Leonardo da Vinci

1. Effect of selected LAB onthe growth ofL. monocytogenes during productionof traditionallyfermented sausages Caklovica, F., Alagic, D., Smajlovic, M., Kozacinski, L., Cvrtila Z., Veskovic-Moracanin, S., Gasparik-Reichardt, J., Zdolec, N. (2005) Effect of selected LAB onL. monocytogenes during production of traditionally fermented sausages., Proc. Research Project “Safety of traditional fermented sausages: Research on protective cultures and bacteriocins”, Workshop for Dissemination p:72-84 Leonardo da Vinci

2. Introduction Fermentation and drying of meat products are probably themost ancient ways of preservation. Sausages made traditionally or with starter cultures can be considered as safe. Namely, reduction of water activity and pH are generally sufficient for that purpose. However, in the last decades new emerging pathogens have been isolated from foods. In raw fermented meat products L. monocytogenes was identified, being resistant to low pH, low aw and other environmental factors and induces serious human health risks.

3. Objective The objective of the present work was to study the survival, growth and loss of viability of L. monocytogenes in artificially inoculated fermented sausages using selected protective cultures (inoculation the sausage with high number of L. monocytogenes, 105 CFU/g) to verify the reduction of L. monocytogenes during sausage processing in different countries.

4. Materials and methods Strains - L. monocytogenes - pathogenic (from Italian partner) - L. sakei strains (isolates from sausages, I-151, I-154 and I-155)

5. Materials and methods Challenge test - sausage batter were prepared according to each country’sstandard practice without commercial starter cultures - the sausage batter was inoculated with about 105 CFU/g of L. monocytogenes and with protective starter cultures (inoculation in laboratory to prevent the contamination), - after stuffing the sausages were fermented and ripened applying traditional protocols of different countries - samples were taken from each batch at day 0, 3, 7, 14 and 28 of fermentation for microbiological and physicochemical analysis

6. Results Bosnia and Herzegovina - The growth of the pathogen in control sausages was observed up to day 3 and after a stationary phase lasted to the day 7. During the rest of fermentation and ripening phase L. monocytogenes count was slightly decreased about 1 log cycle at the end of the ripening (Figure 1) - All three investigated strains of Lb. sakei showed desirable impact on decrease of L. monocytogenes in Bosnian sudzuk in all batches. Growth of pathogen in inoculated sausages showed similar trend during the first seven days. Starting from day 7 the L. monocytogenes count was reduced and absence of the pathogen was observed on day 28 in all batches. - Strain I-151 showed the relatively best effectiveness against L. monocytogenes.

7. Figure 1 Effect of selected strains of Lb. sakei on the growth of L. monocytogenes in Bosnian sudzuk during 28 day fermentation and ripening (average of 3 batches)

8. Results Croatia • The growth of L. monocytogenes in control sausages slightly increased to day 3 and in the rest of fermentation the pathogen count decreased below detection limit (Figure 2).Absence of pathogen on day 28 in control sausage was characteristic only for Croatian one. • Growth of L. monocytogenes showed similar trend during the first 7 days of fermentation in sausages inoculated with the three protective strains. After this L. monocytogenes count was reduced and at the end of fermentation decreased below detection limit. • Strain I-155 showed the relatively best effectiveness against L. monocytogenes on day 14.

9. Figure 2 Effect of selected strains of Lb. sakei on the growth of L. monocytogenes in Croatian traditional sausage during 28 day fermentation and ripening (average of 3 batches)

10. Results Serbia and Montenegro • In control batch mean values of L. monocytogenes decreased continuously and showed about 3 log cycle reduction at the end of fermentation at day 28 (Figure 3). • In the batches with protective cultures, in all three fermentations the number of L. monocytogenes decreased continuously and could not be found even with enrichment procedure. • Strain I-151 showed a slight advantage over the other two strains on 14th day against L. monocytogenes.

11. Figure 3 Effect of selected strains of Lb. sakei on the growth of L. monocytogenes in Sremska sausage during 28 day fermentation and ripening (average of 3 batches)

12. Results Hungary • The growth of pathogen after a stationary phase was reduced only by1,5 log cycle during 28 days of fermentation (Figure 4). • The used protective starter cultures reduced L. monocytogenes count about 2 log cycle by the end of fermentation. No growth of the pathogen was detected at the beginning of fermentation. • The best effectiveness against L. monocytogenes showed strain I-151, too.

13. Figure 4 Effect of selected strains of Lb. sakei on the growth of L. monocytogenes in Hungarian traditional sausage during 28 days fermentation and ripening (average of 3 batches)

14. Table 1 Changes of pH during fermentation and ripening of sausages (mean values of three batches)

15. Table 2 Changes of salt% and aw during fermentation and ripening of sausages(mean values of three batches)

16. Conclusions • With the used protective starter cultures and combinations of antimicrobial treatments (salt, nitrite, pH and aw) the count of inoculated L. monocytogenes was reduced at the end of fermentation by 5 log cycle in Bosnian, Croatian and Serbian sausage. In Hungarian sausage the reduction of L. monocytogenes was only about 2 log cycles. • The reason of this can be the higher pH value of traditional Hungarian sausage compared with other sausages (Table 1) because the salt content was the highest and the water activity was low (Table 2). • No significant differences in antilisterial activity were observed among the strains, but slight advantage of strain I-151 was observed and it may be suggested to use as a protective starter culture.