Biomedical subjects
Publications and source records attributed to F Kiermeier.
The legal controls of dried fruits put forward by the state control laboratories show that contamination by insects, especially mites, is often a reason for complaints. This is evidently due to the rural conditions of production. Because hygiene is of great importance, a much stronger control should be achieved in the producing countries as well as in the importing countries.
From more than 67 reports on food analysis of aflatoxin, patulin and ochratoxin, the results between 1980-1983 were evaluated and tabulated. The following number of samples were investigated: aflatoxin 15 550, patulin 356 and ochratoxin 437. The amount of Control samples for aflatoxins has reached such a high number that rapid methods for sampling, preparation and analysis are necessary. Out of the many foods investigated, only 9-10 might really be jeopardized, whereby the tendency of contamination-reduction was observed.
In contrast with the numerous publications in the USA about filth in food the German literature pays scant attention to this topic. In spite of the wide-spread contamination of imported foods with living insects, their residues are scarcely analyzed for in the Federal Republic of Germany. This report should point out this gap.
Usual TLC-methods for the determination of Aflatoxin M1 in milk and milk products sometimes do not separate interfering substances with similar optical and chemical properties from Aflatoxin M1, thus leading to considerably incorrect results. Even after an additional clean-up on polystyrene column the solutions contain so many compounds that the identification of Aflatoxin M1 is difficult. The resolving power of HPLC yield pure frations, which may be determined by their MS and UV spectra.
In Part I the occurrence of mycotoxins is discussed in general terms with emphasis on how they originate in the food, taking full account of presence as secondary contaminants. Further discussions emphasize the occurrence of aflatoxin in foods with a detailed table containing 106 references. Finally, the discussion goes into the factors which influence the formation of mycotoxins and the consequences for the consumer. Part II is concerned with problems in sampling foods with aflatoxin. Difficulties in sampling arise from the different levels of aflatoxins present and from their instability in foods. Based on proposals of different investigators, sample quantities of more than 1 kg are recommended. Regulatory agencies expect concrete plans for statistical evaluation similar to those used for determination of salmonellae. It is particularly important that a solution should be found for the sampling of packaged foods so that a fair and reliable control is possible.
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Processed cheese was manufactured with different types of phosphates (P1 monophosphate to P4 tetrapolyphosphate) and by various techniques. The limiting viscosity numbers of sodium casein and the casein of processed cheese were determined and the axial ratio calculated. The axial ratio of processed cheese protein was a/b=20,0 and that of sodium casein 10,0 resp. We deduce that the apparent increase of the axial ratio is caused by the depolymerization of casein caused by emulsifying salts. The flow curves of processed cheese were analysed. There is good agreement with the power law of Ostwald tau=k.Dn. Therefore processed cheeses are characterized as pseudoplasts. The influence of melting salts on the flow properties can be described by a differing emulsifying effect (P1 less than or equal P4). Temperature, concentration and salt effects are discussed for the viewpoint of correlation between aggregation-desaggregation processes and dehydration and hydration of casein.
Due to the greatly differing incidence of molds on cheese and thus the extremely differing amounts of aflatoxin reliable sampling in cheeses is difficult, particularly if only one semi-hard of hard cheese is available. Experiments were undertaken now to sample--hopefully without losses and suitable for commercial application--in order to be able to ascertain the possible aflatoxin content of a cheese. Based on storage results with artificially contaminated Provolone and Tilsit cheeses as a pretest determination of aflatoxins in scrapings from a 100 cm2 surface are is recommended. At high levels (20-30 microgram/kg) the sampling of several borings at different locations on the cheese is necessary. The aflatoxin content on the surface is not constant, a fact which can be explained by biochemical reactions and by migration of aflatoxins into deeper layers. Washing of cheeses seems not to be of any significant influence on the aflatoxin-content.
In the literature several contradictionary results have been published on the aflatoxin formation at temperatures below 10 degrees C. Therefore experiments with pastes made from milk and cheese powder artificially contaminated with Aspergillus parasiticus, were performed at temperatures of 1 degree C, 5 degrees C, and 10 degrees C for 28 days at a relative humidity of 90--95%. Even at 1 degree C, the aflatoxins B1, B2, G1, G2, and M1 could be determined quantitatively. The lactose content did not have a significant influence on the aflatoxin values. Even storage of cheese (camembert and cottage cheese) in a 10% salt solution did not inhibit aflatoxin formation at 20 degrees C.
Several contracdictory results in the literature lead us to the conclusion, that the influence of raw milk processing on aflatoxin content should be tested under as close as possible practical conditions. Even though storage at 5 degrees C for 1-3 days does not greatly influence aflatoxin content (11-25% reduction), this might still be important for food inspection. Although experiments on exposure to light resp. oxygen were repeated ten times, the results could not be definitely be shown as due to oxidation. Part of the contradictory literature references also in the case of freeze drying in our opinion caused by the way in which aflatoxins was added. The loss was less pronounced when milk was contaminated naturally than when it was added artificially. Heating of milk, depending on the conditions, caused a decrease of the aflatoxin-content of between 12 and 35% when making butter from naturally contaminated cream 23% (18-28%) of the aflatoxin M1 appeared in the butter, whereas the buttermilk contained the major amount of aflatoxin.
In model experiments on the distribution of aflatoxin M1 in whey and curd, the influence of the different processing steps was investigated. Taking the same weight ratio between whey and curd, the following results were obtained: a) The aflatoxin M1-distribution in whey and curd was not changed with increasing amounts of rennet, thus decreasing the renneting time at constant renneting temperatures. b) With increasing renneting temperatures, however, the toxin's percentage in the curd decreased at constant amounts of rennet, whereas the whey's content remained stable. For the commonly used temperature variations between 28 and 35 degrees C, the toxin content of the cheese varied in the range of about 12%. c) Processing of curd by acidification with different organic acids at constant temperatures did not show any change in the aflatoxin M1 distribution as compared to rennet coagulation. d) Curd processing by means of starter cultures led to a decrease in the aflatoxin M1 in curd only at higher temperatures; the toxin's percentage in whey remained practically the same. e) Washing of the curd with the 2 1/2 volumes of water decreased the aflatoxin M1 content of cheese by 22%.
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Between May 10 and August 9 1976, a total of 197 commercial cheese samples were tested on their aflatoxin M1 content; 136 samples (69%) were positive. The highest value was at 0.23 mug/kg, the average value of all positive samples was at 0.09 mug/kg. In more than half of the positive samples (54%), only traces of aflatoxin M1 could be detected, 21% contained up to 0.1 mug/kg and 14% more than 0.1 mug/kg. In soft cheese definitely less aflatoxin was found (57% positive samples) than in other cheese varieties (83--85% positive samples). Supplemental feeding of concentrates during spring season and pasture grazing of dairy cows resulted in more respectively less aflatoxin-positive cheese samples. This was especially true in soft cheese. The other cheeses with longer ripening periods showed the same effect, however, less pronounced with a clear lag phase. Inspite of many positive identity reactions on aflatoxin M1, a routine control of cheeses so far cannot be recommended because of the negative results on mass spectrometry.
Between March 4 and April 15, 1976, the aflatoxin M1 content of milk shipped to a dairy plant was investigated. Out of 419 samples, 79 (19%) were positive. 33% of these samples showed values of 0.05--0.1 microng/1, 38% of more than 0.1 microng/1 and the highest value was at 0.54 microng/1. Other aflatoxins were not found. From samples of milk producers showing positive reactions, feed samples (n = 105) were taken, 45 of which (43%) were also positive. 40% of the positive samples contained up to 20 microng/kg, 36% were in the range between 20--50 microng/kg and the highest values were at 280 resp. 300 microng/kg, especially in mixed feed. Out of these samples, 47% (DLG Standard IV) resp. 36% (DLG Standard III) were higher than the tolerated maximum level of 20 microng/kg. There is a qualitative relationship between the aflatoxin M1 content in milk and the aflatoxin B1 content in feed. In 61 cases where milk samples were positive, in 41 feed samples (67%) aflatoxin B1 was detected.
The method proposed by the Federal Department of Health for the determination of aflatoxin B1,B2,G1, and G2 was tested for additional determination of aflatoxin M1. With relatively small changes of the original method, all aflatoxins including. B1, B2,G1,g2, and M1 can be determined quantitatively in milk, milk powder, butter, cheese, quark, cream, yoghurt and fruit yoghurt.
The mass-spectroscopical identification of aflatoxines in the eluant after thin-layer-chromatographical seperation represents a problem at amounts of less than 50 ng normally found in foods. Therefore it was necessary to investigate the factors influencing the identification. Among others, influences of plate material and the eluant were found. Based on the results with pure aflatoxines ideas were developed on their possible reactions.
The prior condition for the application of pimaricin is its heat stability (up to 80 degrees C) and the low penetration (around 2.6 mm), so that it is available for the effect on the cheese surface for a long period. With 13 different aflatoxin forming moulds, the inhibition on the mycel development and thereby on the aflatoxin formation was tested; the different strains were inhibited to various degrees. The effect on cheese was not definite, because the native cheese surface remained free of mould for 8 weeks after pimaricin treatment, whereas cheese slices dipped into pimaricin solution were covered with moulds very soon. The aflatoxin formation itself is only inhibited, if the growth of the moulds is inhibited. Therefore the cheeses have to be treated very early before strong growth of the moulds has started. The aflatoxin formation, however only starts at a certain growth period of the moulds, but at a not completed inhibition, a reduced aflatoxin formation has to be taken into consideration.
The problem of aflatoxin determination in processed cheese can be solved by the destruction of the emulsion with 6 m urea solution; the detection limit is 0.1-0.05 ppb B1 respectively G1. Examination of 115 trade samples resulted in 2 positive processed cheese samples only. This result cannot be reffered to the destruction of aflatoxins by the melting process, because neither at melting temperatures of 80-138 degrees C nor by melting salts and pH adjustments considerable losses could be observed, mostly below 5%.