Rapid methods for monitoring the microbiological quality of raw milk

A. Căpriţă et al ./ Journal of Agroalimentary Processes and Technologies 14 (2008)bacteria counts in the past several decades,including ATP estimations (Niza-Ribeiro etal., 2000), direct epifluorescent filtertechnique (Rosmini et al., 2004) andelectrical methods (Yang et al., 2004;Felice et al., 1999). The ATP method andepifluorescent filter method are fast butcomplicated.Electrical methods are simple but requirean expensive instrument. Impedancemicrobiology (Firstenberg-Eden, 1984)was one of the earliest electrical methodsfor the detection of bacteria in foods, andhas been developed as a rapid method thatcan detect bacteria within 24 h (Silley,1996). It is based on the measurement ofchanges in electrical impedance of amedium or a reaction solution resultingfrom the growth of bacteria. The majorityof previous studies on the determination ofbacterial counts have focused on themodification of the medium, aiming at theoptimization of the electrical signals(Easter, 1985) or supporting the selectivegrowth of target bacteria against otherbacteria. The impedance method wasapproved as an official method for thedetection of Salmonella in foods by theAssociation of Analytical Communities(AOAC) (Yang et al., 2004). Recentstudies have focused on the separatemeasurements of impedance change in theelectrode and medium components for therapid detection of bacteria in foods (Yanget al., 2004; Ong et al., 2001) known asimpedance-splitting (IS) methods. Felice etal. (1999) reported an IS method based onthe measurement of the change in theelectrode interface capacitance duringbacteria growth for the quantification ofbacteria in milk.Conventional conductivity and impedancemeasurement techniques have proved to beuseful for bacteria counting in commercialapplications (Felice et al., 1999; Silley,1996). Devices based on these techniquesmonitor microbial metabolism in a growthmedium by immersing electrodes directlyinto the medium and measuring thepermittivity and/or conductivity (Ong etal., 2001).Despite their widespread application, thesetechniques have many disadvantagesincluding polarization of the probeelectrodes, decreased sensitivity of thedevice in more conductive media and thehigh cost of instruments used.Adenosine Tri-Phosphate (ATP) is themost popular biochemical index as it isubiquitous in cellular life forms and can bedetected rapidly using bioluminescencereactions. Bioluminescence is the emissionof light by biological methods usingLuciferase enzyme (Griffiths, 1993).Luciferase, together with its co-factors D-Luciferin and oxygen, produces light in thepresence of ATP according to the followingreaction:Luciferase + D-Luciferin + O 2 + ATP → Luciferase+ oxy-luciferin + CO 2 + + AMP + PPi + LightThe amount of light is proportional to theconcentration of ATP in the originalsample. The ATP concentration in a sampleis, in turn, related to the number and typesof organisms within the sample. Thus arelative index of the amount ofcontamination can be generated usingfirefly bioluminescence within a fewminutes of sampling. Other chemicals canbe used to index the levels of microbialcontamination in addition to ATP.3. Material and methodsThe experiments were carried out on milksamples drawn from 12 dairy cows.The electrical conductance of the milksamples was measured with conductivitymeter type OK-102/1 (Radelkis).The instrument was standardized with KClsolutions of known conductance before use.The cell was washed with 0.01 M KClfollowed by one to two rinses with thesample prior to measurement. Temperaturecorrections were made, as the samples werenot analyzed at 25°C.In our experiments, the ATP determinationwas carried out with an apparatus (BioscanMonitor RHS 055) and products from theBetz Dearborn Company.103