Today the challenge test is the most effective tool for estimating the growth potential and maximum growth rate of microorganisms in foods. In this thesis study, the aim was to examine and compare the population kinetics of various strains of Listeria monocytogenes using beef homogenized baby-foods as substrate. The strains used had previously been isolated from different food sources and they come from a reference strain collection. Their serogroups were determined through PCR methodology at the “Istituto Zooprofilattico Sperimentale” of Teramo. The references for the development of this experimental design took into consideration the standard UNI EN ISO 20976-1:2019, which contains indications for setting up and carrying out challenge tests for all foodborne pathogens in all foods; and the EURL Lm Anses version 4 of 1st July 2021, a technical guidance document on challenge tests and durability studies of ready-to-eat foods related to Listeria monocytogenes. In the beginning, a series of preliminary tests were scheduled to assess the suitability of the matrix to support bacterial growth and to set up the analyses, considering the product's shelf-life. The results obtained confirmed that by applying the same culturing methods consistently, a starting load of L. monocytogenes of 10e8 - 10e9 CFU/ml in the original cultures could be achieved with repeatability, from which inoculations started in 45 test units for each strain, with a concentration of 50-200 CFU/ml. A portion of the test units was kept at a constant temperature of 6°C for 14 days to calculate the maximum growth rate and growth potential. The second half of the test units was subjected to thermal abuse conditions of 7-10°C for 16 days to calculate the growth potential. pH and water activity values were determined on individual control units kept under different storage conditions, to evaluate if the different temperatures used could influence the bacterial growth. Through the analysis of variance (ANOVA), it was observed that the strain 82 exhibited some behavioral differences, with increasing and decreasing concentrations of 27% at 6°C. The different storage temperature influenced the growth potential, with a maximum value of 1.2 log10 at 6°C and 5.63 log10 at 7-10°C in strain 82. The maximum growth rate under 6°C storage conditions showed no significant differences both within the same strain and among the 6 different strains, as their growth was minimal, and µmax could not be calculated.
Il challenge test è lo strumento a oggi più efficace per stimare il potenziale di crescita (growth potential) e il tasso di massima crescita (maximum growth rate, µmax) dei microrganismi negli alimenti. Nello studio oggetto di tesi lo scopo era di valutare e confrontare la cinetica di popolazione di più ceppi di Listeria monocytogenes utilizzando come unico substrato dell’omogeneizzato di manzo. I ceppi impiegati, provenienti da una ceppoteca di riferimento, erano stati precedentemente isolati da diverse matrici alimentari e sottoposti ad analisi per la determinazione del sierogruppo (tramite metodica PCR) presso l’Istituto Zooprofilattico Sperimentale di Teramo. Per tracciare il disegno sperimentale di questo studio sono state prese in considerazione la norma UNI EN ISO 20976-1:2019 che contiene le indicazioni per l'impostazione e l'esecuzione di challenge test per tutti i patogeni di origine alimentare in tutti gli alimenti e la EURL Lm Anses versione 4 del 1° luglio 2021, documento di orientamento tecnico sui challenge test e sugli studi di durabilità di alimenti pronti correlati a Listeria monocytogenes. Preliminarmente ho programmato una serie di prove iniziali per valutare l’idoneità della matrice a supportare la crescita del batterio e per impostare al meglio le analisi tenendo in considerazione la shelf-life del prodotto. I risultati ottenuti hanno permesso di confermare che applicando sempre le stesse modalità colturali si può raggiungere, con ripetibilità, una carica di partenza di L. monocytogenes pari a 10e8 - 10e9 ufc/ml nelle colture originarie. Da esse poi è stato programmato di iniziare con le inoculazioni di 45 omogeneizzati per ogni ceppo. Una parte delle unità di prova sono state mantenute a temperatura di 6°C costanti per 14 giorni col fine di arrivare a calcolare il tasso di massima crescita e il potenziale di crescita. Una seconda metà delle unità di prova è stata mantenuta in condizioni di abuso termico programmato a 7-10°C per 16 giorni per il calcolo del potenziale di crescita. Sulle singole unità di controllo di omogeneizzato sono stati determinati i valori di pH e aw nelle diverse condizioni di mantenimento, col fine di accertare se la temperatura influenzi o meno la crescita batterica con eventuali ripercussioni sulle caratteristiche chimico-fisiche del substrato. Attraverso l’analisi della varianza è emerso che il ceppo 82 alla temperatura di 6°C ha presentato alcune differenze di comportamento, con aumenti e decrementi del 27%. La diversa temperatura di conservazione ha influenzato il potenziale di crescita, con un valore massimo di 1,2 log10 a 6°C e 5,63 log10 a 7-10°C nel ceppo 82. Non sono state individuate differenze significative in condizioni di conservazione a 6 °C tra i dati utili per il calcolo del tasso di massima crescita, sia nello stesso ceppo sia tra i 6 diversi ceppi; la loro crescita si è rilevata minima e non si è potuto calcolare il µmax.
Confronto della dinamica di popolazione di ceppi differenti di Listeria monocytogenes in alimenti, con misurazione del potenziale di crescita e del tasso di crescita massimo.
MARCHIORI, SOFIA
2022/2023
Abstract
Today the challenge test is the most effective tool for estimating the growth potential and maximum growth rate of microorganisms in foods. In this thesis study, the aim was to examine and compare the population kinetics of various strains of Listeria monocytogenes using beef homogenized baby-foods as substrate. The strains used had previously been isolated from different food sources and they come from a reference strain collection. Their serogroups were determined through PCR methodology at the “Istituto Zooprofilattico Sperimentale” of Teramo. The references for the development of this experimental design took into consideration the standard UNI EN ISO 20976-1:2019, which contains indications for setting up and carrying out challenge tests for all foodborne pathogens in all foods; and the EURL Lm Anses version 4 of 1st July 2021, a technical guidance document on challenge tests and durability studies of ready-to-eat foods related to Listeria monocytogenes. In the beginning, a series of preliminary tests were scheduled to assess the suitability of the matrix to support bacterial growth and to set up the analyses, considering the product's shelf-life. The results obtained confirmed that by applying the same culturing methods consistently, a starting load of L. monocytogenes of 10e8 - 10e9 CFU/ml in the original cultures could be achieved with repeatability, from which inoculations started in 45 test units for each strain, with a concentration of 50-200 CFU/ml. A portion of the test units was kept at a constant temperature of 6°C for 14 days to calculate the maximum growth rate and growth potential. The second half of the test units was subjected to thermal abuse conditions of 7-10°C for 16 days to calculate the growth potential. pH and water activity values were determined on individual control units kept under different storage conditions, to evaluate if the different temperatures used could influence the bacterial growth. Through the analysis of variance (ANOVA), it was observed that the strain 82 exhibited some behavioral differences, with increasing and decreasing concentrations of 27% at 6°C. The different storage temperature influenced the growth potential, with a maximum value of 1.2 log10 at 6°C and 5.63 log10 at 7-10°C in strain 82. The maximum growth rate under 6°C storage conditions showed no significant differences both within the same strain and among the 6 different strains, as their growth was minimal, and µmax could not be calculated.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/56134