Determination of aflatoxin M1 in dairy cattle milk in Khartoum State,Sudan

Aflatoxins are a group of mycotoxins that contaminate various types of food and feedstuff leading to health risk in both humans and animals. Aflatoxin M1 (AfM1), the major metabolite of AfB1, was determined in dairy cattle milk samples of Khartoum State of Sudan using high-performance liquid chromatography (HPLC) with fluorescence detection. A total of 44 bulk dairy cattle milk samples were collected and analyzed. The percentage of AfM1 contamination has been found in 42/44 (95.45%) samples with contamination level ranging between 0.22 and 6.90 μg L⁻¹ and average concentration of 2.07 μg L⁻¹. AfM1 contamination in the samples of dairy cattle milk of Khartoum State of Sudan appears to be prevalent and may pose a public health problem at the moment. Awareness must be conveyed to producers, handlers and specialists.     

Estimate of aflatoxin M1 exposure in milk and occurrence in Brazil

The presence of aflatoxin M₁ (AFM₁) was investigated in 125 samples of powdered milk, pasteurized milk and ultra high treated (UHT) milk in the city of São Paulo, and estimates of AFM₁ intake were assessed. The samples were analysed using an immunoaffinity column for cleanup and a HPLC-FLD for determining AFM₁. The quantification limit was 10 ng/kg. AFM₁ was found in 119 (95.2%) at levels ranging from 10 to 200 ng/kg with mean concentration of 31 ng/kg. The average daily intake estimated for AFM₁ was 1 ng/kg bw per day for children and 0.188 ng/kg bw per day for adults.     

Aflatoxin M1 in raw,pasteurized and powdered milk available in the Syrian market

The incidence of contamination of aflatoxin M1 (AFM1) in milk samples collected from the Syrian market was investigated by using the competitive enzyme linked immunosorbent assay (ELISA) technique. A total of 126 samples composed of raw cow milk (74 samples), raw sheep milk (23), raw goat milk (11), pasteurized cow milk (10) and powdered milk (8) showed that 80% of tested samples were contaminated with various levels of AFM1 ranging from >20 to 765 ng/l. Percentages of AFM1-contaminated samples exceeding the American, Syrian and European tolerance limits were 22%, 38% and 52%, respectively.The range of contamination was relatively higher in pasteurized milk than in raw cow and sheep milk. 80% of AFM1-contaminated pasteurized cow milk samples exceeded the European tolerance limit with a range of contamination between 89 and 765 ng/l. Percentages of contaminated raw cow, sheep and goat milk exceeding the European tolerance limit were 59%, 24% and 14%, respectively.Milk powder was almost free of AFM1 contamination with only one sample containing a concentration lower than the European tolerance limit (12 ng/l).Extrapolation of aflatoxin B1 (AFB1) from AFM1 levels of contamination in milk samples indicates that contamination in dairy cattle feeds may range from 0.5 to 47.8 μg/kg.     

Aflatoxin M1 contamination in dairy products marketed in Iran during winter and summer

In the present study, 298 dairy product samples consisting of pasteurized milk (91 samples), yoghurt (68 samples), white cheese (72 samples), butter (31 samples) and ice cream (36 samples) collected from popular markets in four large Iranian cities were examined for aflatoxin M₁ (AFM₁) by thin layer chromatography (TLC) technique. The toxin was detected in 66 (72.5%) pasteurized milk samples (mean: 0.052 μg/l; range: 0.013–0.250 μg/l), 45 (66.1%) yoghurt samples (mean: 0.032 μg/kg; range: 0.015–0.119 μg/kg), 59 (81.9%) white cheese samples (mean: 0.297 μg/kg; range: 0.030–1.200 μg/kg), 8 (25.8%) butter samples (mean: 0.005 μg/kg; range: 0.013–0.026 μg/kg) and 25 (69.4%) ice cream samples (mean: 0.041 μg/kg; range: 0.015–0.132 μg/kg). The concentration of AFM₁ in 36.2%, 20.6%, 30.5%, 9.6% and 27.7% of pasteurized milk, yoghurt, white cheese, butter and ice cream samples, respectively, were higher than Iranian national standard limits. Levels of AFM₁ in samples of pasteurized milk, yoghurt, butter and ice cream collected in winter were significantly higher (P < 0.05) than those collected in summer. In the case of white cheese, level of AFM₁ was higher in winter than in summer, but the difference was not statistically significant (P > 0.05). The results indicated that the contamination of the dairy products in such a level could be a serious public health problem at the moment.     

Assessment of benzoic acid levels in milk in China

Benzoic acid is the most commonly used preservative in foodstuffs. Although benzoic acid is generally recognized as safe (GRAS), adverse effects such as asthma, urticaria, metabolic acidosis and convulsions were observed at low doses in sensitive persons. Some weak clastogenic activity was noted in in vitro assays. The aim of our study was to determine the levels of benzoic acid in milk samples consumed in the city of Guangzhou, China. In this study, 142 samples of pasteurized milk (24), ultra high temperature milk (UHT, n = 45), milk powder (n = 31) and infant formula (n = 42) were analyzed by high performance liquid chromatograph (HPLC) with diode array detector (DAD) from October 2006 to January 2007. Benzoic acid was detected in 109 (76.8%) samples, ranging from 0.51 to 111 mg/kg. The LOD for benzoic acid was, respectively, 0.2 mg/kg in pasteurized and UHT milk, 2 mg/kg in milk powder and infant formula. The mean recoveries of spiked samples at 4 levels were 99.5 ± 3.1% in liquid milk, and 98.5 ± 4.3% in milk powder, respectively. The results show that benzoic acid widely occurs in milk and milk products in China at the low levels. The levels found are not high, and should not affect the general public; however, it may affect the infants and/or sensitive persons. Therefore, it is important to assess the levels of benzoic acid in milk in China. Meanwhile, it should be noted that the lowest percentage of positive samples in pasteurized milk probably results from the difference in the quality of raw milk, processing technical and storage condition of milk products.     

Aflatoxin M1 contamination in pasteurized milk in Shiraz (south of Iran)

The aim of this study was to evaluate Aflatoxin M1 (AFM1) contamination in pasteurized milk samples in Shiraz city (Iran). Six hundred and twenty four pasteurized milk samples from different supermarkets in Shiraz city were collected during 6 months (April to September 2003). AFM1 was found in 100% of the examined milk samples. 17.8% of the samples had AFM1 greater than the maximum tolerance limit (50 ng/l) accepted by European Union. Contamination with AFM1 is a serious problem for public health. To achieve a low level of AFM1 in milk, cows’ feed samples from various cowsheds must be evaluated routinely for aflatoxin and kept away from fungal contamination as much as possible.     

Presence of aflatoxin M1 in milk and infant milk products in Tehran,Iran

Aflatoxins are highly toxic, mutagenic, teratogenic and carcinogenic compounds. The purpose of this survey was to determine natural occurrence and level of AFM₁ in pasteurized liquid milk, infant formula and milk-based cereal weaning food consumed in Tehran, Iran.A total of 328 branded milk products and liquid milk samples were collected and investigated by Enzyme Linked Immuno Sorbent Assay (ELISA).The samples of pasteurized liquid milk (n = 128), infant formula (n = 120) and milk-based cereal weaning food (n = 80) showed that the incidence of contamination with AFM₁ is 96.3%, the presence of AFM₁ in each group was 72.2 ± 23.5, 7.3 ± 3.9 and 16.8 ± 12.5 ng/kg, ranging between 31–113, 1–14 and 3–35 ng/kg, respectively.In general, the amount of AFM₁ in 100 (78%) of liquid milk samples and 24 (33%) of milk-based weaning food was higher than the maximum tolerance limit accepted by European Union, but in all of the infant formula samples was lower (European Communities and Codex Alimentarius has prescribed a limit of 50 ng/kg for AFM₁ in milk and 25 ng/kg in infant milk products).     

Occurrence of aflatoxin M1 in raw milk in South Korea using an immunoaffinity column and liquid chromatography

The level of aflatoxin M₁ (AFM₁) contamination in raw milk produced in South Korea was investigated using immunoaffinity column chromatography and high performance liquid chromatography with fluorescence detector. A total of 100 raw milk samples were collected from 100 cattle ranches located in three different provinces of South Korea. Forty eight out of 100 raw milk samples contained AFM₁ at low level (0.002–0.08 μg/L) with mean value of 0.026 μg/L. Among the AFM₁ contaminated samples, 29 raw milk samples contained only traceable amount of AFM₁ below the limit of LOQ, 0.02 μg/L. None of samples exceeded the maximum level (0.5 μg/L) of Korean regulation for AFM₁ in milk. The limit of detection was 0.002 μg/L. The result of recovery test with 0.5 μg/L AFM₁ in raw milk sample was 96.3% (SD 3.6, n = 5). This is the first pioneering study to investigate the level of AFM₁ in raw milk used in dairy industries in South Korea.     

Aflatoxin M1 and ochratoxin A in human milk in Ribeirão Preto-SP,Brazil

The objective of this study was to determine the extent of aflatoxin M₁ (AFM₁) and ochratoxin A (OTA) contamination in human breast milk in the city of Ribeirão Preto, São Paulo State, Brazil. During 2012, 100 samples of human milk were collected at the local Human Milk Bank. The method comprised, immunoaffinity column purification and isolation, liquid chromatography separation and fluorescence detection. The average percentage recoveries of AFM₁ and OTA spiked at 20 and 50 ng/L in control human milk were 78.1 ± 11.7% and 73.7 ± 9.6%, respectively. The average relative standard deviations of AFM₁ and OTA spiked at the same levels were 11.7 and 9.6% respectively. The limits of detection was 0.3 ng/L for AFM₁ and OTA. The limit of determination was 0.8 ng/L for both mycotoxins. This method was used to analyze 100 human milk samples, of which, two samples were found to contain AFM₁ at level greater than 0.3 ng/L. OTA was detected in 66 samples (66%), wherein 32 were above the limit of detection and 34 were in the range of from 0.8 to 21 ng/L. Results of our study indicate that breast-fed Brazilian infants had only an insignificant exposure to AFM₁ and OTA.     

Levels of aflatoxin M1 in milk,cheese consumed in Kuwait and occurrence of total aflatoxin in local and imported animal feed

A total of 321 milk samples (177 fresh, 105 long-life and 27 powdered milk, and 12 human milk), 40 cheese samples and 84 feed samples were analyzed for aflatoxin M1 (AFM1) and total aflatoxin. The samples were collected randomly during January 2005–March 2007, from Kuwaiti markets. The method used was ELISA technique. Results showed that all fresh milk samples except one were contaminated with AFM1 ranging from 4.9 to 68.7 ng/kg. Eight samples exceeded the EC’s regulatory limit. For the long-life milk samples, the ranges of AFM1 were from below the detection limit to 88.8 ng/kg, with four samples above the action limit of the EC. In the powdered milk samples, AFM1 ranged from 2.04 to 4.14 ng/kg. Of the human milk samples, only five were contaminated, with AFM1 levels ranging from 8.83 to 15.2 ng/kg with a mean 9.7 ng/kg. The cheese samples recorded 80% contamination with AFM1 with a range 23.8–452 and mean of 87.6 ng/kg, with one sample being above the regulatory limit (250 ng/kg) while the feed samples, showed 79.8% contamination with total aflatoxin.     

Occurrence of AFM1 in urine samples of a Brazilian population and association with food consumption

This survey evaluated the presence of AFM₁ in human urine samples from a specific Brazilian population, as well as corn, peanut, and milk consumption measured by two types of food inquiry. Urine samples from donors who live in the city of Piracicaba, State of São Paulo, Brazil were analyzed to detect the presence of aflatoxin M₁ (AFM₁), an aflatoxin B₁ metabolite, which may be used as aflatoxin B₁ exposure biomarker. The AFM₁ analysis was performed using immunoaffinity clean-up and detection by high-performance-liquid chromatography with fluorescence detector. A total of 69 samples were analyzed and 45 of them (65%) presented contaminations ⩾1.8 pg ml⁻¹, which was the limit of quantification (LOQ). Seventy eight percent (n = 54) of the samples presented detectable concentrations of AFM₁ (>0.6 pg ml⁻¹). The AFM₁ concentration among samples above LOQ ranged from 1.8 to 39.9 pg ml⁻¹. There were differences in food consumption profile among donors, although no association was found between food consumption and AFM₁ concentration in urine. The high frequency of positive samples suggests exposure of the populations studied to aflatoxins.     

Simplex and duplex PCR assays for species specific identification of cattle and buffalo milk and cheese

A polymerase chain reaction, amplifying a fragment of the mitochondrial DNA D loop region was developed for species specific detection of cattle and buffalo milk. The method was simultaneously extended for detection of HTST pasteurized milk samples and cheeses of bovine and buffalo origin. A common forward primer was used with two different species specific reverse primers that resulted amplification of a 126 bp and 226 bp products for cattle and buffalo, respectively, in simplex as well as in multiplex polymerase chain reaction. The primers successfully amplified DNA extracted by conventional protocol from minimal amount of raw milk, heat treated milk and cheese of either bovine or buffalo origin. The primers showed a high degree of specificity. The sensitivity of the assay was excellent with detection level of 0.1 percent adulteration of cow and buffalo milk or cheese (0.15 ng buffalo and 0.04 ng cattle DNA). The assay represents a sensitive and simple method for identification of adulteration in milk and cheese.     

Regional monitoring plan regarding the presence of aflatoxin M1 in pasteurized and UHT milk in Italy

The aim of the study involved evaluation of the presence of aflatoxin M₁ in milk for sale in a specific North West Italian region, Piedmont. The study, conducted from November 2003 to July 2005, was linked to the specific emergency situation which arose due to the climatic conditions during the summer of 2003 which encouraged the development of aflatoxin B₁ in items used for animal feed. This in turn led to the transfer of the metabolite, aflatoxin M₁, into the milk. In total some 316 milk samples were collected during the commercial phase by the official control bodies and analysed. The analysis involved the use of high pressure liquid chromatography (HPLC) combined with fluorimetric measurement, and purification of the extracts using immunoaffinity columns. The results indicated only 2 non conforming samples (0.6%), with limits higher than those set out in the regulations (0.05 μg L⁻¹). In addition, the analyses revealed, in 5 samples (1.6%), threshold values of 0.05 μg L⁻¹. From the data obtained it can be seen that the “aflatoxins” problem only marginally affected Piedmont Region though the trend for average monthly values suggests a return to the use of contaminated animal feed as soon as official controls are less intensive.     

Characteristics of Batzos cheese made from raw,pasteurized and/or pasteurized standardized goat milk and a native culture

Experimental cheeses were made from raw (R), raw with starter (RS), pasteurized with starter (PS) and standardized, pasteurized with starter (PSS) goat milk to study the influence of the starter and pasteurization on the quality of Batzos cheese. Lactococcus lactis subsp lactis strains from raw milk cheese used as starter proliferated significantly (P < 0.05) only in PSS cheese. Heat treatment lowered (P < 0.05) the levels of most microbial groups. However, TC and LAB counts were higher (P < 0.05) in PSS cheese and this was accompanied by lower (P < 0.05) pH; thus, a higher rate of decrease of undesirable microorganisms in PSS cheese was recorded. Degradation of αs-casein was in R > RS and in PS > PSS, while a small reduction of β-casein during ripening and storage was recorded. The proportions of aminoacids and lipolysis products increased throughout ripening and storage.     

A survey on the occurrence of aflatoxin M1 in milk samples in Ardabil,Iran

Mycotoxins are natural food and feed toxic contaminants produced as a result of fungal growth on agricultural materials during storage and/or transportation. The purpose of this study was survey on the occurrence of aflatoxin M1 (AFM1) in milk samples from Ardabil, Iran.Ninety samples were collected from local shops of the Ardabil city during different seasons in order to address representative data on AFM1 in milk consumed by people in this city. AFM1 was analyzed by Enzyme Linked Immuno Sorbent Assay (ELISA).All of the samples (100%) were contaminated with AFM1 in concentration levels ranged from 2.9 to 85 ng/kg. In 30 samples (33% of total samples) the AFM1 level was higher than 50 ng/kg, the maximum residue limit (MRL) accepted by European Union for AFM1 in milk. The difference of AFM1 level between seasons was statistically significant (P < 0.05).The highest and lowest levels of contamination were detected in winter and summer seasons which were 17.4 and 56.3 ng/kg, respectively.This is the first report on the occurrence of AFM1 in milk samples of Ardabil, one of the main milk-production regions in Iran. The contamination with AFM1, in this area is most probable because of contaminated feed consumed by cattle, which needs prompt effort to reduce the risk of contamination.     

Chemical composition of fermented milk (roub and mish) in Sudan

Two batches of whole milk, skim milk and yoghurt that used for processing of roub and mish from Khartoum Dairy Company (Butana factory) were analyzed. Similarly roub and mish (fermented milk), obtained from the same samples, were also collected and analyzed during this study. Similarly, a total of four experiments as an attempt to process “Roub and mish” in the factory’s laboratory were tried following the same procedures of the factory for two batches. While in the other two pasteurization for whole milk were done before manufacturing. Comparison of chemical constituents (total solids %, total proteins %, fat %, lactose %, ash %, and acidity), and pH were estimated in those products.The total solids %, the total proteins % and the fat % recorded lower means for the control samples, compared to those of the factory for whole milk; yoghurt; mixture; and roub. However, the control samples for mish revealed a higher mean than those produce commercially by the factory. Moreover, the higher values obtained for ash and acidity for skim milk and fermented products, in the control samples, reflected the adulteration done at the factory. Similarly, Lactose revealed higher means for whole milk; skim milk; and mixture for the control samples. While yoghurt; roub; and mish showed a higher mean values for the factory samples. This might be due to the fermentation action by the bacteria.The results indicated that there were significance differences in total solids %, ash %, and lactose % (P ⩽ 0.05) between the different groups. Similarly there is significant increase in pH, fat % (P ⩽  0.001) and acidity (P ⩽ 0.01) between the different groups. However, a non-significance difference was found for the other measurements.When comparing roub and mish a non significant differences were obtained for the measurements. Between mixture and roub significance variations were reported for ash %, lactic acid % (P ⩽ 0.05) and lactose (P  ⩽ 0.001). Also, significance variations were found in ash % (P  ⩽ 0.05), and lactose % (P ⩽ 0.001), when comparing mixture and mish. This could be due to fermentation and addition of spices.     

Occurrence of aflatoxin M1 in pasteurised,UHT milk and milk powder from goat origin

In the present study, 36 samples of pasteurised, ultra-high-temperature (UHT) treated and goat milk powder traded in the city of Campinas, Brazil, were analysed for aflatoxin M₁ (AFM₁), from October to December 2004 and March to May 2005. Results showed 25 (69.4%) positive samples for AFM₁ at levels of 0.011–0.161 μg L⁻¹ of milk, which were below the tolerance limit of 0.500 μg L⁻¹ as adopted for AFM₁ in milk by Brazilian regulations. Mean levels of AFM₁ in pasteurised, UHT and goat milk powder were 0.072 ± 0.048, 0.058 ± 0.044 and 0.056 ± 0.031 μg L⁻¹, respectively. It is concluded that the incidence of AFM₁ in goat milk traded in Campinas is high, but at levels that probably leads to a non-significant human exposure to AFM₁ by consumption of goat milks.     

A versatile splitless injection GC-FID method for the determination of mineral oil paraffins in vegetable oils and dried fruit

A splitless injection GC-FID method for determining mineral paraffins in food was set up. The method has been developed on vegetable oils and subsequently adjusted and applied to dried fruits, a food matrix not yet investigated from this point of view. The method avoids the saponification step foreseeing only a clean-up on silica gel SPE, and allows the quantification of mineral paraffins with a limit of quantification (LOQ) of 15 mg kg⁻¹ and a detection limit (LOD) of 5 mg kg⁻¹ in the oils. LOQ and LOD for the analysis of dried fruit samples are respectively 1.0 and 0.3 mg kg⁻¹.Eighteen samples of dried fruit were analysed (apricots, plums, raisins, coconut, dates, mango, pineapple). Two samples of apricots showed high values of contamination, 23.0 and 28.2 mg kg⁻¹ of mineral paraffins, while all the other samples contained less than 6.4 mg kg⁻¹ of mineral paraffins and in four samples mineral paraffins were undetectable.     

A sensitive and validated method for determination of melamine residue in liquid milk by reversed phase high-performance liquid chromatography with solid-phase extraction

A sensitive and validated method for the determination of melamine residue in liquid milk is developed using reversed phase high-performance liquid chromatography-diode array detection (RP-HPLC-DAD) with solid-phase extraction (SPE). The conditions of the extraction, SPE and HPLC were investigated and optimized. The linearity is satisfactory in the range of 0.1–50 μg/mL with a correlation coefficient of 0.9998. Under the optimal conditions, the method limit of detection (LOD) and method limit of quantification (LOQ) were 18 μg/kg and 60 μg/kg, respectively. The recovery of melamine for milk samples spiked with 0.10–3 mg/kg was in the range of 85.5–99.3% with the RSDs (n = 3) of 2.3–3.7%. The intra-day assay precision (RSD) was 5.6% for five replicates of quality control milk sample at 2 mg/kg level. Confirmation of the identities of melamine was achieved by monitoring the two transitions in multiple-reaction monitoring (MRM) mode, and has been applied successfully for the determination of melamine residue in liquid milk samples. The confirmatory method can permit the detection of melamine residues at levels as low as 60 μg/kg in different liquid milks.     

A limited survey of aflatoxin M1 in milk from Indonesia by ELISA

This paper presents a limited survey of aflatoxin M1 (AFM1) in Indonesian milk. AFM1 concentrations of 113 fresh milk samples, collected in 2006 from farms in five different areas of the Yogyakarta Province were analysed. The fresh milk samples were taken directly from dairy farms before pasteurisation. Enzyme-linked immunosorbent assay (ELISA) was used for the analysis of milk samples. Results show that in 48 samples (42.5%) the AFM1 concentrations were less than 5 ng/L and in 31 samples (27.4%) AFM1 was found between 5 and 10 ng/L. In 34 samples (30.1%) the concentrations were above 10 ng/L. None of contaminated samples exceeded the European Union regulation limits of 25 and 50 ng AFM1/L for infant and adult consumption, respectively. Since AFM1 is derived from aflatoxin B1 (AFB1) contained in cow feedstuffs, based on the contamination levels of AFM1 found in this study, the exposure of animals to AFB1 seems to be low.