Behaviour of L. monocytogenes in sliced, vacuum-packed mortadella.

This study evaluated the growth of naturally occurring L. monocytogenes in sliced, vacuum-packed mortadella samples during storage at 5°C until the expiration date. Tukey's test indicated that counts of L. monocytogenes on 0, 10, 20, 30 and 40 days of storage were significantly different (p<0.05), indicating growth during shelf life. In three trials, the mean increase was 1.72 log cycles. Vacuum packing and storage under refrigeration were not effective in controlling the growth of L. monocytogenes in sliced mortadella, indicating that good manufacturing practices and implemented HACCP programs are essential to assure safety of this product.

Listeria monocytogenes is ubiquitous and almost all kinds of foods may be contaminated with this bacterium. Equipment and utensils used in the food industry are considered important sources of contamination, and it is generally accepted that total elimination of L. monocytogenes from all foods is impracticable and may even be impossible. An important characteristic of L. monocytogenes is the capability to survive and grow under refrigeration, which is usually an obstacle for the growth of most foodborne pathogens. This fact is extremely relevant, mainly when ready-to-eat (RTE) products are considered, as they may have not been submitted to a listericidal step during processing and/or may have been contaminated after processing. When present, growth of Listeria may be favored by refrigeration in detriment of other microorganisms (10,12,13)

Epidemiological evidences indicate that food products that present the greatest risk for listeriosis dissemination are those that support the growth of the pathogen, have long shelf-life under refrigeration and are ready-to-eat (9,18).

In general, contamination of meat products occurs as primary contamination of the meat but also via ingredients and environment (3). A previous study has shown that L. monocytogenes does not survive the cooking process of mortadella (1) indicating that presence of the pathogen in vacuum-packed sliced product is due to cross-contamination during slicing/packaging.

Information on L. monocytogenes behavior in RTE products is important for the development of new strategies to control this pathogen in the product. Thus, the objective of the present study was to evaluate the capability of naturally occurring L. monocytogenes to grow in sliced, vacuum-packed mortadella stored under refrigeration.

Sliced, vacuum packed mortadella samples were obtained from a meat industry in packs of 250 g on the day of packaging. This industry received the mortadella in pieces of 5 kg in the original casing from another meat plant and sliced and packed the product using a fully automatized procedure. According to the label, the shelf life of the sliced product was 40 days under refrigeration, without specifying the temperature. The samples were placed in isothermal containers, transported to the laboratory and were tested immediately (day zero) or stored at 5°C.

Quantification of L. monocytogenes was done on days zero, 10, 20, 30 and 40, using the method described by Farber et al. (6) in a Most Probable Number technique format (14). In each sampling day, three packs of 250 g were analyzed. Fifty grams of mortadella were asseptically removed from each package and transferred to plastic bags containing 450 mL of Listeria Enrichment Broth, UVM Formulation (LEB) (Oxoid, England). After homogenization, the mixture was submitted to four decimal dilutions (equivalent to 10g, 1g, 0.1g and 0.01g of product) in LEB, in triplicates, and incubated at 30°C for 24h. For identification of L. monocytogenes, the methodology of Farber et al. (6) was carried out. The experiments were run three times (trials A, B and C).

Results were submitted to variance analysis and Tukey’s test, using Statistic for Windows, release 5.0 (Stat Soft Inc, 1984–1995).

Table 1 shows the mean populations of L. monocytogenes (MPNg-1) in the sliced, vacuum-packed mortadella samples, in the three trials, and the mean counts for each of the five days of analysis (days 0, 10, 20, 30 and 40). Final populations at the end of the shelf life were 89.1, 23.4 and 102.3 MPN.g-1 for trials A, B and C, respectively. Variance analysis of the results indicated that differences in these populations were not significant (p>0.05). Tukey’s test, applied to the general means, showed that differences in populations on day 10 compared to day 0, on day 20 compared to day 10, on day 20 compared to day 30 and on day 40 compared to day 30 were significant (p < 0.05), indicating multiplication of L. monocytogenes during the shelf life of the product stored under refrigeration (5°C).

Table 1

Mean populations of L. monocytogenes in sliced, vacuum-packed mortadella, during storage under refrigeration for 40 days.

	Mean population (MPN.g-1)*			General mean population**
Day	Trial A	Trial B	Trial C	(MPN.g-1)
0	0.03	1.00	0.25	0.40a***
10	0.14	0.72	11.20	4.00a
20	95.50	0.50	41.70	45.90b
30	12.60	1.60	2.40	5.50a
40	89.10	23.40	102.30	71.6b

mean of three determinations

mean of trials A, B and C

same letters indicate values that are statistically similar (p>0.05)

Similar populations of L. monocytogenes at the end of shelf life were reported elsewhere.Examining sliced vacuum-packed frankfurters the authors found counts around 100 – 200 CFU. g-1 of the product (16,17).

It is interesting to note that in two out of three trials the population of L. monocytogenes on the 30th day of storage was lower than that observed on the 20th day. This decrease in L. monocytogenes population may be explained by the presence of autoctonous lactic acid bacteria (LAB), well-known for their capability of inhibiting growth of L. monocytogenes during food storage (4). Contamination with LAB can occur during slicing and packaging procedures, mainly when hygiene conditions are precarious.

Table 2 shows that L. monocytogenes population increase (mean MPN.g-1) during storage at 5oC for 40 days was equal to 76.4, 22.1 and 89.0 for trials A, B and C, respectively. General mean increase of L. monocytogenes was 62.5 MPN. g-1. According to several reports, L. monocytogenes population can increase up to 6 logs in vacuum-packed meat products stored under refrigeration (3,7,8,11,17). However these studies were conducted with spiked products and the conditions required for the growth of microorganisms are not necessarily the same as for naturally contaminated foodstuffs. In artificially contaminated products, the microbial cells are in optimal physiological conditions, favoring the competition with the normal microbiota of the product.

Table 2

Increment in population of L. monocytogenes in sliced, vacuum-packed mortadella during storage under refrigeration for 40 days

Interval in days	Increment (MPN.g-1)*			General increment
	Trial A	Trial B	Trial C	(MPN.g-1)
0 - 10	0.10	-0.28	10.95	3.60
10 - 20	95.4	-0.22	30.50	41.9
20 - 30	-82.9	1.10	-39.3	-40.4
30 - 40	76.5	21.8	99.9	66.1
Total increment	76.4	22.1	89.0	62.5

mean of three determinations.

Refrigeration between 4 and 8°C of sliced, vacuum-packed meat products does not seem to be an obstacle to L. monocytogenes growth, as observed in the present study and also by Rosso et al. (15). Temperatures below 2°C are recommended for the control of L. monocytogenes in these products; however, these temperatures are not commonly achieved at retail level. It should be also taken into account that L. monocytogenes tolerates low O2 tension, being able to survive and grow in anaerobic conditions, even at low temperatures (13), as encountered in vacuum-packed mortadella stored under refrigeration.

The low initial L. monocytogenes population in the products found in this study has also been observed by other authors (5,16,17). However, these low initial numbers are not an assurance of low risk for the product since the pathogen may grow and reach risky levels (≥100g-1) (13).

The method employed for enumeration of L. monocytogenes in a food product has strong influence in the results (2). Once initial population of the pathogen can be as low as 0.03 MPN g-1 (Table 1), one may not find L. monocytogenes in the product if the method lacks sensitivity.

In the present study L. monocytogenes was found in all sliced mortadella samples. This result differs from the one observed in a previous study (1), carried out with non-sliced mortadella, where the pathogen was detected in 26.7% of the samples. These findings indicate that slicing has an important role in the contamination process.

In view of the fact that L. monocytogenes can reach risky levels in vacuum-packed sliced mortadella stored under refrigeration, appropriate control measures are needed to minimize contamination of the product and/or inhibit the growth of the pathogen. Implementation of good hygiene practices (GHP), SSOPs and good manufacturing practices (GMP), together with HACCP is of uppermost importance to minimize environmental and product contamination. Reduction of shelf life, reformulation of the product by adding inhibitory ingredients, post-packing listericidal treatment (e.g. irradiation or ultra-high pressure) and active packaging are some options for the control growth of the pathogen in sliced meat products such as mortadella.