [78].

Risk assessment parameters for AFB1, AFL and AFM1 have been compared [79]. The virtually safe dose for AFL was 1.7 times higher than that for AFB1.

The incidence of hepatocellular carcinoma in rats and fish dosed with AFL was lower than that in animals treated with AFB1 at the same dosage. AFL in milk might still be a health hazard, particularly for infants whose staple diet is milk-based. AFM1 was not the most abundant AF, and the risk increases when the AFL contamination in milk is added. AFM1 is possibly carcinogenic to humans and was classified as Group 2B by the IARC (1997) [69].

AFM1 has been found in Mexican milk [25], so its presence in cheese was not unexpected, where most AFs were metabolized to AFL. Autumn milk was significantly more contaminated with AFL (p<0.0002). AFB1 had no significant correlation with season, and it is not clear if the presence of vegetable oil helped to decrease the AFL contamination [26]. AFB1 was generally present in milk at trace levels (0.05 mg L-1 to 0.42 mg L-1) in 5.2% of the 290 samples [26] and is not considered a health risk, but cheese had more concentrated amounts (0.04 to 49.2 ng g-1), with an average of 11.2 ng g-1 in the 30 samples and can be considered a health risk.

The hydroxylates AFP1 and AFL are not accepted as toxicologically important in many countries. Polish and European Union legislations (Commission Regulation No. 152/98) agree that all food should be free from AF [80]. Oltipraz was shown to reduce AFB1 adduct biomarkers [81] and inhibit AFM1 production by bovine hepatocytes [82], so it can be used to lower the risk related to cheese consumption. It is necessary to balance the availability of milk in relation to the health risk, not only for cancer but also for other diseases, such as immune suppression, hepatitis and cirrhosis. This fact makes mycotoxin regulation difficult and very incomplete.

AFs are recurrent and occasionally unavoidable contaminants of milk, cereals and oilseeds, and their thermal stability rules out both pasteurization and ultrapasteurization as effective control methods. The best control strategy is to keep raw materials and feed under obligatory mycotoxin regulation. In the case of cheese, it is recommended not to add maize flour during the manufacturing process.

Conclusion

Although the legislation regarding maximum tolerance levels has attempted to decrease the level of AFM1 contamination in cheeses and although there is no direct evidence of human toxicity resulting from the consumption of cheese contaminated with AFs, the problem of ingesting AFB1 and AFL is still present in fresh cheeses, such as the artisanal Oaxaca cheese.

Acknowledgments

The authors thank the Instituto Tecnológico de Veracruz for the cheese sampling and the Instituto de Biología, Universidad Nacional Autónoma de México (IBUNAM) for the data analysis. The authors also thank IBUNAM´s personnel: Noemí Chávez from the Secretaría Técnica, and Joel Villavicencio, Jorge López, Alfredo Wong, Celina Bernal, Diana Martínez and Julio César Montero provided valuable assistance with imaging, computer analysis and design. Additionally, we thank Georgina Ortega Leite and Gerardo Arévalo for library information.

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