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Determination of Listeria monocytogenes growth potential on new fresh salmon preparations
Food Control 21 (2010) 1415–1418
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Short communication
Determination of Listeria monocytogenes growth potential on new fresh salmon preparations Graziella Midelet-Bourdin *, Stéphanie Copin, Guylaine Leleu, Pierre Malle Agence Française de Sécurité Sanitaire des Aliments, Laboratoire d’Etudes et de Recherches sur les produits de la pêche, Boulogne sur Mer, France
a r t i c l e
i n f o
Article history: Received 24 September 2009 Received in revised form 17 March 2010 Accepted 28 March 2010
Keywords: Listeria monocytogenes Salmon Growth
a b s t r a c t The aim of this work was to evaluate the increase of Listeria monocytogenes on new salmon preparations (salt–sugar–pepper–dill salmon) in comparison with that obtained on cold-smoked salmon. Salmon preparations were inoculated with L. monocytogenes and were analyzed during storage at 4 °C then 8 °C. At 8 °C, the bacteria growth was of 4.53 log CFU g 1 in cold-smoked salmon and of 2.06 log CFU g 1 in salt–sugar–pepper–dill salmon without background microflora. The growth of L. monocytogenes was different in new salmon preparation because the mixture salt–sugar–pepper had an anti-Listeria activity and its presence could inhibitory to the growth. It is difficult to generalize findings observed with coldsmoked salmon to a new salmon preparation. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction New salmon-based products have been available for some years in the fresh food departments of supermarkets and food shops, including raw salmon carpaccio, and raw or smoked salmon marinated in herbs. Salmon marinated in herbs is ready-to-eat product and is regularly consumed in Sweden and in other countries of Northern Europe. Many reports have investigated Listeria monocytogenes in smoked salmon (studies of prevalence, characterization of strains, risk assessment) (Beaufort et al., 2007; Lappi, Ho, Gall, & Wiedmann, 2004), but few studies have been performed with new fresh salmon-based products (Lindqvist & Westoo, 2000; Loncarevic, Tham, & Danielsson-Tham, 1996). Despite prevalence rates ranging from 0% to over 50% in salmon preparations, levels of contamination are generally low (<100 CFU g 1) (Beaufort et al., 2007). However, these levels can exceed 100 CFU g 1 during or at the end of storage (Midelet-Bourdin, Leleu, & Malle, 2007). Bacteria such as L. monocytogenes adapt well to this type of food matrix (Huss, Jorgensen, & Vogel, 2000) and can grow over a wide range of temperature, pH and water activity (Ross, Dalgaard, & Tienungoon, 2000). We studied the increases of L. monocytogenes on new fresh salmon products (salmon with herbs, carpaccio of raw salmon) as a function of the storage temperature (4 °C and 8 °C) and of background microflora and in comparison with that obtained on cold-smoked salmon. As noted in standard NF V01* Corresponding author. Address: AFSSA LERPPê, Boulevard Bassin Napoléon, F-62200 Boulogne sur Mer, France. Tel.: +33 (0) 3 21 99 25 00; fax: +33 (0) 3 21 99 17 25. E-mail address: [email protected] (G. Midelet-Bourdin). 0956-7135/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2010.03.009
009, when data are collected in variable conditions such as temperature, it is not possible to calculate lag times and the growth rates (lmax). These data can, however, be used to determine the growth potential over a given period of time. Growth potentials defined as changes in log CFU g 1 between phases of storage were determined as indicated in the French Food Safety Agency (AFSSA) recommendation (2005) and in standard NF V01-009.
2. Materials and methods 2.1. Bacterial strains and growth conditions As recommended by French Food Safety Agency (Agence Française de Sécurité Sanitaire des Aliments, 2001, 2005), we worked with the reference strain, L. monocytogenes CIP 78.38, serovar 4b– e, of unknown origin from the collection of the Pasteur Institute (Paris, France), and a field strain, L. monocytogenes E 1012 PT1, serovar 1/2a–3a (AFSSA collection, Boulogne sur mer, France), isolated from smoked salmon. Cultures were maintained in brain heart infusion (BHI) broth (AES, Combourg, France) with 18% (vol/vol) glycerol at 80 °C. Before testing, both strains were first subcultured in BHI at 37 °C for 24 h and 100 ll of suspension in 100 ml of BHI were incubated at 8 °C for 10 days (bacteria were in the stationary growth phase at the end of the storage condition). The suspension was then washed twice by centrifugation at 2800 g for 5 min at 20 °C in 30 ml of physiological saline (8.5 g of NaCl per liter of water). The pellet was resuspended in 9 ml of physiological saline and the bacterial concentration was adjusted to 3 108 CFU ml 1 (OD650 nm = 0.15).
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Table 1 Physicochemical parameters for salmon products. RS: raw salmon, SCSS: salt-cold-smoked salmon, SS: salt salmon, SSDS: salt–sugar–pepper–dill salmon, SSS: salt–sugar–pepper salmon. Salmon product
RS SCSS SS SSDS SSS
Physicochemical parameters aw
Salt content (g per 100 g)
Water content (g per 100 g)
Salt content in water phase (WPS) (g per 100 ml)
Phenol content (%)
0.9997 0.9997 0.9997 0.9997
2.80 3.19 3.1 3.65
62.82 62.80 61.8 61.57
4.46 5.08 5.02 5.93
0.94
2.2. Salmon products (raw, salted, salt-cold-smoked, salt–sugar– pepper and salt–sugar–pepper–dill) Products were prepared from Atlantic salmon (Salmo salar) by a company in the north of France. The first product was raw salmon carpaccio: raw sliced salmon fillets. The second product was salted salmon: raw salmon fillets treated with dry salt, and then sliced. The third product was salt-cold-smoked salmon: raw salmon fillets treated with dry salt, smoked at below 30 °C using beech wood, then sliced. The fourth product was salt–sugar–pepper salmon: raw salmon fillets covered with a mixture of dry salt, pepper and sugar, and then sliced. The fifth product was salt–sugar–pepper– dill salmon: raw salmon fillets covered with a mixture of dry salt, pepper, sugar and dill, and then sliced. The physicochemical parameters of these products were presented in Table 1. The phenol content was determinate by the method of (Grondin & Guillard, 2001). Salt contents (in g/100 g) were divided by water contents to obtain water phase salt (WPS) in g/100 ml (Cornu et al., 2006). The water activity, aw, was calculated from WPS by the equation aw = 1 0.0052471WPS 0.00012206WPS2. Salting and smoking times depended on salmon size. The salmon slices were vacuumpackaged in portions of about 150 g.
Sugar content (g per 100 g)
Dill content (g per 100 g)
5.3 5.5
1.8
L. monocytogenes was counted onto plates of Palcam agar (Oxoid, Dardilly, France) after 48 h incubation at 37 °C. Monitoring of background microflora as well as detection and quantification of naturally present L. monocytogenes were respectively performed according to the standards AFNOR XPV08-062 (quantification of background microflora or mesophilic bacteria on plate count agar incubated for 72 h at 30 °C), NF V 08050 and EN ISO 11290 (detection of L. monocytogenes on PALCAM and Oxford media incubated for 48 h at 37 °C). 2.5. Statistical analysis As noted in standard NF V01-009, when data are collected in variable conditions such as temperature, it is not possible to calculate lag times and the growth rates (lmax). These data can, however, be used to determine the growth potential over a given period of time. Growth potentials defined as changes in log CFU g 1 between phases of storage were determined as indicated in the AFSSA recommendation (2005) and in standard NF V01-009. The data were analyzed using analysis of variance. All calculations were performed using Statgraphics Plus 5 software (Sigma plus, Paris, France), and P < 0.05 was accepted as statistically significant.
2.3. Inoculation of salmon products One series of vacuum-packaged salmon was treated with ionizing radiation (beta) at a controlled minimal dose of 10.2 kGy (Ionisos, Orsay, France) to obtain a bacteria-free product. A second series of vacuum-packaged salmon was used in challenge tests on exiting the factory, as concentrations of initial background microflora were low (around 103 CFU g 1). A third series of vacuum-packaged salmon was stored for 15 days at 8 °C to enable background microflora to grow so that initial bacterial concentrations were high (greater than 107 CFU g 1). Fifty microliters of L. monocytogenes suspension were spread on the surface of salmon slices (25 g) to yield final concentrations of 1000 CFU g 1, 100 CFU g 1 and 10 CFU g 1. After inoculation, the salmon slices were again vacuum-packaged and stored as follows: 7 days at 4 °C ± 1 °C (manufacture of products and transport to the point of sale), 7 days at 8 °C ± 1 °C (point of sale), 2 h at 20 °C ± 1 °C (break in cold storage corresponding to transport from point of sale to the consumer’s home), and 16 days at 8 °C ± 1 °C (consumer’s home). 2.4. Microbiological analyses During time and temperature cycling, three slices from three vacuum-packed bags of salmon products were analyzed on the day of inoculation and throughout storage of the inoculated products (two to three times a week). Inoculated salmon was homogenized in peptone water (25.5 g of peptone per liter of water, pH = 7 [Biomérieux, Marcy l’Etoile, France]) using a blender. A 60-min revivification at 20 °C was performed (EN ISO 11290-2) then
3. Results and discussion Naturally occurring L. monocytogenes were not detected in the control samples. The two strains of L. monocytogenes studied showed similar growth, and no significant difference in growth potential was observed between strains in all test conditions (P = 0.3628). The data from the two individual strains was pooled for the statistical analyses below. The interaction of salmon preparation nature and background microflora had an impact on the growth of L. monocytogenes (P = 0.0002) (Fig. 1a–e). The increases of L. monocytogenes were significantly higher in raw, salted and salt-cold-smoked salmon without background microflora than in the same preparations with low initial levels of background microflora. This tendency was reversed for salt–sugar–pepper salmon and salt–sugar–pepper–dill salmon. The results of water and salt contents of salmon preparations were very similar with those observed by Dalgaard and Jorgensen (1998), Niedziela, MacRae, Ogden, and Nesvadba (1998), Leroi, Joffraud, Chevalier, and Cardinal (2001), Cornu et al. (2006) and didn’t explain these difference. We were carried out some antimicrobial resistance tests with dry salt, dry salt–sugar–pepper, dry salt–sugar–pepper–dill (results not shown) and we observed L. monocytogenes inhibition zones around dry salt–sugar–pepper and dry salt–sugar–pepper–dill. The mixture salt–sugar–pepper had an activity anti-L. monocytogenes and the background microflora had an impact on the growth of L. monocytogenes in salt–sugar–pepper salmon and salt–sugar– pepper–dill salmon. In fact, the presence of low initial levels of
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Fig. 1. Increase (log CFU g 1) of L. monocytogenes in function of background microflora (BM) levels, salmon preparation (a) RS: raw salmon, (b) SCSS: salt-cold-smoked salmon, (c) SS: salt salmon, (d) SSDS: salt–sugar–pepper–dill salmon, (e) SSS: salt–sugar–pepper salmon, and temperature storage ( P1: Phase 1: 4 °C for 7 days, P2: Phase 2: 8 °C for 7 days, P3: Phase 3: 20 °C for 2 h, P4: Phase 4: 8 °C for 16 days).
background microflora in these salmon allowed a growth of L. monocytogenes more important in comparison with that obtained without microflora. In salt–sugar–pepper salmon and salt–sugar–pepper–dill salmon, we did not know the composition of the background microflora and the interaction into this microflora, L. monocytogenes and the mixture salt–sugar–pepper. In cold-smoked salmon, Gimenez and Dalgaard (2004) and Leroi, Joffraud, Chevalier, and Cardinal (1998) showed the impact of the background microflora, which has been dominated by lactic acid bacteria, on the growth of L. monocytogenes but no works showed the effect of the microflora of salt–sugar–pepper salmon on the growth of L. monocytogenes. In all salmon preparations with high initial levels of background microflora, no significant growth of L. monocytogenes was observed (the potential growth were inferior to 2 log CFU g 1). The presence of background microflora markedly
affected the growth of L. monocytogenes, as did the ingredients and processing of the salmon products. The presence of high initial levels of background microflora stopped the growth of the inoculated L. monocytogenes strains during challenge tests in all salmon products. In contrast, the presence of low initial levels of background microflora allowed L. monocytogenes to grow in raw, salted and salt-cold-smoked salmon during challenge tests, but at a lower level than in the same products without background microflora. When the majority microflora reached its maximum population, the minority microflora stopped growing and entered the stationary phase at levels lower than they would have reached if they had been inoculated alone or as the majority culture. The presence of background microflora on products can lead to growth cessation of pathogenic bacteria, a phenomenon known as the Jameson effect (Jameson, 1962; Ross et al., 2000). The interaction temperature
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storage and background microflora (P = 0.0000) and the interaction salmon preparation nature and temperature storage (P = 0.0068) had a significantly impact on the growth of L. monocytogenes. Growth potentials of L. monocytogenes were inferior to 1.3 log CFU g 1 in the first phase (4 °C for 7 days) and in the third phase (20 °C for 2 h) of challenge-test by taking into account background microflora levels and salmon preparations. In contrast, in the second phase (8 °C for 7 days) and in the fourth phase (8 °C for 16 days), the growth of potentials were significantly high by taking into account background microflora levels and salmon preparations. For the two phase at 8 °C, the increase was important in raw (4.88 log CFU g 1), salt-cold smoked (4.53 log CFU g 1) and salted (6.42 log CFU g 1) salmon compared to salt–sugar–pepper salmon (2.65 log CFU g 1) and salt–sugar–pepper–dill (2.06 log CFU g 1) salmon without background microflora. In presence of low initial level of background microflora, the growth potentials were between 2.9 log CFU g 1 and 4.92 log CFU g 1 as a function of salmon preparation. The storage at 8 °C allowed the L. monocytogenes population to increase in all salmon preparations with low or whitout background microflora. Jemmi and Keusch (1992) reported that the growth of L. monocytogenes was greater at 8 °C than at 4 °C during storage of about 20 days. We and others have reported that L. monocytogenes grows in naturally and experimentally contaminated products stored at 4 °C to 10 °C, and that growth is slower at lower storage temperatures (Cornu et al., 2006; Cortesi, Sarli, Santoro, Murru, & Pepe, 1997; Hudson & Mott, 1993; Hwang, 2007). Our study showed that it is thus necessary to take into account the temperature storage, background microflora, compounds and spices added to the product when considering bacterial growth. Indeed, products without background microflora (by heat treatment or ionization) can be dangerous for the health of the consumer if a recontamination of the product by L. monoctogenes takes place. We observed that no background microflora in the product allows a fast and important development of L. monocytogenes at the beginning of the product storage. On the other hand, the presence of the background microflora opposes the development of L. monocytogenes. Indeed, a significant amount of background microflora can lead to stop the growth of L. monocytogenes (Jameson effect) but the products are likely to be inedible (very bad sensory properties). The experience carried out on the salmon with low level of background correspond to the products immediately after processing if this one respected the good practices of hygiene during the manufacture. This background microflora has the advantage of limiting the development of L. monocytogenes, without however degrading the sensory quality of the product if the low temperature is respected. In conclusion, these data are important for the manufacturers, the retailers, the consumers and the public health officials because they make it possible to make sure of the reliability of the products taking into consideration risk related to L. monocytogenes. Indeed, in the alert system of food, the knowledge of the development of the population of L. monocytogenes in this product will make it possible the public health officials to determine if the batches suspected can represent a danger to the human health.
Acknowledgement The authors thank Céline Sart, Mylène Gobert, Nicolas Radomski, and Rémi Cappelaere for their technical assistance. References Beaufort, A., Rudelle, S., Gnanou-Besse, N., Toquin, M. T., Kerouanton, A., Bergis, H., et al. (2007). Prevalence and growth of Listeria monocytogenes in naturally contaminated cold-smoked salmon. Letters in Applied Microbiology, 44(4), 406–411. Cornu, M., Beaufort, A., Rudelle, S., Laloux, L., Bergis, H., Miconnet, N., et al. (2006). Effect of temperature, water-phase salt and phenolic contents on Listeria monocytogenes growth rates on cold-smoked salmon and evaluation of secondary models. International Journal of Food Microbiology, 106(2), 159–168. Cortesi, M. L., Sarli, T., Santoro, A., Murru, N., & Pepe, T. (1997). Distribution and behavior of Listeria monocytogenes in three lots of naturally-contaminated vacuum-packed smoked salmon stored at 2 and 10 °C. International Journal of Food Microbiology, 37(2–3), 209–214. Dalgaard, P., & Jorgensen, L. V. (1998). Predicted and observed growth of Listeria monocytogenes in seafood challenge tests and in naturally contaminated coldsmoked salmon. International Journal of Food Microbiology, 40(1–2), 105–115. Gimenez, B., & Dalgaard, P. (2004). Modelling and predicting the simultaneous growth of Listeria monocytogenes and spoilage micro-organisms in cold-smoked salmon. Journal of Applied Microbiology, 96(1), 96–109. Grondin, C., & Guillard, A. S. (2001). Analyse des phénols dans les produits fumés: Utilisation de la microextraction en phase solide (SPME). Ann. Fals. Exp. Chim., 94(954), 63–68. Hudson, J. A., & Mott, S. J. (1993). Growth of Listeria monocytogenes, Aeromonas hydrophila and Yersinia enterocolitica on cold-smoked salmon under refrigeration and mild temperature abuse. Food Microbiology, 10, 61–68. Huss, H. H., Jorgensen, L. V., & Vogel, B. F. (2000). Control options for Listeria monocytogenes in seafoods. International Journal of Food Microbiology, 62(3), 267–274. Hwang, C. A. (2007). Effect of salt, smoke compound, and storage temperature on the growth of Listeria monocytogenes in simulated smoked salmon. Journal of Food Protection, 70(10), 2321–2328. Jameson, J. E. (1962). A discussion of the dynamics of salmonella enrichment. Journal of Hygiene, Cambridge, 60, 193–207. Jemmi, T., & Keusch, A. (1992). Behavior of Listeria monocytogenes during processing and storage of experimentally contaminated hot-smoked trout. International Journal of Food Microbiology, 15(3–4), 339–346. Lappi, V. R., Ho, A., Gall, K., & Wiedmann, M. (2004). Prevalence and growth of Listeria on naturally contaminated smoked salmon over 28 days of storage at 4 °C. Journal of Food Protection, 67(5), 1022–1026. Leroi, F., Joffraud, J. J., Chevalier, F., & Cardinal, M. (1998). Study of the microbial ecology of cold-smoked salmon during storage at 8 °C. International Journal of Food Microbiology, 39(1–2), 111–121. Leroi, F., Joffraud, J. J., Chevalier, F., & Cardinal, M. (2001). Research of quality indices for cold-smoked salmon using a stepwise multiple regression of microbiological counts and physico-chemical parameters. Journal of Applied Microbiology, 90(4), 578–587. Lindqvist, R., & Westoo, A. (2000). Quantitative risk assessment for Listeria monocytogenes in smoked or gravad salmon and rainbow trout in Sweden. International Journal of Food Microbiology, 58(3), 181–196. Loncarevic, S., Tham, W., & Danielsson-Tham, M. L. (1996). Prevalence of Listeria monocytogenes and other Listeria spp. in smoked and ‘gravad’ fish. Acta Veterinaria Scandinavica, 37(1), 13–18. Midelet-Bourdin, G., Leleu, G., & Malle, P. (2007). Evaluation of the international reference methods NF EN ISO 11290-1 and 11290-2 and an in-house method for the isolation of Listeria monocytogenes from retail seafood products in France. Journal of Food Protection, 70(4), 891–900. Niedziela, J.-C., MacRae, M., Ogden, I., & Nesvadba, P. (1998). Control of Listeria monocytogenes in salmon; antimicrobial effect of salting, smoking and specific smoke compounds. Lebensm-Wissu-Technol, 31, 155–161. Ross, T., Dalgaard, P., & Tienungoon, S. (2000). Predictive modelling of the growth and survival of Listeria in fishery products. International Journal of Food Microbiology, 62(3), 231–245.
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Short communication
Determination of Listeria monocytogenes growth potential on new fresh salmon preparations Graziella Midelet-Bourdin *, Stéphanie Copin, Guylaine Leleu, Pierre Malle Agence Française de Sécurité Sanitaire des Aliments, Laboratoire d’Etudes et de Recherches sur les produits de la pêche, Boulogne sur Mer, France
a r t i c l e
i n f o
Article history: Received 24 September 2009 Received in revised form 17 March 2010 Accepted 28 March 2010
Keywords: Listeria monocytogenes Salmon Growth
a b s t r a c t The aim of this work was to evaluate the increase of Listeria monocytogenes on new salmon preparations (salt–sugar–pepper–dill salmon) in comparison with that obtained on cold-smoked salmon. Salmon preparations were inoculated with L. monocytogenes and were analyzed during storage at 4 °C then 8 °C. At 8 °C, the bacteria growth was of 4.53 log CFU g 1 in cold-smoked salmon and of 2.06 log CFU g 1 in salt–sugar–pepper–dill salmon without background microflora. The growth of L. monocytogenes was different in new salmon preparation because the mixture salt–sugar–pepper had an anti-Listeria activity and its presence could inhibitory to the growth. It is difficult to generalize findings observed with coldsmoked salmon to a new salmon preparation. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction New salmon-based products have been available for some years in the fresh food departments of supermarkets and food shops, including raw salmon carpaccio, and raw or smoked salmon marinated in herbs. Salmon marinated in herbs is ready-to-eat product and is regularly consumed in Sweden and in other countries of Northern Europe. Many reports have investigated Listeria monocytogenes in smoked salmon (studies of prevalence, characterization of strains, risk assessment) (Beaufort et al., 2007; Lappi, Ho, Gall, & Wiedmann, 2004), but few studies have been performed with new fresh salmon-based products (Lindqvist & Westoo, 2000; Loncarevic, Tham, & Danielsson-Tham, 1996). Despite prevalence rates ranging from 0% to over 50% in salmon preparations, levels of contamination are generally low (<100 CFU g 1) (Beaufort et al., 2007). However, these levels can exceed 100 CFU g 1 during or at the end of storage (Midelet-Bourdin, Leleu, & Malle, 2007). Bacteria such as L. monocytogenes adapt well to this type of food matrix (Huss, Jorgensen, & Vogel, 2000) and can grow over a wide range of temperature, pH and water activity (Ross, Dalgaard, & Tienungoon, 2000). We studied the increases of L. monocytogenes on new fresh salmon products (salmon with herbs, carpaccio of raw salmon) as a function of the storage temperature (4 °C and 8 °C) and of background microflora and in comparison with that obtained on cold-smoked salmon. As noted in standard NF V01* Corresponding author. Address: AFSSA LERPPê, Boulevard Bassin Napoléon, F-62200 Boulogne sur Mer, France. Tel.: +33 (0) 3 21 99 25 00; fax: +33 (0) 3 21 99 17 25. E-mail address: [email protected] (G. Midelet-Bourdin). 0956-7135/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2010.03.009
009, when data are collected in variable conditions such as temperature, it is not possible to calculate lag times and the growth rates (lmax). These data can, however, be used to determine the growth potential over a given period of time. Growth potentials defined as changes in log CFU g 1 between phases of storage were determined as indicated in the French Food Safety Agency (AFSSA) recommendation (2005) and in standard NF V01-009.
2. Materials and methods 2.1. Bacterial strains and growth conditions As recommended by French Food Safety Agency (Agence Française de Sécurité Sanitaire des Aliments, 2001, 2005), we worked with the reference strain, L. monocytogenes CIP 78.38, serovar 4b– e, of unknown origin from the collection of the Pasteur Institute (Paris, France), and a field strain, L. monocytogenes E 1012 PT1, serovar 1/2a–3a (AFSSA collection, Boulogne sur mer, France), isolated from smoked salmon. Cultures were maintained in brain heart infusion (BHI) broth (AES, Combourg, France) with 18% (vol/vol) glycerol at 80 °C. Before testing, both strains were first subcultured in BHI at 37 °C for 24 h and 100 ll of suspension in 100 ml of BHI were incubated at 8 °C for 10 days (bacteria were in the stationary growth phase at the end of the storage condition). The suspension was then washed twice by centrifugation at 2800 g for 5 min at 20 °C in 30 ml of physiological saline (8.5 g of NaCl per liter of water). The pellet was resuspended in 9 ml of physiological saline and the bacterial concentration was adjusted to 3 108 CFU ml 1 (OD650 nm = 0.15).
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Table 1 Physicochemical parameters for salmon products. RS: raw salmon, SCSS: salt-cold-smoked salmon, SS: salt salmon, SSDS: salt–sugar–pepper–dill salmon, SSS: salt–sugar–pepper salmon. Salmon product
RS SCSS SS SSDS SSS
Physicochemical parameters aw
Salt content (g per 100 g)
Water content (g per 100 g)
Salt content in water phase (WPS) (g per 100 ml)
Phenol content (%)
0.9997 0.9997 0.9997 0.9997
2.80 3.19 3.1 3.65
62.82 62.80 61.8 61.57
4.46 5.08 5.02 5.93
0.94
2.2. Salmon products (raw, salted, salt-cold-smoked, salt–sugar– pepper and salt–sugar–pepper–dill) Products were prepared from Atlantic salmon (Salmo salar) by a company in the north of France. The first product was raw salmon carpaccio: raw sliced salmon fillets. The second product was salted salmon: raw salmon fillets treated with dry salt, and then sliced. The third product was salt-cold-smoked salmon: raw salmon fillets treated with dry salt, smoked at below 30 °C using beech wood, then sliced. The fourth product was salt–sugar–pepper salmon: raw salmon fillets covered with a mixture of dry salt, pepper and sugar, and then sliced. The fifth product was salt–sugar–pepper– dill salmon: raw salmon fillets covered with a mixture of dry salt, pepper, sugar and dill, and then sliced. The physicochemical parameters of these products were presented in Table 1. The phenol content was determinate by the method of (Grondin & Guillard, 2001). Salt contents (in g/100 g) were divided by water contents to obtain water phase salt (WPS) in g/100 ml (Cornu et al., 2006). The water activity, aw, was calculated from WPS by the equation aw = 1 0.0052471WPS 0.00012206WPS2. Salting and smoking times depended on salmon size. The salmon slices were vacuumpackaged in portions of about 150 g.
Sugar content (g per 100 g)
Dill content (g per 100 g)
5.3 5.5
1.8
L. monocytogenes was counted onto plates of Palcam agar (Oxoid, Dardilly, France) after 48 h incubation at 37 °C. Monitoring of background microflora as well as detection and quantification of naturally present L. monocytogenes were respectively performed according to the standards AFNOR XPV08-062 (quantification of background microflora or mesophilic bacteria on plate count agar incubated for 72 h at 30 °C), NF V 08050 and EN ISO 11290 (detection of L. monocytogenes on PALCAM and Oxford media incubated for 48 h at 37 °C). 2.5. Statistical analysis As noted in standard NF V01-009, when data are collected in variable conditions such as temperature, it is not possible to calculate lag times and the growth rates (lmax). These data can, however, be used to determine the growth potential over a given period of time. Growth potentials defined as changes in log CFU g 1 between phases of storage were determined as indicated in the AFSSA recommendation (2005) and in standard NF V01-009. The data were analyzed using analysis of variance. All calculations were performed using Statgraphics Plus 5 software (Sigma plus, Paris, France), and P < 0.05 was accepted as statistically significant.
2.3. Inoculation of salmon products One series of vacuum-packaged salmon was treated with ionizing radiation (beta) at a controlled minimal dose of 10.2 kGy (Ionisos, Orsay, France) to obtain a bacteria-free product. A second series of vacuum-packaged salmon was used in challenge tests on exiting the factory, as concentrations of initial background microflora were low (around 103 CFU g 1). A third series of vacuum-packaged salmon was stored for 15 days at 8 °C to enable background microflora to grow so that initial bacterial concentrations were high (greater than 107 CFU g 1). Fifty microliters of L. monocytogenes suspension were spread on the surface of salmon slices (25 g) to yield final concentrations of 1000 CFU g 1, 100 CFU g 1 and 10 CFU g 1. After inoculation, the salmon slices were again vacuum-packaged and stored as follows: 7 days at 4 °C ± 1 °C (manufacture of products and transport to the point of sale), 7 days at 8 °C ± 1 °C (point of sale), 2 h at 20 °C ± 1 °C (break in cold storage corresponding to transport from point of sale to the consumer’s home), and 16 days at 8 °C ± 1 °C (consumer’s home). 2.4. Microbiological analyses During time and temperature cycling, three slices from three vacuum-packed bags of salmon products were analyzed on the day of inoculation and throughout storage of the inoculated products (two to three times a week). Inoculated salmon was homogenized in peptone water (25.5 g of peptone per liter of water, pH = 7 [Biomérieux, Marcy l’Etoile, France]) using a blender. A 60-min revivification at 20 °C was performed (EN ISO 11290-2) then
3. Results and discussion Naturally occurring L. monocytogenes were not detected in the control samples. The two strains of L. monocytogenes studied showed similar growth, and no significant difference in growth potential was observed between strains in all test conditions (P = 0.3628). The data from the two individual strains was pooled for the statistical analyses below. The interaction of salmon preparation nature and background microflora had an impact on the growth of L. monocytogenes (P = 0.0002) (Fig. 1a–e). The increases of L. monocytogenes were significantly higher in raw, salted and salt-cold-smoked salmon without background microflora than in the same preparations with low initial levels of background microflora. This tendency was reversed for salt–sugar–pepper salmon and salt–sugar–pepper–dill salmon. The results of water and salt contents of salmon preparations were very similar with those observed by Dalgaard and Jorgensen (1998), Niedziela, MacRae, Ogden, and Nesvadba (1998), Leroi, Joffraud, Chevalier, and Cardinal (2001), Cornu et al. (2006) and didn’t explain these difference. We were carried out some antimicrobial resistance tests with dry salt, dry salt–sugar–pepper, dry salt–sugar–pepper–dill (results not shown) and we observed L. monocytogenes inhibition zones around dry salt–sugar–pepper and dry salt–sugar–pepper–dill. The mixture salt–sugar–pepper had an activity anti-L. monocytogenes and the background microflora had an impact on the growth of L. monocytogenes in salt–sugar–pepper salmon and salt–sugar– pepper–dill salmon. In fact, the presence of low initial levels of
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Fig. 1. Increase (log CFU g 1) of L. monocytogenes in function of background microflora (BM) levels, salmon preparation (a) RS: raw salmon, (b) SCSS: salt-cold-smoked salmon, (c) SS: salt salmon, (d) SSDS: salt–sugar–pepper–dill salmon, (e) SSS: salt–sugar–pepper salmon, and temperature storage ( P1: Phase 1: 4 °C for 7 days, P2: Phase 2: 8 °C for 7 days, P3: Phase 3: 20 °C for 2 h, P4: Phase 4: 8 °C for 16 days).
background microflora in these salmon allowed a growth of L. monocytogenes more important in comparison with that obtained without microflora. In salt–sugar–pepper salmon and salt–sugar–pepper–dill salmon, we did not know the composition of the background microflora and the interaction into this microflora, L. monocytogenes and the mixture salt–sugar–pepper. In cold-smoked salmon, Gimenez and Dalgaard (2004) and Leroi, Joffraud, Chevalier, and Cardinal (1998) showed the impact of the background microflora, which has been dominated by lactic acid bacteria, on the growth of L. monocytogenes but no works showed the effect of the microflora of salt–sugar–pepper salmon on the growth of L. monocytogenes. In all salmon preparations with high initial levels of background microflora, no significant growth of L. monocytogenes was observed (the potential growth were inferior to 2 log CFU g 1). The presence of background microflora markedly
affected the growth of L. monocytogenes, as did the ingredients and processing of the salmon products. The presence of high initial levels of background microflora stopped the growth of the inoculated L. monocytogenes strains during challenge tests in all salmon products. In contrast, the presence of low initial levels of background microflora allowed L. monocytogenes to grow in raw, salted and salt-cold-smoked salmon during challenge tests, but at a lower level than in the same products without background microflora. When the majority microflora reached its maximum population, the minority microflora stopped growing and entered the stationary phase at levels lower than they would have reached if they had been inoculated alone or as the majority culture. The presence of background microflora on products can lead to growth cessation of pathogenic bacteria, a phenomenon known as the Jameson effect (Jameson, 1962; Ross et al., 2000). The interaction temperature
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storage and background microflora (P = 0.0000) and the interaction salmon preparation nature and temperature storage (P = 0.0068) had a significantly impact on the growth of L. monocytogenes. Growth potentials of L. monocytogenes were inferior to 1.3 log CFU g 1 in the first phase (4 °C for 7 days) and in the third phase (20 °C for 2 h) of challenge-test by taking into account background microflora levels and salmon preparations. In contrast, in the second phase (8 °C for 7 days) and in the fourth phase (8 °C for 16 days), the growth of potentials were significantly high by taking into account background microflora levels and salmon preparations. For the two phase at 8 °C, the increase was important in raw (4.88 log CFU g 1), salt-cold smoked (4.53 log CFU g 1) and salted (6.42 log CFU g 1) salmon compared to salt–sugar–pepper salmon (2.65 log CFU g 1) and salt–sugar–pepper–dill (2.06 log CFU g 1) salmon without background microflora. In presence of low initial level of background microflora, the growth potentials were between 2.9 log CFU g 1 and 4.92 log CFU g 1 as a function of salmon preparation. The storage at 8 °C allowed the L. monocytogenes population to increase in all salmon preparations with low or whitout background microflora. Jemmi and Keusch (1992) reported that the growth of L. monocytogenes was greater at 8 °C than at 4 °C during storage of about 20 days. We and others have reported that L. monocytogenes grows in naturally and experimentally contaminated products stored at 4 °C to 10 °C, and that growth is slower at lower storage temperatures (Cornu et al., 2006; Cortesi, Sarli, Santoro, Murru, & Pepe, 1997; Hudson & Mott, 1993; Hwang, 2007). Our study showed that it is thus necessary to take into account the temperature storage, background microflora, compounds and spices added to the product when considering bacterial growth. Indeed, products without background microflora (by heat treatment or ionization) can be dangerous for the health of the consumer if a recontamination of the product by L. monoctogenes takes place. We observed that no background microflora in the product allows a fast and important development of L. monocytogenes at the beginning of the product storage. On the other hand, the presence of the background microflora opposes the development of L. monocytogenes. Indeed, a significant amount of background microflora can lead to stop the growth of L. monocytogenes (Jameson effect) but the products are likely to be inedible (very bad sensory properties). The experience carried out on the salmon with low level of background correspond to the products immediately after processing if this one respected the good practices of hygiene during the manufacture. This background microflora has the advantage of limiting the development of L. monocytogenes, without however degrading the sensory quality of the product if the low temperature is respected. In conclusion, these data are important for the manufacturers, the retailers, the consumers and the public health officials because they make it possible to make sure of the reliability of the products taking into consideration risk related to L. monocytogenes. Indeed, in the alert system of food, the knowledge of the development of the population of L. monocytogenes in this product will make it possible the public health officials to determine if the batches suspected can represent a danger to the human health.
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