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

A. Căpriţă et al ./ Journal of Agroalimentary Processes and Technologies 14 (2008)For all the stages, followedprotocols are those prescribed by thiscompany. The photometer produces someRelative Light Unit (RLU) during theenzymatic reaction. The Bioscan MonitorRHS 055 employs a sampling pen, whichfacilitates precise sampling, eliminating theneed to mix and measure reagents, andfunctions as the measurement cell for theluminometer. A total ATP sampling penconsists of the following parts: -a stick foraccurate sampling of the liquid to be tested,coated with an extractant, which releaseATP from cellular material; the stick alsotransfers the sample into the cuvette; -acuvette filled with test buffer for dilution,buffering and neutralization of the sample;-a reagent chamber sealed with aluminiumfoil, containing freeze-dried and stabilizedluciferin/luciferase reagent.4. Results and DiscussionIn our previous studies (Caprita et al., 2007)we observed the best correlation betweenRLU and the SCC and CFU sum in raw milk(r=0.9590). The electrical conductivity valuesof the same milk samples show that the RLUand the electrical conductivity values arehighly correlated (r = 0.9553).These two rapid methods can be used inmonitoring the microbiological quality of rawmilk. The electrical conductivity assay has thedisadvantage of being induced also by milkacidification (Caprita, 2007).El. cond. (mS/cm)5.35.25.154.94.84.74.6y = 1.7171x - 2.6371R 2 = 0.91274.54.1 4.2 4.3 4.4 4.5 4.6 4.7log RLUFigure 1. The correlation between RLU and the electrical conductivity4. ConclusionsThe bioluminescence assay of ATP and theelectrical conductivity assay are very rapid,non-polluting methods.There is positive correlation betweenmilk ATP and electrical conductivity(r= 0.9553).Both methods can be used for monitoringthe microbiological quality of raw milk.ReferencesCaprita, A., Caprita, R., Vintila, T. (2007). TheATP assay, a method for measuring biologicalactivity in raw milk. Proceedings of 5thInternational Congress on Food Technology,Thessaloniki, 9-11 mar., vol. 2, 500-505.Caprita A., Caprita, R. (2007). Physico-chemicalchanges during milk fermentation.Proceedings of the International Conference„Agricultural and Food Sciences Processesand Technologies”, Sibiu, 26-27 apr., 171-174.104

A. Căpriţă et al ./ Journal of Agroalimentary Processes and Technologies 14 (2008)Easter, M. C., Gibson, D. M. (1989). Detectionof microorganisms by electricalmeasurements. In M. R. Adams and C. F.A. Hope, Rapids Methods in foodMicrobiology. Amsterdam: Elsievier Sci.Publ.Felice, C.J., Madrid, R.E., Olivera, J.M.,Rotger, V.I., Valentinuzzi, M.E., (1999).Impedance microbiology: quantification ofbacterial content in milk by means ofcapacitance growth curves. J. Microbiol.Methods. 35, 37–42.Firstenberg-Eden, R., Zindulis, J. (1984).Electrochemical changes in media due tomicrobial growth. J. Microbiol. Methods,2, 103–115.Griffiths, M. W. (1993). Applications ofbioluminescence in the dairy industry. J.Dairy Sci., 76, 3118-3125.Niza-Ribeiro, J., Louza, A. C., Santos, P.,Lima, M. (2000). Monitoring themicrobiological qualityof raw milk through the use of an ATPbioluminescence method. Food Control, 11,209–216.Rosmini, M. R., Signorini, M. L., Schneider, R.,Bonazza, J. C. (2004). Evaluation of twoalternative techniques for counting mesophilicaerophilic aerobic bacteria in raw milk. FoodControl. 15, 39–44.Ong, K. G., Wang, J., Singh, R. S., Bachas, L. G.,Grimes, C. A. (2001). Monitoring of bacteriagrowth using a wireless, remote queryresonant-circuit sensor: application toenvironmental sensing. Biosens. Bioelectron.16, 305–312.Silley, P., Forsythe, S., (1996). Impedancemicrobiology—a rapid change formicrobiologists. J. Appl. Bacteriol. 80, 233–243.Yang, L., Li, Y., Griffis, C. L., Johnson, M. G.,(2004). Interdigitated microelectrode (IME)impedance sensor for the detection of viable,Salmonella typhimurium. Biosens.Bioelectron. 19, 1139–1147.105