Validation and stability studies on the MBS method for microbiological control of food samples

Abstract

Food safety and quality are issues of vital importance nowadays. The increased potential threat for human health has led to continuous research for innovative processes and systems for food control, in order to obtain a significant improvement of food quality and, at the same time, a considerable reduction of biological contamination. For this reason, in recent years it’s become necessary to develop faster and more reliable analysis systems that allows to obtain immediate results making possible a more rigorous prevention of microbiological hazards. D.L. 31/01 and Regulation EC 2073/2005 specify that, in addition to reference methods for food microbiological analysis, alternative analytical methods can be used after validation. To validate an alternative method means to compare it with a reference method, in order to define specificity, sensitivity and reproducibility of the two methods in comparison. The innovative MBS method meets these requirements: the MBS method has the prerequisite of being a fast method that can detect bacterial contamination and, in most cases, provide results within the same day. The MBS method measures the catalytic activity of redox enzymes of the main metabolic pathways of bacteria, allowing an unequivocal correlation between the observed enzymatic activity and the number of viable cells present in the samples. The time taken to change colour is inversely related with the log of bacterial concentration; like an enzymatic reaction, the greater the number of bacteria, the faster the colour change. The results reported in this thesis further support the previous findings concerning the existence of a stringent correlation between metabolic activity of bacteria and the number of viable cells. Comparing the MBS method to other analytical methods currently in use, the following considerations come to light. With traditional count plate method bacteria replication can be observed with the naked eye, but greater expertise in the operators and operational complexity are required. On the other hand, alternative methods often turn out to be very expensive also requiring highly equipped laboratories. The use of immunological or genetic probes (with the assistance of PCR to increase sensitiveness) had a great impact in microbial analysis. Indeed they are very quick and sensitiveness can be improved by using automated o semi-automated systems. The disadvantages are not only related to the need for specialized personnel and equipment, but also for a high limit of sensitiveness (immunological methods) and/or complexity and high costs of analysis (genetic methods). In addition the exact quantification of the number of bacteria over a large range of concentrations is not always possible. Colorimetric methods currently available are mainly based upon microorganisms secondary metabolism measuring, but they are very expensive and can not be used outside of specialized laboratory. The work in this thesis concerns studies on the stability of MBS reagents, used for food microbiological analysis. Stability studies represent a critical point to assess the quality of a substance. The correct storage is very important to maintain the products characteristics for the entire period of validity. This can only be achieved thanks to a complete and correct stability program. Accelerated and natural aging tests were performed at various times on the different MBS reagents. Microbiological and physical-chemical parameters were then evaluated. Accelerated aging tests have been carried out maintaining the reagents in an oven in humidity saturated atmosphere and comparing different storage conditions. Thanks to these studies, informations were obtained on the best storage conditions of reagents, their shelf life, and on how to improve stability: compositions of the reagents was slightly modified in order to accomplish these results. Another crucial information furnished by these test was the importance of providing protection from humidity, which has resulted in improved stability. In this regard it has been observed that both the presence of Vaseline and the use of a humidity protective packaging were essential to ensure as long as possible the functionality of the reagents. After this research, several tests were carried out on different tipologies of food samples using the Multi Reader, a modular optical device made by the Department of Physics of the University of Tor Vergata. The Multi Reader is a device used for the analysis: vials can be thermostated, the time taken for them to change colour is automatically detected and the amount of microorganisms present in the sample is calculated. Studies were carried out to set up the Multi Reader software parameters for the automatic detection of the colour change occurring in the presence of bacteria. The last part of the work has concerned the validation of the Micro Biological Survey (MBS) method for food microbiological analysis. Validation aims to compare the results obtained with an alternative method, in this case the MBS method, with the results obtained with the reference method verifying the equivalence between the two methods by looking at linearity, accuracy and selectivity. The results were statistically analysed and compared according to the ISO/IEC 17025 (2005) and ISO 16140 (2003) standards, verifying the equivalence between the reference method and the MBS method. All the performance parameters indicated a total equivalence between the two methods for detection and counting of TVC and E. coli in artificially contaminated and in naturally contaminated food samples. In this study, five different food matrices, cheese, vegetables, white meat, red meat and fruit are analyzed. The validation of the MBS method strongly supports its use as an alternative method for food analysis. The linearity over a range of bacterial concentrations was excellent. The selectivity was more than satisfactory with the absence of false negatives and false positives. The accuracy, evaluated on 125 naturally contaminated samples, showed a high correlation between the MBS method and the reference methods. For the above reported reasons, I have demonstrated that the MBS method can represent a worthy aid in food screening without replacing the analysis carried out with traditional methods which are very precise though often long and complex. In conclusion, this thesis is an applied research project in which the sperimentation essentially aims to develop and validate a kit for microbiological analysis of food. It is therefore important to underline the industrial relevance of these studies, representing a best practice example of a successfully technology transfer project.