The hazard analysis critical control point) (HACCP) system is set up with the aim of ensuring the harmlessness of food along the process chain, from the producer to the ultimate consumer. To set up this system, it is necessary to perform an analysis to identify the dangers and the critical points of the process in real time. Living organisms are detected on the principle of measurement of cellular energy in the form of adenosine triphosphate (ATP), which is produced by all living cells and of particularly by microorganisms. The principle of the bioluminescence of firefly luciferase is applied, whereby a reduced form of luciferin, together with a molecule of oxygen and ATP in the presence of the enzyme luciferase, produces a reaction in which oxyluciferine is released, together with AMP. This is accompanied by a simultaneous release of photons, which are then quantified. The emission spectra recorded with the firefly luciferin/luciferase system is obtained with absolute sensitivity. The method of ATP assays here proposed is adapted for measuring the total content of different adenylates in the cell, i.e. the adenylate pool. ATP concentration is then measured through bioluminescence, i.e. the light is measured by a photomultiplier tube detector and the signal is converted to relative light units (RLU). Thus, RLU have a relationship with the amount of ATP present in the sample, which is not the case with traditional methods. Adequate levels of sensitivity are calculated with appropriate controls and the readings are translated into a statistical designation of positive or negative result. There are numerous illustrations of HACCP applications using biochemiluminescence (BCL) techniques as detection technology in food industry production lines and in control of critical points in real time in industrial applications.
A new ATP bioluminescence-based method was developed to determine the effectiveness of nisin on a sensitive strain of Lactococcus cremoris. The principle of the method is to quantify the release of adenylic-nucleotides (AN) by a sensitive strain under the action of the bacteriocin, with the complex luciferin-luciferase. Nisin-induced leakage of AN included ATP from a sensitive L. cremoris to the external medium immediately after the contact with the bacteria. The growth of L. cremoris was correlated with the extracellular AN content. The extracellular ATP and AN concentration exhibited a linear correlation to the logarithm of the nisin concentration. For the determination of the effectiveness threshold, the concentration of AN was more sensitive and more reliable than the direct quantification of ATP. The effectiveness threshold, corresponding to a 100% inhibition of L. cremoris growth, was obtained for a null concentration of intracellular nucleotides, i.e. for a AN(tot):AN(ext) ratio = 1. For an initial concentration of 1.4 x 10(7) bacteria/mL, the nisin effectiveness threshold is 3.4 +/- 0.01 mg nisin/L. It is possible to detect effectiveness threshold concentration by taking into account the physiological state of the cells.
Dried yam (Dioscorea spp.) chips are widely consumed in Bénin but are often attacked by molds. Invasion of food by Aspergillus flavus may lead to aflatoxin contamination. We report here the result of a survey on the sanitary quality of dried yam chips in Bénin. During July and August 2000, 50 dried yam chips samples were collected from different points in the marketing chain; 10 samples were collected from each of 5 stages: producers, wholesalers, retailers, dried yam-based food sellers and consumers. Aflatoxin content was assayed by the bio-luminescence method (1) after methanol/water extraction. Aflatoxins were detected in all dried yam chip samples, with levels ranging from 2.2 to 200 ppb and a mean value of 14 ppb. An aflatoxin concentration higher than the European Union's maximum residue limit (MRL) of 4 ppb was found in 98% of the samples (N = 50), while 6% had an aflatoxin concentration higher than the World Health Organization's MRL of 20 ppb. Molds were analyzed from two samples, each with aflatoxin levels around 5 ppb, on colony unit medium specific for A. flavus (2). Aspergillus spp. were detected in the inner part of dried yam chips of both samples, with a mean level of 9,000 CFU/g. Fusarium colonies were also present but were not identified to species. References: (1) D. Champiat and J. Larpent. Bio-chimie-luminescence: Principes et Applications. Masson, Paris, France. 1993. (2) P. J. Cotty. Mycopathologia 125:157, 1994.
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