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Technological characterization of a bacteriocin-producing Lactobacillus sakei and its use in fermented sausages production
International Journal of Food Microbiology 110 (2006) 232 – 239 www.elsevier.com/locate/ijfoodmicro
Technological characterization of a bacteriocin-producing Lactobacillus sakei and its use in fermented sausages production Rosalinda Urso a , Kalliopi Rantsiou a , Carlo Cantoni b , Giuseppe Comi a , Luca Cocolin a,⁎ a
b
Dipartimento di Scienze degli Alimenti, Università degli studi di Udine, via Marangoni 97, 33100, Udine, Italy Dipartimento di Scienze e Tecnologie Veterinarie per la Sicurezza degli Alimenti, Università degli studi di Milano, via Celoria 10, 20121, Milan, Italy Received 1 April 2005; received in revised form 22 December 2005; accepted 3 April 2006
Abstract The aim of this paper was the technological characterization of a Lactobacillus sakei strain, able to produce the bacteriocin sakacin P, that was originally isolated from naturally fermented sausages. Experiments were conducted in situ, using MRS-based medium, and in situ, when the strain was inoculated as starter culture in real sausage fermentation. The results obtained underlined that the strain was able to grow in conditions that are commonly used in the production line, and only lactose and high concentrations of NaCl (5% w/v) reduced the capability for bacteriocin production. When inoculated in sausages, the strain showed a good performance, being able to colonize rapidly the ecosystem. A high number of isolates, capable of producing sakacin P, were already isolated after the third day of fermentation, and persisted throughout the course of the fermentation. The inoculated strain also affected other microbial colonization trends; in fact the total bacterial count and fecal enterococci showed a rapid decrease at the end of the fermentation. Moreover, during sensory evaluation, the final sausage product received high scores for the parameters of tenderness and juiciness, with medium acidity and low rancidity. Lastly, the panelists preferred the sausages produced with the L. sakei characterized in this study when compared to a fermented sausage produced with a commercial starter. © 2006 Elsevier B.V. All rights reserved. Keywords: L. sakei; Bacteriocin; Bioprotection; Starter culture; Sausage fermentation
1. Introduction The safety and preservation of dry sausages is based on hurdle technology. Hurdle technology represents the deliberate combination of existing parameters (temperature, aw, pH, Eh, preservatives, etc.) with novel preservation techniques (gas packaging, bioconservation, bacteriocins, ultrahigh pressure treatment, etc.) in order to establish a series of more selective preservative factors (hurdle) that the spoilage and pathogenic microorganisms should not be able to overcome (Hugas, 1998; Leistner and Gorris, 1995). In the last decade, a new approach to food stabilization, called biopreservation, based on the antagonism displayed by one microorganism towards another, was established, linking lactic acid bacteria (LAB), other protective cultures and bioprotection af⁎ Corresponding author. Tel.: +39 0432 590 759; fax: +39 0432 590 719. E-mail address: [email protected] (L. Cocolin). 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.04.015
forded by natural products. According to Stiles (1996), biopreservation refers to extended storage life and enhanced safety of food using natural or controlled microflora and (or) associated antibacterial products. LAB play an important role in meat fermentations, as they induce flavor and texture changes, together with a preservative effect, resulting in an increase of the shelf life of the product. The fermentation of carbohydrates inherent in the starting material leads to the production of lactic acid, which decreases the pH in fermented sausages. The lower external pH disturbs the homeostasis of the pathogens and spoilage bacteria, and restricts their growth (Leistner, 2000). Moreover, microbial interference by LAB can also arise due to the production of bacteriocins. Adding a pure culture of a viable bacteriocin-producing LAB strain thus represents an example of biopreservation. This indirect way of incorporating bacteriocins in meat products depends on the capability of the added strain to grow and produce the bacteriocin
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during the fermentation process. The production of a certain bacteriocin under laboratory conditions does not confirm its effectiveness during a food production process. When evaluating a bacteriocin-producing culture for sausage fermentation or biopreservation, it is important to consider that meat and meat products are complex systems, therefore, the influence of formula and fermentation technology on the performance of bacteriocin-producing strains needs to be tested (Hugas, 1998). Several studies have been conducted in order to assay the potential of bacteriocin-producing strains to function in meat systems. Most studies, which have been focused on the in situ behavior of bacteriocins against food pathogens, like Listeria monocytogenes, have been conducted using bacteriocinogenic strains of Pediococcus acidilactici or pediocin (Berry et al., 1991; Foegeding et al., 1992; Nielsen et al., 1990; Schillinger et al., 1991; Winkowski et al., 1993), Lactobacillus rhamnous and Lactobacillus plantarum (Tyopponen et al., 2003), Lactobacillus sakei (Liserre et al., 2002) and Lactococcus lactis subsp. lactis. These strains were inoculated singly (Coffey et al., 1998; Benkerroum et al., 2003) or in association with Lactobacillus curvatus (Benkerroum et al., 2005). Moreover the use of a bacteriocinproducing starter culture consisting of Lb. plantarum and Lb. curvatus was recently tested in the production of ostrich meat salami (Dicks et al., 2004). Within the frame of the European project “Safety of traditional fermented sausages: research on protective cultures and bacteriocins”, contract n. ICA4-CT-2002-10037, we isolated a strain of Lb. sakei that possessed antimicrobial activity towards L. monocytogenes. We determined that the bacteriocin produced was sakacin P (Urso et al., 2004). In this paper, the technological characterization of the strain, and its bacteriocin production, was carried out, as well as the evaluation of its ability to predominate a sausage fermentation. The main microbiological parameters, such as total bacterial counts (TBC), LAB counts, coagulase negative cocci (CNC), total enterobacteria and Escherichia coli, fecal enterococci, yeast and mould counts, as well as the presence of the pathogens Staphylococcus aureus, Salmonella spp. and L. monocytogenes, were followed and the sensory profile of the final product was compared with sausages produced using another commercial starter cultures. 2. Materials and methods 2.1. Bacterial strain A sakacin P producer, Lb. sakei, previously isolated from Friuli Venezia Giulia region fermented sausages (Urso et al., 2004), was routinely grown in MRS broth (Oxoid, Milan, Italy) at 30 °C for 24 h. 2.2. Technological characterization of Lb. sakei The growth of Lb. sakei, and its capability of producing bacteriocin was tested in media conditions resembling the fermented sausage production line. Temperatures of 10, 14, 18 and 25 °C, pH values of 6.0, 5.7 and 5.4, NaCl concentrations of 2, 3.5 and 5% (w/v), glucose concentrations of 0.5, 1.0, 1.5% (w/
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v), lactose concentrations of 0.25, 0.5 and 1% (w/v) and sucrose concentrations of 0.5, 1.0 and 1.5% (w/v) were selected as variables. Growth was followed by measuring the optical density (OD) of the cultures at 600 nm with the SmartSpec™ 3000 spectrophotometer (Biorad, Milan, Italy), while quantification of the bacteriocin was performed by the critical dilution method, as suggested by Barefoot and Klaenhammer (1983), using the indicator strain L. monocytognes NCTC 10527. Strain growths at different temperatures, at different pH and containing different concentrations of NaCl were carried out in MRS broth, using the commercial ready-to-use medium (Oxoid). To monitor the growth in the presence of different concentrations of sugars, a sugar-free MRS broth was prepared, since the commercial formula contains 20 g/l glucose. Peptone (10 g/l), meat extract (8 g/l), yeast extract (4 g/l), potassium phosphate monoacid (2 g/l), sodium acetate trihydrate (5 g/l), triammonium citrate (2 g/l), magnesium sulfate heptahydrate (0.2 g/l), manganese sulfate tetrahydrate (0.05 g/l) and Tween 80 (1 ml/l) were mixed and the pH adjusted to 6.2 ± 0.2 with 1 M KOH. Sugars were added at the appropriate concentration and the media filter sterilized through 0.22 μm filter-top bottles (Millipore, Milan, Italy). An active culture (18 h in MRS broth) of Lb. sakei was used as inoculum (1% v/v). The experiments were performed twice and samples were collected in duplicates. 2.3. Fermented sausage technology and sampling procedures Fermented sausages were prepared in a local meat factory using traditional techniques. A 200 kg batch consisting of ground pork meat (60 kg), lard (40 kg), a mix of sodium chloride (2.5 kg) and black pepper (70 g), sugars (1.5 kg), and nitrite/nitrate (200 ppm) was inoculated with a total cell count of Lb. sakei of 1.5 × 1011, resulting in a final concentration in the mix of about 7.5 × 105 cells/g. After mixing, stuffing of natural casings produced fresh sausages which were 25 cm long and 5 cm in diameter. The ripening was performed as follows: the first stage consisted of 2 days drying with the relative humidity (RH) of 85% and a temperature of 22 °C that was then decreased to 12 °C, with a rate of 2 °C per day with a RH between 60 and 90%. The ripening was then carried out for 38 days at 12 °C in storerooms with 65–85% RH. No smoke was applied at any stage. The fermented sausages at 0, 3, 5, 7, 14, 21, 30 and 45 days of processing were subjected to microbiological analysis. Three samples were collected and used for the analyses. 2.4. pH measurement Potentiometric measurements of pH were made using a pin electrode of a pH-meter (Radiometer Copenhagen pH M82, Cecchinato, Italy) inserted directly into the sausage. Three independent measurements were made on each sample. Mean and standard deviations were calculated. 2.5. Microbiological analysis The fermented sausages were subjected to microbiological analysis to monitor the dynamic changes in the populations
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responsible for the ripening, but also to determine their hygienic quality. In particular, 25 g of each sample was transferred into a sterile stomacher bag and 225 ml of saline/peptone water (8 g/ l NaCl, 1 g/l bacteriological peptone, Oxoid) were added and mixed for 1 min and 30 s in a stomacher machine (PBI, Milan, Italy). Further decimal dilutions were made and the following
analyses were carried out on duplicate agar plates: a) TBC on Peptone Agar (8 g/l bacteriological peptone, 15 g/l bacteriological agar, Oxoid) incubated for 48–72 h at 30 °C; b) LAB count on MRS agar (Oxoid) incubated with a double layer at 30 °C for 48 h; c) coagulase negative cocci (CNC) on Mannitol Salt Agar (Oxoid) incubated at 30 °C for 48 h; d) total enterobacteria and
Fig. 1. Growth curves of the bacteriocin-producing Lb. sakei strain at different temperatures. Acidification and the bacteriocin production (arbitrary units/ml) are reported as well. The results reported are the mean of duplicate samples.
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on MRS agar and after an overnight incubation at 30 °C in MRS broth, stored at − 20 °C with 30% (v/v) glycerol. 2.6. Characterization of the isolated strains for the production of bacteriocin
Fig. 2. Critical dilution method for the quantification of the bacteriocin produced by Lb. sakei at 14 and 10 °C. Abbreviations: ND, broth not diluted; 1:2, broth diluted 2 times; 1:4, broth diluted 4 times; 1:8, broth diluted 8 times; 1:16, broth diluted 16 times.
The capability of the isolated strains to produce sakacin P was assessed by both physiological and molecular methods. The agar well diffusion assay (AWDA) using L. monocytognes NCTC 10527 as the indicator strain, was performed as described by Schillinger and Lücke (1987). Moreover, the sppA gene, encoding for the SakP protein, was targeted in the isolated strains using specific PCR primers, sakP_F and sakP_R, as previously described by Remiger et al. (1996). 2.7. Sensory evaluation
E. coli on Coli-ID medium (Bio Merieux, Rome, Italy) incubated with a double layer at 37 °C for 24–48 h; e) fecal enterococci on Kanamycin Aesculin agar (Oxoid) incubated at 42 °C for 24 h; f) S. aureus on Baird Parker medium (Oxoid) with added egg yolk tellurite emulsion (Oxoid) incubated at 37 °C for 24– 48 h; g) yeasts and moulds on Malt Extract Agar (Oxoid) supplemented with tetracycline (1 μg/ml, Sigma, Milan, Italy) incubated at 25 °C for 48–72 h. For L. monocytogenes, the ISO/ DIS method (1990) was used, while for Salmonella spp. the ISO/DIS method (1991) was applied. After counting, mean and standard deviations were calculated. At each sampling point, 30 LAB strains from MRS plates were randomly selected, streaked
A group of 12 people evaluated the sensory characteristics of the sausages studied. It is important to underline that the panelists were not experts and they were represented by students and employees of the Department of Food Science, University of Udine. They were asked to score different parameters of the sausages on the basis of the intensity perceived, as previously described by Comi et al. (2005). Moreover, a duo–trio test was performed to understand if the panelists could differentiate between the product obtained with Lb. sakei and a sausage produced with a commercial starter under the same fermentation conditions.
Fig. 3. Growth curves of the bacteriocin-producing strain in MRS medium with different concentrations of sugars. Arbitrary units are reported as well. The results reported are the mean of duplicate samples.
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3. Results
3.2. Growth at differing pH, NaCl, glucose, lactose and sucrose concentration
3.1. Growth at different temperatures The results of the microbial growth profiles at different temperatures are shown in Fig. 1. As reported in the materials and methods (Section 2.2), growth was monitored by measurement of the OD at 600 nm, and a value of 0.5 was equal to about 108 cells/ ml, as determined by plate counts (data not shown). The best growth and bacteriocin production was observed at 25 °C, were already at 20 h the bacteriocin quantity reached the maximum values measured. The acidification was deep, in fact the strain was able to decrease the pH at values of 4. The experiments conducted at 18, 14 and 10 °C showed the main differences with the previous analyzed temperature. As shown in Fig. 1, with the decrease of temperature, the lag phases of the culture became longer and at 10 °C only a weak growth rate was observed. At 14 and 18 °C the cells reached values higher than 108 cells/ml and the acidification was good. Moreover, also the production of the bacteriocin reached maximum values. Only at 10 °C were the growth and acidification not optimal, although the production of bacteriocin reached levels comparable with the other temperatures tested. It seems that the differences observed in the growth at 10 °C did not influence the bacteriocin production when measured with the critical dilution method. However, the effect of the temperature becomes more obvious when observing the inhibition diameters of the bacteriocin dilutions at 14 and 10 °C (Fig. 2). Based on the results obtained and considering its use in the production line, a temperature of 18 °C was chosen to carry out the other characterization experiments.
The results obtained from the laboratory experiments for the determination of the optimal conditions for the production of bacteriocins are reported in Figs. 3 and 4. The main parameters affecting the growth and the production of bacteriocins were determined to be the concentration of NaCl and lactose. As shown, with the increase of the concentration of salt, from 2 to 3.5% a decrease in the production of the bacteriocin was observed. Lb. sakei was able to grow, but the quantity of bacteriocin produced was very low, as determined by the arbitrary units measured. When the concentration of NaCl increased to 5%, the strain showed only a little growth and no bacteriocin production. Concerning the lactose, in all the conditions tested, it did not support a significant growth of the strain, and also the production of bacteriocin was not satisfactory. No significant differences were observed for the other sugars tested. Different pH values or different concentrations of glucose and sucrose did not affect the growth and the production of the bacteriocin, although sucrose was the sugar that allowed the highest values of OD to be reached. 3.3. Microbial dynamics and pH of the fermented sausages inoculated with Lb. sakei As shown in Fig. 5, LAB populations, potentially represented by the inoculated strain, showed a rapid increase in the counts, and already at three days they reached values between 108–109 colony forming unit (cfu)/g sausage mixture, that remained stable
Fig. 4. Growth curves of the bacteriocin-producing strain in MRS medium with different concentrations of NaCl and diverse pH. Arbitrary units are reported as well. The results reported are the mean of duplicate samples.
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the first 20 days of fermentation. TBC started to decrease after that time, whereas the fecal enterococci remained at high counts until the last day of fermentation where they showed a steep drop from 105 to 102 cfu/g. Yeast and E. coli counts were low, whereas the total enterobacteria decreased gradually during fermentation, displaying at the end of the fermentation values of 102–103 cfu/g. S. aureus was always below 50 cfu/g, as well as mould counts. Salmonella spp. were always absent in 25 g of product and L. monocytogenes isolates visible at zero days, were absent in the following stages of fermentation. The isolated LAB strains were tested for their capability of producing bacteriocins, in order to understand if Lb. sakei populations took over the course of the fermentation. Of the 30 strains isolated at each sampling point, we found a very low number of producing strains (3) at day zero, while for the rest of the fermentation we had at least 20 isolates that were positive. Only at 21 days that 8 strains were found to be producers, a fact that may be explained considering the random isolation of the colonies from the MRS plates. At 30 days we found 26 strains to be positive of the bacteriocin production. The same results were obtained when the strains were subjected to sakacin P amplification using the specific primers described by Remiger et al. (1996). The pH showed the classical trend typical of the sausage fermentation: within the first 7 days of fermentation a deep acidification was observed. The values from 5.9 decreased to 5.3. After that time, the pH started to increase reaching a final value of about 6. 3.4. Sensory evaluation In Fig. 6 the sensory profile of the sausages is presented. As shown, the panel found the sausages with a medium acidity and smell characteristics, very low in rancidity with very good juiciness and tenderness. The only objections were the badly scared surface and overall coherence. The samples were ranked as medium quality overall. Out of a panel of 12 persons, 8 recognized the product when compared with a reference and 9 preferred the sausage produced with the Lb. sakei strain rather than the one produced with a commercial starter. These results
Fig. 5. Trends of the populations monitored during the fermentation of the sausages inoculated with Lb. sakei. Abbreviations: LAB, lactic acid bacteria; CNC, coagulase negative cocci, TBC, total bacterial count.
throughout the fermentation. Sausages were characterized by a low impact of the CNC, which remained below 106 cfu/g, while an increase of the TBC and fecal enterococci was observed within
Fig. 6. Sensory evaluation of the sausages produced by using the Lb. sakei as a starter culture.
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underline how the use of Lb. sakei resulted in a product with specific characteristics that not only made it distinguishable when compared with others, but also possessed a better organoleptic profile. 4. Discussion In meat fermentations, the use of starter cultures is a commonly used procedure. It assures the safety of the product and stability of the aromatic and sensory profile of the sausages produced by a specific industry. Despite the advantages introduced by the use of commercial starters, meat processors often are not completely satisfied with the starter-mediated fermentations. Often times the products are characterized by high acidity with a poor aroma and taste. This is attributed to the lack of suitable strains able to produce moderate acidulation and at the same time more complex aromatic profile. It is essential to point out that, commercial starters and their optimal conditions for use are recommended on the basis of fermentation trials conducted mainly in the Northern Europe. For these reasons, their performance could be different if applied to different products, such as low-acid fermented sausages typical of the Mediterranean area. Many processors choose the alternative of natural fermentations, driven by indigenous strains, with the goal of obtaining products with milder taste and richer aroma. The aim of this paper was to characterize a “wild” strain of Lb. sakei, isolated from naturally fermented sausages, that exhibits antimicrobial activity towards L. monocytogenes. L. monocytogenes is a Gram-positive non-sporulating foodborne pathogen, that in spite of the various natural hurdles inherent in dry sausage manufacturing, is able to survive during this process (Gahan et al., 1996; Varabioff, 1992). The risk of L. monocytogenes would be further reduced by utilizing specific bacteriocin-producing cultures with desirable technological characteristics, such as Lb. sakei. The trials carried out in this study were designed firstly to understand if the strain was able to produce the bacteriocin under conditions resembling the fermentation process, and secondly if it could be used as a starter culture during the production of fermented sausages. Using MRS broth based media Lb. sakei showed a different growth behavior when different temperatures were tested. Between 25 and 10 °C a progressive extension of the lag phase was observed, and at 10°C the growth was weak. Acidification followed the trend of the cell numbers. While at 25, 18 and 14 °C, the pH values reached at the end of the period were close to 4, at 10 °C it did not go below 5. Despite variations in growth behavior, the different temperatures did not affect the capability of the strain to produce the bacteriocin or its quantity. As shown in Fig. 1, at all the temperatures tested the bacteriocin or its quantity reached 360 arbitrary units (AU)/ml, using the critical dilution method. However, in our opinion, this result should be considered carefully, since the inhibition areas, as determined by the agar diffusion assay, decreased significantly from 14 to 10 °C (Fig. 2). A more careful quantification of the produced bacteriocin would be a simple measurement of the diameters of the inhibition of the undiluted broth. To carry out the additional tests for the technological characterization, the temperature of 18 °C was selected, since it is the principal one used during fermentation process. In our in vitro
media based system the best growth was obtained using sucrose in the MRS medium, where the OD values were highest. Glucose was another sugar which enabled Lb. sakei to grow well. Bacteriocin production was satisfactory on sucrose plus glucose where the maximum values of 360 AU/ml were reached after only 12 h. In contrast to this, when lactose was added to the medium, the OD values were always below 1, and bacteriocin production decreased by 50%. No differences were observed for the different values of pH, while a strong impact was determined when using different concentrations of salt. Already at 3.5% NaCl, a drastic decrease in the capability to produce sakacin P was detected, and by 5% both growth and bacteriocin production were severely compromised. These results are in agreement with Verluyten et al. (2004), who reported a decrease in the production of curvacin A from Lb. curvatus LHT1174 when augmenting the growth medium with NaCl. However, Gänzle et al. (1997) reported an increase in the activity of the sakacin P at low pH and NaCl up to 3%, but this evidence could be explained considering that the authors tested the activity of the purified bacteriocin. In the second phase of our study, Lb. sakei was used as a starter culture in real sausage fermentation. It was able to establish itself during the course of the fermentation process and this is supported by both the LAB counts reached (109 cfu/ml) and by the evidence that at each sampling point, for a large number of isolates (>70%, data not shown), it was possible to target the sakacin P gene by specific PCR. The influence of the starter on the microbial ecology of the fermented sausages, as determined by traditional plating, was shown to significantly decrease the counts of TBC and fecal enterococci after day 21 of the fermentation. This trend was in contrast with previously published results on the same type of fermented sausages, produced by the same production facility, but without the addition of starter cultures (Comi et al., 2005). In this case, both TBC and fecal enterococci remained stable until the end of the fermentation, with numbers up to 108–109 cfu/ml and 105 cfu/ml, respectively. The lower counts we observed could be a direct consequence of the Lb. sakei inoculation. Moreover, L. monocytogenes isolates detected at zero days were eradicated. The production of bacteriocin may play a role in the inactivation of L. monocytogenes present in the unfermented sausages. The strain of Lb. sakei tested as starter culture was characterized by a good performance during the fermentation. The final product obtained, when subjected to sensory analysis, was described with high scores on tenderness, juiciness and smell. It possessed medium acidity and it showed a very low rancidity. Low scores were given to the scared surface and coherence, but this fact should be considered to be more related with the ingredients used for production, such as fat quality, rather than with the performance of the strain inoculated. It is interesting to note that the majority of the panelists were able to recognize the product when compared with a sausage produced using a commercial starter, thereby demonstrating the capability of the Lb. sakei to characterize, by the sensory point of view, the final product. Moreover the sausage produced with the starter developed here was preferred to the one obtained with the commercial starter. Selecting strains indigenous to specific traditional products, studying their technological characteristics and eventually
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developing them as starter cultures to be used for the particular products offers an alternative to the implementation of commercialized starters that not do always give satisfactory results. The use of product-specific starter cultures is in line with the efforts of food processors to preserve the organoleptic and sensory characteristics of traditional products. Acknowledgements This research has been funded by the E. C. within the framework of the specific research and technological development program “Confirming the International Role of the Community Research”, (Contract n. ICA4-CT-2002-10037). Authors express their gratitude to the technical staff of the industry that provided the fermented sausages studied. References Barefoot, S.F., Klaenhammer, T.R., 1983. Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus. Applied and Environmental Microbiology 45, 1808–1815. Benkerroum, N., Daoudi, A., Kamal, M., 2003. Behaviour of Listeria monocytogenes in raw sausages (merguez) in presence of bacteriocinproducing lactococcal strain as a protective culture. Meat Science 63, 479–484. Benkerroum, N., Daoundi, A., Hamraoui, T., Ghalfi, H., Thiry, C., Duroy, M., Evrart, P., Roblain, D., Thonart, P., 2005. Lyophilized preparations of bacteriocinogenic Lactobacillus curvatus and Lactococcus lactis subsp. lactis as potential protective adjuncts to control Listeria monocytogenes in dry-fermented sausages. Journal of Applied Microbiology 98, 56–63. Berry, E.D., Hutkins, R.W., Mandigo, R.W., 1991. The use of bacteriocinproducing Pediococcus acidilactici to control post-processing Listeria monocytogenes contamination of frankfurters. Journal of Food Protection 54, 681–686. Coffey, A., Ryan, M., Ross, R.P., Hill, C., Arendt, E., Schwarz, G., 1998. Use of a broad-host-range bacteriocin-producing Lactococcus lactis transconjugant as an alternative starter for salami manufacture. International Journal of Food Microbiology 43, 231–235. Comi, G., Urso, R., Iacumin, L., Rantsiou, K., Cattaneo, P., Cantoni, C., Cocolin, L., 2005. Characterization of naturally fermented sausages produced in the North East of Italy. Meat Science 69, 381–392. Dicks, L.M.T., Mellett, F.D., Hoffman, L.C., 2004. Use of bacteriocinproducing starter cultures of Lactobacillus plantarum and Lactobacillus curvatus in production of ostrich meat salami. Meat Science 66, 703–708. Foegeding, P.M., Thomas, A.B., Pilkington, D.H., Klaenhammer, T.R., 1992. Enhanced control of Listeria monocytogenes by in situ produced pediocin during dry fermented sausage production. Applied and Environmental Microbiology 58, 884–890. Gahan, C., O'Briscoll, B., Hill, C., 1996. Acid adaptation of Listeria monocytogenes enhance survival in acidic foods and during milk fermentation. Applied and Environmental Microbiology 62, 3128–3132.
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Technological characterization of a bacteriocin-producing Lactobacillus sakei and its use in fermented sausages production Rosalinda Urso a , Kalliopi Rantsiou a , Carlo Cantoni b , Giuseppe Comi a , Luca Cocolin a,⁎ a
b
Dipartimento di Scienze degli Alimenti, Università degli studi di Udine, via Marangoni 97, 33100, Udine, Italy Dipartimento di Scienze e Tecnologie Veterinarie per la Sicurezza degli Alimenti, Università degli studi di Milano, via Celoria 10, 20121, Milan, Italy Received 1 April 2005; received in revised form 22 December 2005; accepted 3 April 2006
Abstract The aim of this paper was the technological characterization of a Lactobacillus sakei strain, able to produce the bacteriocin sakacin P, that was originally isolated from naturally fermented sausages. Experiments were conducted in situ, using MRS-based medium, and in situ, when the strain was inoculated as starter culture in real sausage fermentation. The results obtained underlined that the strain was able to grow in conditions that are commonly used in the production line, and only lactose and high concentrations of NaCl (5% w/v) reduced the capability for bacteriocin production. When inoculated in sausages, the strain showed a good performance, being able to colonize rapidly the ecosystem. A high number of isolates, capable of producing sakacin P, were already isolated after the third day of fermentation, and persisted throughout the course of the fermentation. The inoculated strain also affected other microbial colonization trends; in fact the total bacterial count and fecal enterococci showed a rapid decrease at the end of the fermentation. Moreover, during sensory evaluation, the final sausage product received high scores for the parameters of tenderness and juiciness, with medium acidity and low rancidity. Lastly, the panelists preferred the sausages produced with the L. sakei characterized in this study when compared to a fermented sausage produced with a commercial starter. © 2006 Elsevier B.V. All rights reserved. Keywords: L. sakei; Bacteriocin; Bioprotection; Starter culture; Sausage fermentation
1. Introduction The safety and preservation of dry sausages is based on hurdle technology. Hurdle technology represents the deliberate combination of existing parameters (temperature, aw, pH, Eh, preservatives, etc.) with novel preservation techniques (gas packaging, bioconservation, bacteriocins, ultrahigh pressure treatment, etc.) in order to establish a series of more selective preservative factors (hurdle) that the spoilage and pathogenic microorganisms should not be able to overcome (Hugas, 1998; Leistner and Gorris, 1995). In the last decade, a new approach to food stabilization, called biopreservation, based on the antagonism displayed by one microorganism towards another, was established, linking lactic acid bacteria (LAB), other protective cultures and bioprotection af⁎ Corresponding author. Tel.: +39 0432 590 759; fax: +39 0432 590 719. E-mail address: [email protected] (L. Cocolin). 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.04.015
forded by natural products. According to Stiles (1996), biopreservation refers to extended storage life and enhanced safety of food using natural or controlled microflora and (or) associated antibacterial products. LAB play an important role in meat fermentations, as they induce flavor and texture changes, together with a preservative effect, resulting in an increase of the shelf life of the product. The fermentation of carbohydrates inherent in the starting material leads to the production of lactic acid, which decreases the pH in fermented sausages. The lower external pH disturbs the homeostasis of the pathogens and spoilage bacteria, and restricts their growth (Leistner, 2000). Moreover, microbial interference by LAB can also arise due to the production of bacteriocins. Adding a pure culture of a viable bacteriocin-producing LAB strain thus represents an example of biopreservation. This indirect way of incorporating bacteriocins in meat products depends on the capability of the added strain to grow and produce the bacteriocin
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during the fermentation process. The production of a certain bacteriocin under laboratory conditions does not confirm its effectiveness during a food production process. When evaluating a bacteriocin-producing culture for sausage fermentation or biopreservation, it is important to consider that meat and meat products are complex systems, therefore, the influence of formula and fermentation technology on the performance of bacteriocin-producing strains needs to be tested (Hugas, 1998). Several studies have been conducted in order to assay the potential of bacteriocin-producing strains to function in meat systems. Most studies, which have been focused on the in situ behavior of bacteriocins against food pathogens, like Listeria monocytogenes, have been conducted using bacteriocinogenic strains of Pediococcus acidilactici or pediocin (Berry et al., 1991; Foegeding et al., 1992; Nielsen et al., 1990; Schillinger et al., 1991; Winkowski et al., 1993), Lactobacillus rhamnous and Lactobacillus plantarum (Tyopponen et al., 2003), Lactobacillus sakei (Liserre et al., 2002) and Lactococcus lactis subsp. lactis. These strains were inoculated singly (Coffey et al., 1998; Benkerroum et al., 2003) or in association with Lactobacillus curvatus (Benkerroum et al., 2005). Moreover the use of a bacteriocinproducing starter culture consisting of Lb. plantarum and Lb. curvatus was recently tested in the production of ostrich meat salami (Dicks et al., 2004). Within the frame of the European project “Safety of traditional fermented sausages: research on protective cultures and bacteriocins”, contract n. ICA4-CT-2002-10037, we isolated a strain of Lb. sakei that possessed antimicrobial activity towards L. monocytogenes. We determined that the bacteriocin produced was sakacin P (Urso et al., 2004). In this paper, the technological characterization of the strain, and its bacteriocin production, was carried out, as well as the evaluation of its ability to predominate a sausage fermentation. The main microbiological parameters, such as total bacterial counts (TBC), LAB counts, coagulase negative cocci (CNC), total enterobacteria and Escherichia coli, fecal enterococci, yeast and mould counts, as well as the presence of the pathogens Staphylococcus aureus, Salmonella spp. and L. monocytogenes, were followed and the sensory profile of the final product was compared with sausages produced using another commercial starter cultures. 2. Materials and methods 2.1. Bacterial strain A sakacin P producer, Lb. sakei, previously isolated from Friuli Venezia Giulia region fermented sausages (Urso et al., 2004), was routinely grown in MRS broth (Oxoid, Milan, Italy) at 30 °C for 24 h. 2.2. Technological characterization of Lb. sakei The growth of Lb. sakei, and its capability of producing bacteriocin was tested in media conditions resembling the fermented sausage production line. Temperatures of 10, 14, 18 and 25 °C, pH values of 6.0, 5.7 and 5.4, NaCl concentrations of 2, 3.5 and 5% (w/v), glucose concentrations of 0.5, 1.0, 1.5% (w/
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v), lactose concentrations of 0.25, 0.5 and 1% (w/v) and sucrose concentrations of 0.5, 1.0 and 1.5% (w/v) were selected as variables. Growth was followed by measuring the optical density (OD) of the cultures at 600 nm with the SmartSpec™ 3000 spectrophotometer (Biorad, Milan, Italy), while quantification of the bacteriocin was performed by the critical dilution method, as suggested by Barefoot and Klaenhammer (1983), using the indicator strain L. monocytognes NCTC 10527. Strain growths at different temperatures, at different pH and containing different concentrations of NaCl were carried out in MRS broth, using the commercial ready-to-use medium (Oxoid). To monitor the growth in the presence of different concentrations of sugars, a sugar-free MRS broth was prepared, since the commercial formula contains 20 g/l glucose. Peptone (10 g/l), meat extract (8 g/l), yeast extract (4 g/l), potassium phosphate monoacid (2 g/l), sodium acetate trihydrate (5 g/l), triammonium citrate (2 g/l), magnesium sulfate heptahydrate (0.2 g/l), manganese sulfate tetrahydrate (0.05 g/l) and Tween 80 (1 ml/l) were mixed and the pH adjusted to 6.2 ± 0.2 with 1 M KOH. Sugars were added at the appropriate concentration and the media filter sterilized through 0.22 μm filter-top bottles (Millipore, Milan, Italy). An active culture (18 h in MRS broth) of Lb. sakei was used as inoculum (1% v/v). The experiments were performed twice and samples were collected in duplicates. 2.3. Fermented sausage technology and sampling procedures Fermented sausages were prepared in a local meat factory using traditional techniques. A 200 kg batch consisting of ground pork meat (60 kg), lard (40 kg), a mix of sodium chloride (2.5 kg) and black pepper (70 g), sugars (1.5 kg), and nitrite/nitrate (200 ppm) was inoculated with a total cell count of Lb. sakei of 1.5 × 1011, resulting in a final concentration in the mix of about 7.5 × 105 cells/g. After mixing, stuffing of natural casings produced fresh sausages which were 25 cm long and 5 cm in diameter. The ripening was performed as follows: the first stage consisted of 2 days drying with the relative humidity (RH) of 85% and a temperature of 22 °C that was then decreased to 12 °C, with a rate of 2 °C per day with a RH between 60 and 90%. The ripening was then carried out for 38 days at 12 °C in storerooms with 65–85% RH. No smoke was applied at any stage. The fermented sausages at 0, 3, 5, 7, 14, 21, 30 and 45 days of processing were subjected to microbiological analysis. Three samples were collected and used for the analyses. 2.4. pH measurement Potentiometric measurements of pH were made using a pin electrode of a pH-meter (Radiometer Copenhagen pH M82, Cecchinato, Italy) inserted directly into the sausage. Three independent measurements were made on each sample. Mean and standard deviations were calculated. 2.5. Microbiological analysis The fermented sausages were subjected to microbiological analysis to monitor the dynamic changes in the populations
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responsible for the ripening, but also to determine their hygienic quality. In particular, 25 g of each sample was transferred into a sterile stomacher bag and 225 ml of saline/peptone water (8 g/ l NaCl, 1 g/l bacteriological peptone, Oxoid) were added and mixed for 1 min and 30 s in a stomacher machine (PBI, Milan, Italy). Further decimal dilutions were made and the following
analyses were carried out on duplicate agar plates: a) TBC on Peptone Agar (8 g/l bacteriological peptone, 15 g/l bacteriological agar, Oxoid) incubated for 48–72 h at 30 °C; b) LAB count on MRS agar (Oxoid) incubated with a double layer at 30 °C for 48 h; c) coagulase negative cocci (CNC) on Mannitol Salt Agar (Oxoid) incubated at 30 °C for 48 h; d) total enterobacteria and
Fig. 1. Growth curves of the bacteriocin-producing Lb. sakei strain at different temperatures. Acidification and the bacteriocin production (arbitrary units/ml) are reported as well. The results reported are the mean of duplicate samples.
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on MRS agar and after an overnight incubation at 30 °C in MRS broth, stored at − 20 °C with 30% (v/v) glycerol. 2.6. Characterization of the isolated strains for the production of bacteriocin
Fig. 2. Critical dilution method for the quantification of the bacteriocin produced by Lb. sakei at 14 and 10 °C. Abbreviations: ND, broth not diluted; 1:2, broth diluted 2 times; 1:4, broth diluted 4 times; 1:8, broth diluted 8 times; 1:16, broth diluted 16 times.
The capability of the isolated strains to produce sakacin P was assessed by both physiological and molecular methods. The agar well diffusion assay (AWDA) using L. monocytognes NCTC 10527 as the indicator strain, was performed as described by Schillinger and Lücke (1987). Moreover, the sppA gene, encoding for the SakP protein, was targeted in the isolated strains using specific PCR primers, sakP_F and sakP_R, as previously described by Remiger et al. (1996). 2.7. Sensory evaluation
E. coli on Coli-ID medium (Bio Merieux, Rome, Italy) incubated with a double layer at 37 °C for 24–48 h; e) fecal enterococci on Kanamycin Aesculin agar (Oxoid) incubated at 42 °C for 24 h; f) S. aureus on Baird Parker medium (Oxoid) with added egg yolk tellurite emulsion (Oxoid) incubated at 37 °C for 24– 48 h; g) yeasts and moulds on Malt Extract Agar (Oxoid) supplemented with tetracycline (1 μg/ml, Sigma, Milan, Italy) incubated at 25 °C for 48–72 h. For L. monocytogenes, the ISO/ DIS method (1990) was used, while for Salmonella spp. the ISO/DIS method (1991) was applied. After counting, mean and standard deviations were calculated. At each sampling point, 30 LAB strains from MRS plates were randomly selected, streaked
A group of 12 people evaluated the sensory characteristics of the sausages studied. It is important to underline that the panelists were not experts and they were represented by students and employees of the Department of Food Science, University of Udine. They were asked to score different parameters of the sausages on the basis of the intensity perceived, as previously described by Comi et al. (2005). Moreover, a duo–trio test was performed to understand if the panelists could differentiate between the product obtained with Lb. sakei and a sausage produced with a commercial starter under the same fermentation conditions.
Fig. 3. Growth curves of the bacteriocin-producing strain in MRS medium with different concentrations of sugars. Arbitrary units are reported as well. The results reported are the mean of duplicate samples.
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3. Results
3.2. Growth at differing pH, NaCl, glucose, lactose and sucrose concentration
3.1. Growth at different temperatures The results of the microbial growth profiles at different temperatures are shown in Fig. 1. As reported in the materials and methods (Section 2.2), growth was monitored by measurement of the OD at 600 nm, and a value of 0.5 was equal to about 108 cells/ ml, as determined by plate counts (data not shown). The best growth and bacteriocin production was observed at 25 °C, were already at 20 h the bacteriocin quantity reached the maximum values measured. The acidification was deep, in fact the strain was able to decrease the pH at values of 4. The experiments conducted at 18, 14 and 10 °C showed the main differences with the previous analyzed temperature. As shown in Fig. 1, with the decrease of temperature, the lag phases of the culture became longer and at 10 °C only a weak growth rate was observed. At 14 and 18 °C the cells reached values higher than 108 cells/ml and the acidification was good. Moreover, also the production of the bacteriocin reached maximum values. Only at 10 °C were the growth and acidification not optimal, although the production of bacteriocin reached levels comparable with the other temperatures tested. It seems that the differences observed in the growth at 10 °C did not influence the bacteriocin production when measured with the critical dilution method. However, the effect of the temperature becomes more obvious when observing the inhibition diameters of the bacteriocin dilutions at 14 and 10 °C (Fig. 2). Based on the results obtained and considering its use in the production line, a temperature of 18 °C was chosen to carry out the other characterization experiments.
The results obtained from the laboratory experiments for the determination of the optimal conditions for the production of bacteriocins are reported in Figs. 3 and 4. The main parameters affecting the growth and the production of bacteriocins were determined to be the concentration of NaCl and lactose. As shown, with the increase of the concentration of salt, from 2 to 3.5% a decrease in the production of the bacteriocin was observed. Lb. sakei was able to grow, but the quantity of bacteriocin produced was very low, as determined by the arbitrary units measured. When the concentration of NaCl increased to 5%, the strain showed only a little growth and no bacteriocin production. Concerning the lactose, in all the conditions tested, it did not support a significant growth of the strain, and also the production of bacteriocin was not satisfactory. No significant differences were observed for the other sugars tested. Different pH values or different concentrations of glucose and sucrose did not affect the growth and the production of the bacteriocin, although sucrose was the sugar that allowed the highest values of OD to be reached. 3.3. Microbial dynamics and pH of the fermented sausages inoculated with Lb. sakei As shown in Fig. 5, LAB populations, potentially represented by the inoculated strain, showed a rapid increase in the counts, and already at three days they reached values between 108–109 colony forming unit (cfu)/g sausage mixture, that remained stable
Fig. 4. Growth curves of the bacteriocin-producing strain in MRS medium with different concentrations of NaCl and diverse pH. Arbitrary units are reported as well. The results reported are the mean of duplicate samples.
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the first 20 days of fermentation. TBC started to decrease after that time, whereas the fecal enterococci remained at high counts until the last day of fermentation where they showed a steep drop from 105 to 102 cfu/g. Yeast and E. coli counts were low, whereas the total enterobacteria decreased gradually during fermentation, displaying at the end of the fermentation values of 102–103 cfu/g. S. aureus was always below 50 cfu/g, as well as mould counts. Salmonella spp. were always absent in 25 g of product and L. monocytogenes isolates visible at zero days, were absent in the following stages of fermentation. The isolated LAB strains were tested for their capability of producing bacteriocins, in order to understand if Lb. sakei populations took over the course of the fermentation. Of the 30 strains isolated at each sampling point, we found a very low number of producing strains (3) at day zero, while for the rest of the fermentation we had at least 20 isolates that were positive. Only at 21 days that 8 strains were found to be producers, a fact that may be explained considering the random isolation of the colonies from the MRS plates. At 30 days we found 26 strains to be positive of the bacteriocin production. The same results were obtained when the strains were subjected to sakacin P amplification using the specific primers described by Remiger et al. (1996). The pH showed the classical trend typical of the sausage fermentation: within the first 7 days of fermentation a deep acidification was observed. The values from 5.9 decreased to 5.3. After that time, the pH started to increase reaching a final value of about 6. 3.4. Sensory evaluation In Fig. 6 the sensory profile of the sausages is presented. As shown, the panel found the sausages with a medium acidity and smell characteristics, very low in rancidity with very good juiciness and tenderness. The only objections were the badly scared surface and overall coherence. The samples were ranked as medium quality overall. Out of a panel of 12 persons, 8 recognized the product when compared with a reference and 9 preferred the sausage produced with the Lb. sakei strain rather than the one produced with a commercial starter. These results
Fig. 5. Trends of the populations monitored during the fermentation of the sausages inoculated with Lb. sakei. Abbreviations: LAB, lactic acid bacteria; CNC, coagulase negative cocci, TBC, total bacterial count.
throughout the fermentation. Sausages were characterized by a low impact of the CNC, which remained below 106 cfu/g, while an increase of the TBC and fecal enterococci was observed within
Fig. 6. Sensory evaluation of the sausages produced by using the Lb. sakei as a starter culture.
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underline how the use of Lb. sakei resulted in a product with specific characteristics that not only made it distinguishable when compared with others, but also possessed a better organoleptic profile. 4. Discussion In meat fermentations, the use of starter cultures is a commonly used procedure. It assures the safety of the product and stability of the aromatic and sensory profile of the sausages produced by a specific industry. Despite the advantages introduced by the use of commercial starters, meat processors often are not completely satisfied with the starter-mediated fermentations. Often times the products are characterized by high acidity with a poor aroma and taste. This is attributed to the lack of suitable strains able to produce moderate acidulation and at the same time more complex aromatic profile. It is essential to point out that, commercial starters and their optimal conditions for use are recommended on the basis of fermentation trials conducted mainly in the Northern Europe. For these reasons, their performance could be different if applied to different products, such as low-acid fermented sausages typical of the Mediterranean area. Many processors choose the alternative of natural fermentations, driven by indigenous strains, with the goal of obtaining products with milder taste and richer aroma. The aim of this paper was to characterize a “wild” strain of Lb. sakei, isolated from naturally fermented sausages, that exhibits antimicrobial activity towards L. monocytogenes. L. monocytogenes is a Gram-positive non-sporulating foodborne pathogen, that in spite of the various natural hurdles inherent in dry sausage manufacturing, is able to survive during this process (Gahan et al., 1996; Varabioff, 1992). The risk of L. monocytogenes would be further reduced by utilizing specific bacteriocin-producing cultures with desirable technological characteristics, such as Lb. sakei. The trials carried out in this study were designed firstly to understand if the strain was able to produce the bacteriocin under conditions resembling the fermentation process, and secondly if it could be used as a starter culture during the production of fermented sausages. Using MRS broth based media Lb. sakei showed a different growth behavior when different temperatures were tested. Between 25 and 10 °C a progressive extension of the lag phase was observed, and at 10°C the growth was weak. Acidification followed the trend of the cell numbers. While at 25, 18 and 14 °C, the pH values reached at the end of the period were close to 4, at 10 °C it did not go below 5. Despite variations in growth behavior, the different temperatures did not affect the capability of the strain to produce the bacteriocin or its quantity. As shown in Fig. 1, at all the temperatures tested the bacteriocin or its quantity reached 360 arbitrary units (AU)/ml, using the critical dilution method. However, in our opinion, this result should be considered carefully, since the inhibition areas, as determined by the agar diffusion assay, decreased significantly from 14 to 10 °C (Fig. 2). A more careful quantification of the produced bacteriocin would be a simple measurement of the diameters of the inhibition of the undiluted broth. To carry out the additional tests for the technological characterization, the temperature of 18 °C was selected, since it is the principal one used during fermentation process. In our in vitro
media based system the best growth was obtained using sucrose in the MRS medium, where the OD values were highest. Glucose was another sugar which enabled Lb. sakei to grow well. Bacteriocin production was satisfactory on sucrose plus glucose where the maximum values of 360 AU/ml were reached after only 12 h. In contrast to this, when lactose was added to the medium, the OD values were always below 1, and bacteriocin production decreased by 50%. No differences were observed for the different values of pH, while a strong impact was determined when using different concentrations of salt. Already at 3.5% NaCl, a drastic decrease in the capability to produce sakacin P was detected, and by 5% both growth and bacteriocin production were severely compromised. These results are in agreement with Verluyten et al. (2004), who reported a decrease in the production of curvacin A from Lb. curvatus LHT1174 when augmenting the growth medium with NaCl. However, Gänzle et al. (1997) reported an increase in the activity of the sakacin P at low pH and NaCl up to 3%, but this evidence could be explained considering that the authors tested the activity of the purified bacteriocin. In the second phase of our study, Lb. sakei was used as a starter culture in real sausage fermentation. It was able to establish itself during the course of the fermentation process and this is supported by both the LAB counts reached (109 cfu/ml) and by the evidence that at each sampling point, for a large number of isolates (>70%, data not shown), it was possible to target the sakacin P gene by specific PCR. The influence of the starter on the microbial ecology of the fermented sausages, as determined by traditional plating, was shown to significantly decrease the counts of TBC and fecal enterococci after day 21 of the fermentation. This trend was in contrast with previously published results on the same type of fermented sausages, produced by the same production facility, but without the addition of starter cultures (Comi et al., 2005). In this case, both TBC and fecal enterococci remained stable until the end of the fermentation, with numbers up to 108–109 cfu/ml and 105 cfu/ml, respectively. The lower counts we observed could be a direct consequence of the Lb. sakei inoculation. Moreover, L. monocytogenes isolates detected at zero days were eradicated. The production of bacteriocin may play a role in the inactivation of L. monocytogenes present in the unfermented sausages. The strain of Lb. sakei tested as starter culture was characterized by a good performance during the fermentation. The final product obtained, when subjected to sensory analysis, was described with high scores on tenderness, juiciness and smell. It possessed medium acidity and it showed a very low rancidity. Low scores were given to the scared surface and coherence, but this fact should be considered to be more related with the ingredients used for production, such as fat quality, rather than with the performance of the strain inoculated. It is interesting to note that the majority of the panelists were able to recognize the product when compared with a sausage produced using a commercial starter, thereby demonstrating the capability of the Lb. sakei to characterize, by the sensory point of view, the final product. Moreover the sausage produced with the starter developed here was preferred to the one obtained with the commercial starter. Selecting strains indigenous to specific traditional products, studying their technological characteristics and eventually
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developing them as starter cultures to be used for the particular products offers an alternative to the implementation of commercialized starters that not do always give satisfactory results. The use of product-specific starter cultures is in line with the efforts of food processors to preserve the organoleptic and sensory characteristics of traditional products. Acknowledgements This research has been funded by the E. C. within the framework of the specific research and technological development program “Confirming the International Role of the Community Research”, (Contract n. ICA4-CT-2002-10037). Authors express their gratitude to the technical staff of the industry that provided the fermented sausages studied. References Barefoot, S.F., Klaenhammer, T.R., 1983. Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus. Applied and Environmental Microbiology 45, 1808–1815. Benkerroum, N., Daoudi, A., Kamal, M., 2003. Behaviour of Listeria monocytogenes in raw sausages (merguez) in presence of bacteriocinproducing lactococcal strain as a protective culture. Meat Science 63, 479–484. Benkerroum, N., Daoundi, A., Hamraoui, T., Ghalfi, H., Thiry, C., Duroy, M., Evrart, P., Roblain, D., Thonart, P., 2005. Lyophilized preparations of bacteriocinogenic Lactobacillus curvatus and Lactococcus lactis subsp. lactis as potential protective adjuncts to control Listeria monocytogenes in dry-fermented sausages. Journal of Applied Microbiology 98, 56–63. Berry, E.D., Hutkins, R.W., Mandigo, R.W., 1991. The use of bacteriocinproducing Pediococcus acidilactici to control post-processing Listeria monocytogenes contamination of frankfurters. Journal of Food Protection 54, 681–686. Coffey, A., Ryan, M., Ross, R.P., Hill, C., Arendt, E., Schwarz, G., 1998. Use of a broad-host-range bacteriocin-producing Lactococcus lactis transconjugant as an alternative starter for salami manufacture. International Journal of Food Microbiology 43, 231–235. Comi, G., Urso, R., Iacumin, L., Rantsiou, K., Cattaneo, P., Cantoni, C., Cocolin, L., 2005. Characterization of naturally fermented sausages produced in the North East of Italy. Meat Science 69, 381–392. Dicks, L.M.T., Mellett, F.D., Hoffman, L.C., 2004. Use of bacteriocinproducing starter cultures of Lactobacillus plantarum and Lactobacillus curvatus in production of ostrich meat salami. Meat Science 66, 703–708. Foegeding, P.M., Thomas, A.B., Pilkington, D.H., Klaenhammer, T.R., 1992. Enhanced control of Listeria monocytogenes by in situ produced pediocin during dry fermented sausage production. Applied and Environmental Microbiology 58, 884–890. Gahan, C., O'Briscoll, B., Hill, C., 1996. Acid adaptation of Listeria monocytogenes enhance survival in acidic foods and during milk fermentation. Applied and Environmental Microbiology 62, 3128–3132.
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