Immunoaffinity Column Extraction Research Articles

Distribution and stability of aflatoxin M1 during production and storage of yoghurt

Yoghurt from cow's milk artificially contaminated with aflatoxin M1 (AFM1) at levels of 0.050 and 0.100 g l-1 was fermented to reach pHs 4.0 and 4.6. Yoghurt fermented to pH 4.6 was also used for preparing strained yoghurt. Yoghurts were stored at 4°C for up to 4 weeks. Analysis of AFM1 in milk, yoghurt, strained yoghurt and yoghurt whey was carried out using immunoaffinity column extraction and liquid chromatography coupled with fluorometric detection. AFM1 levels in yoghurt samples showed a significant decrease (p < 0.01) compared with those initially added to milk. Growth of culture lactic acid bacteria was not affected in the AFM1 contaminated yoghurts, with the exception of Streptococcus thermophilus that showed a significantly (p < 0.01) lower increase in the yoghurt containing the toxin at high concentration. Following fermentation, AFM1 was significantly lower (p < 0.01) in yoghurts with pH 4.0 than in yoghurts with pH 4.6 at both contamination levels. During refrigerated storage, AFM1 was rather more stable in yoghurts with pH 4.6 than with pH 4.0. The percentage loss of the initial amount of AFM1 in milk was estimated at about 13 and 22% by the end of the fermentation, and 16 and 34% by the end of storage for yoghurts with pHs 4.6 and 4.0, respectively. The percentage distribution ratio of AFM1 in strained yoghurt/yoghurt whey of the initial toxin present in the yoghurt was about 90/10 and 87/13 for the lower and the higher contamination levels, respectively.

Occurrence of aflatoxin M1 in raw and market milk commercialized in Greece

From December 1999 to May 2000, 114 samples of pasteurized, ultrahigh temperature-treated (UHT) and concentrated milk were collected in supermarkets, whereas 52 raw milk samples from cow, sheep and goat were obtained from different milk producers all over Greece. Sample collection was repeated from December 2000 to May 2001 and concerned 54 samples of pasteurized milk, 23 samples of bulk-tank raw milk and 55 raw milk samples from cow, sheep and goat. The total number of samples analysed for aflatoxin M1 (AFM1) contamination by immunoaffinity column extraction and liquid chromatography was 297. In the first sampling, the incidence rates of AFM1 contamination in pasteurized, UHT, concentrated and cow, sheep and goat raw milk were 85.4, 82.3, 93.3, 73.3, 66.7 and 40%, respectively, with only one cow raw milk and two concentrated milk samples exceeding the EU limit of 50 ng l-1. In the second sampling, the incidence rates of AFM 1 contamination in pasteurized, bulk-tank and cow, sheep and goat raw milk were 79.6, 78.3, 64.3, 73.3 and 66.7%, respectively, with only one cow and one sheep raw milk samples exceeding the limit of 50 ng l-1. The results suggest that the current regulatory status in Greece is effective.

Survey of the occurrence of aflatoxin M1 in dairy products marketed in Italy: second year of observation

During 1996, 161 samples of milk, 92 samples of dry milk for infant formula and 120 samples of yoghurt, were randomly collected in supermarkets and drug stores in four big Italian cities, and checked for aflatoxin M1 (AFM1) by immunoaffinity column extraction and HPLC. AFM1 was detected in 125 (78%) of milk samples (ranging from < 1 ng/l to 23.5 ng/l; mean level: 6.28 ng/l), in 49 (53%) of dry milk samples (ranging from < 1 ng/l to 79.6 ng/kg; mean level: 32.2 ng/kg) and in 73 (61%) of yoghurt samples (ranging from < 1 ng/kg to 32.1 ng/kg; mean level: 9.06 ng/kg). Altogether, only four samples of dry milk were over the legal limits established by the EC in 1999. It is concluded that during 1996, despite the widespread occurrence of AFM1, mean contamination levels in dairy products sold in Italy were not a serious human health hazard.

Distribution and stability of aflatoxin M1 during processing, ripening and storage of Telemes cheese

Telemes cheeses were produced using milk that was artificially-contaminated with aflatoxin M1 at the levels of 0.050 and 0.100 microg/l. The cheeses produced in the two cheese-making trials were allowed to ripen for 2 months and stored for an additional 4 months to simulate commercial production of Telemes cheese. Concentrations of aflatoxin M1 in whey, curd, brine, and the produced cheeses were determined at intervals by liquid chromatography and fluorometric detection coupled with immunoaffinity column extraction. Concentrations of aflatoxin M1 in the produced curds were found to be 3.9 and 4.4 times higher than those in milk, whereas concentrations in whey were lower than those in curd and milk. Aflatoxin M1 was present in cheese at higher concentrations at the beginning than at the end of the ripening/storage period, and it declined to concentrations 2.7 and 3.4 times higher than those initially present in milk by the end of the sixth month of storage. Concentrations of aflatoxin M1 in brine started low and increased by the end of the ripening/storage period but only a portion of the amounts of aflatoxin M1 lost from cheese was found in the brine. Results showed that Telemes cheeses produced from milk containing aflatoxin M1 at a concentration close to either the maximum acceptable level of 0.05 microg/l set by the European union (EU) or at double this value, will contain the toxin at a level that is much lower or slightly higher, respectively, than the maximum acceptable level of 0.250 microg of aflatoxin M1/kg cheese set by some countries.

Occurrence of fumonisin B1 and B2 in beer*

A total of 29 nationally distributed brands of beer, representing 25 domestic US and four imported brands, were purchased in retail outlets in Lincoln, Nebraska and analysed for concentrations of fumonisin B1 (FB1) and B2(FB2). Immunoaffinity column extraction and cleanup of fumonisins from the beer samples, coupled with detection and analysis by gradient high performance liquid chromatography (HPLC), provided a limit of quantitation for each toxin of 0.3ng/ml. Of the brands of beer sampled, 86% were positive for FB and 41% were positive for FB2. No beer contained a detectable quantity of FB without a detectable quantity of FB1. The total fumonisin (FB1 + FB2) content of positive samples ranged from 0.3 to 12.7ng/ml, with a mean concentration for all positive samples of 4.0 ± 3.4ng/ml (n = 25). Considering that the level of fumonisin contamination of corn in recent harvest years has been minimal, the results of this limited survey could represent levels associated with current agricultural and brewing practices.

Survey of the Occurrence of Aflatoxin M1 in Dairy Products Marketed in Italy

During 1995, 159 samples of milk, 97 samples of dry milk for infant formula, and 114 samples of yogurt were randomly collected in supermarkets and drug stores in four large Italian cities and checked for aflatoxin M1 (AFM1) by immunoaffinity column extraction and HPLC. AFM1 was detected in 136 (86%) of the milk samples (in amounts ranging from <1 ng/liter to 108.5 ng/liter; mean level: 10.19 ng/liter), in 81 (84%) of the dry milk samples (in amounts ranging from < 1 ng/kg to 101.3 ng/kg; mean level: 21.77 ng/kg), and in 91 (80%) of the yogurt samples (in amounts ranging from <1 ng/liter to 496.5 ng/liter; mean level: 18.08 ng/liter). Altogether, only two samples of milk, two samples of yogurt, and one sample of dry milk had levels of AFM1 exceeding the Swiss legal limits, which are the most restrictive in the world. AFM1 contamination levels in milk and yogurt samples collected in the period of November to April were ca. four times as high as those in samples collected in the period of May to October. It is concluded that during 1995, despite the widespread occurrence of AFM1, the mean contamination levels in dairy products sold in Italy were not a serious human health hazard.

Chemiluminescent enzyme immunoassay of cholecystokinin in rat plasma using an immuno-affinity column as a pretreatment

The cholecystokinin (CCK) plasma concentration of a basal level in a normal rat was at a hardly detectable level (a few pg/ml) even using the highly sensitive chemiluminescent enzyme immunoassay. In this paper, we prepared an anti-CCK F(ab')2 fragment which was made by pepsin digestion from the anti-CCK antiserum. The anti-CCK F (ab')2 fragment was immobilized to the Sepharose 4B gel activated BrCN and packed to column. We have extracted and concentrated CCK in the rat plasma using this column and the CCK assayed by the chemiluminescent enzyme immunoassay (CL-EIA) of CCK. The immuno-affinity column extraction system was more accurate and precise than a reversed phase gel extraction system. The mean recovery of CCK from the rat pooled plasma using the immuno-affinity column was 62.2% (n = 12) by the proposed CL-EIA. The mean +/- S. E. (n = 3) of the CCK concentration in the fasted rat plasma is 3.54 +/- 0.10 pg/ml. The plasma CCK concentrations of normal rats and those of camostat administrated rats, could be measured by the proposed CL-EIA using the immuno-affinity column as a pretreatment.