EVALUATION OF THE MICROBIOCIDAL EFFECTIVENESS OF A DOMESTIC REFRIGERATION CONTAMINATION CONTROL SYSTEM. Microbiology laboratory

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1 EVALUATION OF THE MICROBIOCIDAL EFFECTIVENESS OF A DOMESTIC REFRIGERATION CONTAMINATION CONTROL SSTEM. Microbiology laboratory Circulation Restricted report Veneto Agricoltura Microbiology Laboratory ork Group Thiene (VI) - Italy Dino Spolaor Biotechnology and Agro alimentary Sector Manager dino.spolaor@venetoagricoltura.org Angiolella Lombardi Microbiology and Biotechnology Laboratory Manager angiolella.lombardi@venetoagricoltura.org Maria Lucia Dal Santo, Elisa Fioravanzo Laboratory Technicians Materials delivered by the Client: 16/01/2006: Two normal fridge-freezers one of which had been modified with the fitting of a CoPlasma microbial contamination control device. Scope of the evaluation Assessment of the effectiveness of the CoPlasma system for containing the growth of some microbial species that, from a hygiene/sanitation point of view, could contaminate various types of foodstuffs stored in domestic refrigerators. The system should create no perceptible alterations in preserved foodstuffs. Scope Tests were carried out using the two refrigerators. Both refrigerators were used in parallel so that data obtained from the Test unit could be compared with that from the Control unit. Resources involved Two refrigerators as outlined above Temperature monitoring devices inside the refrigerators Various types of foodstuffs on which to conduct sensory-based assessments A cross section of differing undesirable bacterial strains most commonly found in animal and/or vegetable foodstuffs Via dell Università 14 Agripolis Legnaro (Pd) Tel Fax Cod. Fisc Laboratories: Thiene (VI). Via S. Gaetano, 74. Tel. 0445/ Fax 0445/ istituto.thiene@venetoagricoltura.org sito web:

2 Additional test information Ozone concentration and negative ion measurements taken from inside the refrigerators Temperature readings from inside the refrigerators DESCRIPTION OF THE TESTS First phase: Preparing the equipment The refrigerators were set up in the microbiology laboratory. Temperature was monitored for 24 hours (once every 20 minutes) before the tests began. The difference between the two refrigerators was 0.44 C and this falls within the 1 C agreed tolerance limit. Temperature ( C) Test Refrigerator (with CoPlasma device) Control Refrigerator (without CoPlasma dev.) Difference C Average Standard deviation Second phase: Sensory assessment of foodstuffs The foodstuffs were chosen in compliance wit document 01 rev. 2 Project IT_05 sent on 18/01/2006 and these items were selected because they were best suited to a standard sensory test protocol. For results of the sensory analysis tests, refer to the Sensory Analysis Laboratory report. Third phase: Evaluation of microbicidal effects In compliance with document 01 rev. 2 Project IT_05, the effectiveness of microbicidal treatment was measured on two microbial species (Listeria innocua BCCM LMG and E. Coli BCCM LMG 2092) kept at 80 C in the Microbial Collection at the Institute for Agro alimentary Quality and Technology. The two microbial strains used in the test are regarded as Type Strain of the species tested and are registered in the International Microbial Collection belonging to the FCC (orld Federation for Culture Collection). Preparing and inoculating cultures Cultures of the same strains were cultivated overnight at 30 C in Tryptic Soy Broth diluted in Ringer s solution. The surface of the Tryptic Soy Agar (TSA) was inoculated with 0.1 ml using a plastic sterile spatula. Pag. 2 of 7

3 Treatment of dishes within the refrigerators After being inoculated respectively with Listeria innocua and Escherichia coli, the uncovered dishes were placed in a regular pattern on the earthed shelves of both the Test and Control refrigerators using the following time and procedure parameters. Dish incubation On completion of each treatment, the dishes were incubated at 37 C for time periods appropriate to each microbial species and were then subjected to counting. Contamination criteria The aim of depositing a known amount of microbial cells on the surfaces of the medium in the Petri dishes was to simulate, as closely as possible, the microbial contamination found inside a refrigerator (walls, shelves), on foodstuff packaging or that found directly on the surfaces of uncovered foodstuffs. For this reason, selective culture media were not used as they could have partially inhibited normal microbial development. The conditions for the potential development of micro-organisms on a nutrient agar medium are without doubt more favourable than those of accidental contamination in relation to the types of surfaces mentioned above. For this reason, they allow more thorough evaluation of the decontamination system being assessed. Test plan So as to conduct a thorough study of the varying factors that might influence the effectiveness of the CoPlasma system, the adopted test plan was based on the randomised distribution of Petri dishes using the Latin Square layout. This methodology allowed the differing effects on treatment to be assessed including shelf position, the distance between ion emitters, and treatment times. An ascending order coding system was used to identify treatment times with for the shortest time and, and for longer times. Randomised dish distribution diagram 1 Refrigerator shelves Negative ions emitters A B C D Pag. 3 of 7

4 As can be seen in the diagram, eight dishes were placed in two rows of four on each shelf. The front and rear rows were laid out in the same manner and are thus replicas. The Test and Control dishes were placed in the refrigerators for the differing times shown below depending upon their codes and the micro-organisms subjected to treatment. Micro-organism Listeria innocua 4 hours 8 hours 12 hours 24 hours Escherichia coli 5 hours 12 hours 25 hours 31 hours Treatment for Escherichia coli was prolonged as they are a Gram Negative species with cellular envelopes that provide increased resistance to anti-microbial treatment and have proven to be more resistant in previous tests. RESULTS Statistical evaluation using the analysis of variables Using an analysis of variables of the data collected, allowed observations to be made regarding the even distribution of treatment in all the test chambers. Significant differences showed up in the analysis of variables tables but only in relation to treatment times. Effects created by shelf position and distance from emitters had no bearing. To better understand these elements, refer to the Analysis of Variables shown below of one of the tests carried out. From the statistical point of view, all the treatments carried out on Listeria and Escherichia have the same results. VARIANCE DEVIATION DoF VARIANCE F Significance F(0,05) F(0,01) ANALSIS TOTAL Treatment *** time effect Emitter NS distance effect Refrigerator NS shelf effect ERROR Key: *** = Highly Significant; NS = Not Significant; DoF = Degrees of Freedom Pag. 4 of 7

5 Evaluation of microbicidal effects on Listeria This photo shows a Control Petri dish (on the left) and a Treated dish. The decrease in the number of Listeria cells subjected to decontamination treatment on the refrigerator shelf can be easily seen. A graph showing the results of counting carried out on dishes at differing times during treatment shows more clearly and objectively the effectiveness of the decontamination system fitted to the Test refrigerator. The test carried out on Listeria innocua used Petri dishes inoculated with a number of vital bacterial cells in excess of 500 (CFU/dish). During the entire treatment period, these remained at uncountable levels on the dishes in the Control refrigerator. For this reason, a constant value of 500 was assigned for the entire 24-hour period. In the Test refrigerator however, a rapid reduction in the number of bacterial cells was observed from 4 hours after treatment began. Listeria innocua inactivation 600 Average readings FCU/ Petri dish TREATED CONTROL 0 0 h 4 h 8 h 12 h 24 h Treatment time Pag. 5 of 7

6 Evaluation of microbicidal effects on Escherichia A similar trend was observed with Escherichia coli even if the microbicidal effect took place later than with Listeria due to its known higher resistance to microbicidal treatment. 300 Escherichia coli inactivation Average readings FCU/ Petri dish TREATED CONTROL 0 5 h 12 h 25 h 31 h Treatment time Evaluation of microbicidal effects of 24-hour treatment A further test was carried out to validate the data collected for the statistical tests. This involved inoculating 36 dishes, 32 of which were placed in the Test Refrigerator and 4 in the Control unit. The plates had been inoculated with 57 ± 10 CFU/dish (estimated on the dishes in the Control Refrigerator). After a 24-hour treatment, the difference between Test and Control was 2.56 log. The table below shows the counts obtained after 24 hours of treatment from the 32 dishes positioned on all the refrigerator shelves. SHELVES Listeria innocua (24-hour treatment on 57 +/- 10 CFU/ Petri DISH) DISTANCE FROM ION EMITTER A B C D Pag. 6 of 7

7 CONCLUSIONS The tests used appropriate statistically significant data to demonstrate that the decontamination system fitted can, even after just 24 hours, reduce the presence of potentially pathogenic micro-organisms by a factor of more than 2 logarithms. The positioning of the ion emitters permitted effective ionisation of the environment inside the refrigerator as demonstrated by the results of the analysis of variables. e felt it appropriate, given the data acquired, to repeat several of the tests described above but his time with the refrigerator shelves not earthed. The tests carried out confirmed that the technique used for assessing microbicidal effectiveness on dishes containing nutrient agar were well suited for providing data that could be statistically processed and that they definitely offer better surface simulation conditions than tests carried out directly on foodstuffs. Too often in the past, problems have been encountered relating to quantifying contamination on foodstuffs because they are all very different in terms of shape, size, acidity and matrix type. It is to be said that the system we examined should not be seen as a means of killing bacteria on food that, as a matter of good hygiene practice, should only be stored in a refrigerator if properly packaged. A bactericidal action however, that is restricted to food surfaces may be deemed possible if one considers the effectiveness observed on agar surfaces. The utility of the system lies in its ability to create a purer environment that prevents contamination between contact surfaces and foodstuffs. Such events happen all too often when good practice is not observed. It is well known that microbial species belonging to the Listeria genus have become a hygiene safety problem due to their ability to proliferate on condensation and on cooled surfaces from which refrigerated foodstuffs can then be easily contaminated. Pag. 7 of 7