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Inactivation of Serratia liquefaciens on dry-cured ham by high pressure processing
Food Microbiology 35 (2013) 34e37
Contents lists available at SciVerse ScienceDirect
Food Microbiology journal homepage: www.elsevier.com/locate/fm
Inactivation of Serratia liquefaciens on dry-cured ham by high pressure processing N. Belletti, M. Garriga, T. Aymerich, S. Bover-Cid* IRTA, Food Safety Programme, Finca Camps i Armet, s/n, E-17121 Monells, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 September 2012 Received in revised form 22 January 2013 Accepted 1 March 2013 Available online 14 March 2013
To quantify the inactivation of Serratia liquefaciens exerted by high pressure processing (HPP), slices of dry-cured ham were inoculated and processed combining different levels of technological parameters: pressure (347e852 MPa), time (2.3e15.8 min) and temperature (7.6e24.4 C) according to a central composite design. Bacterial inactivation, as logarithmic reduction, indicated that S. liquefaciens was relatively sensitive to HPP. Six log reductions were achieved in a total of 10 trials combining pressures of 600 MPa or above with different holding times and temperatures. The inactivation of S. liquefaciens was analysed through the multiple regression analysis to generate a second order polynomial equation. Pressure and time were the two factors which significantly determined the inactivation of S. liquefaciens on dry-cured ham. Temperature did not significantly affect the lethality of the process. The response surface methodology was used to determine optimum process conditions to maximize the inactivation of S. liquefaciens in the experimental range tested. The maximum inactivation of S. liquefaciens in dry-cured ham was achieved by combining a pressure of 650 MPa with a holding time of 8 min. Combinations above these values (i.e. 750 MPa for 13 min) would not significantly improve the lethality of the process. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: High pressure processing Process criteria Serratia liquefaciens Dry-cured ham Response surface methodology
1. Introduction High pressure processing (HPP) is gaining more and more acceptance due to its successful commercial applications on jams, fruit juices, sauces, smoothies, guacamole, ready-to-eat meat products, and whole fresh oysters (Patterson et al., 2007). HPP is principally used as a final hygienization measure after production and/or packaging operations to improve the safety and extend the shelf life of food products. One of the main benefits of HPP is that it allows the inactivation of vegetative cells of spoilage and pathogenic microorganisms at a low or moderate temperature, without compromising the sensorial and nutritional food quality attributes (Chen and Hoover, 2003; Hugas et al., 2002). When using HPP, pressure level, holding time and treatment temperature, are the processing factors which determine the inactivation of microorganisms. Bacterial inactivation is also affected by other factors, including those related to the microorganism (type of strain, growth phase, etc) as well as to the food composition (pH, aw, etc) (Rendueles et al., 2011). Thus, to properly inhibit undesirable microorganisms and at the same time retain sensory product attributes, HPP parameters should be tailored on the basis of the type of food to be processed (Erkmen and Dogan, 2004; Patterson, 2005).
* Corresponding author. Tel.: þ34 972 630052x1446, fax: þ34 972 630373. E-mail address: [email protected] (S. Bover-Cid). 0740-0020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fm.2013.03.001
Enterobacteriaceae are considered to be microbial qualityrelated organisms in the development of HACCP systems for drycured ham. Their presence has been related to important causes of spoilage in Iberian and other kinds of dry-cured ham, the socalled ‘‘bone taint’’ or ‘‘deep spoilage’’ (Martín et al., 2008). Species of the genera Serratia, Enterobacter, Proteus, Leclercia, and Hafnia have frequently been isolated from spoiled hams. In particular, strains of Enterobacteriaceae isolated in spoiled Spanish dry-cured hams belonged to the Serratia liquefaciens and Proteus vulgaris species. Both species are reported to be proteolytic and non-pathogenic (Losantos et al., 2000). Furthermore, Serratia, together with Proteus, is the genera most commonly present on working surfaces in the meat processing industry (Stiles, 1981). S. liquefaciens is a major spoilage agent in fresh, cooked or curedmeat. Its proteolytic activity causes the production of off odours and off flavours. Moreover, its ability to produce biogenic amines (e.g. putrescine and cadaverine) in meat products has been reported (Silla-Santos, 1996). Therefore, the control of the presence of Serratia in foods in general and in high added value meat products, such as sliced dry-cured ham, can contribute to guarantee good quality standards for production. Based upon these premises, the aim of this study was to investigate the effect of HPP on the inactivation of a S. liquefaciens strain in a RTE meat product (sliced dry-cured ham). The modelling approach, through a response surface methodology, was applied to quantify the influence of each technological factor of the
N. Belletti et al. / Food Microbiology 35 (2013) 34e37
pressurization process (i.e. pressure, time and temperature) on the logarithmic reductions of S. liquefaciens loads following HPP.
35
Pressure come up rate was on average 220 MPa/min. Pressure release was almost immediate. Samples for each treatment were prepared and sampled at least twice.
2. Materials and methods 2.3. Microbiological determinations 2.1. Bacterial strain and culture preparation S. liquefaciens (CTC1690) was previously isolated from dry-cured ham and was selected as the most baroresistant strain from five S. liquefaciens strains from our own collection (CTC1045, CTC1985, CTC1691, CTC1692). To prepare the inoculum culture, 100 ml of a stock culture (stored in 20% glycerol at 80 C) were transferred to 10 ml Brain Heart Infusion (BHI, from DB, NJ, USA) broth and incubated for 7 h at 37 C. Subsequently, an aliquot was transferred (1/100 dilution) to fresh BHI and incubated overnight (18 h) at 37 C. This culture was centrifuged (model J2-MC, Beckman Instruments S.A. Madrid, Spain) at 10,000 rpm for 10 min at 4 C, the pellet was resuspended in sterile water to achieve a concentration of the cell suspension ca. 109 cfu/ml. 2.2. Sample preparation and experimental design Dry-cured ham (aw ¼ 0.88 and pH ¼ 5.84), was aseptically deboned and sliced (in ca. 1 mm thick slices). Sample preparation was performed under a laminar flow cabin (Telstar BIO-II-A, Telstar S.A., Terrassa, Spain). The S. liquefaciens CTC1690 cell suspension was inoculated at 1% (v/w) onto each slice obtaining a final concentration of ca. 107 cfu/g. The inoculum was uniformly distributed over the surface of the slice using a sterile disposable spreader until it was absorbed (max. 2 min). Inoculated samples were vacuumpackaged and treated under different HPP conditions according to a Central Composite Design (CCD) of three factors (Table 1): pressure (347, 450, 600, 750 and 852 MPa), holding time (2.3, 5.0, 9.0, 13.0 and 15.8 min) and initial fluid temperature (7.6, 11.0, 16.0, 21.0 and 24.4 C). The experimental ranges considered in this work were chosen on the basis of industrial relevance and the technological feasibility, as published in previous works (Bover-Cid et al., 2011, 2012). Wave 6000 (Hyperbaric, Burgos, Spain) and Thiot ingenierie e Hyperbaric (Bretenoux, France e Burgos, Spain) HPP units were used for pressures up to and above 600 MPa, respectively. Table 1 Combinations of intensity of pressure, holding time and fluid temperature according to the CCD and correspondent log-inactivation of Serratia liquefaciens CTC1690 achieved after high pressure processing. Run
Pressure (MPa)
Time (min)
Temperature ( C)
Inactivation (log (N/N0))
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
450 450 750 750 450 450 750 750 600 600 600 600 600 600 600 600 852 347 600 600
5.0 13.0 5.0 13.0 5.0 13.0 5.0 13.0 9.0 9.0 9.0 9.0 9.0 9.0 15.7 2.3 9.0 9.0 9.0 9.0
11.0 11.0 11.0 11.0 21.0 21.0 21.0 21.0 16.0 16.0 16.0 16.0 16.0 16.0 16.0 16.0 16.0 16.0 24.4 7.6
2.55 3.68 6.48 6.78 2.55 3.43 6.79 6.94 4.98 5.39 6.23 6.41 6.56 6.26 6.42 3.01 6.63 1.40 5.65 4.74
a
Mean (standard deviation).
(0.17)a (0.18) (0.63) (0.21) (0.11) (0.46) (0.15) (0.07) (0.50) (0.02) (0.59) (0.22) (0.00) (0.43) (0.98) (0.21) (0.01) (0.16) (0.69) (0.35)
The enumeration of S. liquefaciens cells was carried out on inoculated samples before and after HPP treatment. The dry-cured ham was homogenized (1/10) in 0.1% Bacto Peptone (Difco Laboratories, Detroit, MI, USA) with 0.85% NaCl (Merck, Darmstadt, Germany) in a Masticator Classic (IUL S.A., Barcelona, Spain) for 60 s. The homogenate was serially diluted in the same diluent and pour-plated using violet red bile glucose agar (VRBGA, Difco); plates were incubated at 37 C for 24 h. 2.4. Statistical analysis and mathematical modelling Inactivation of S. liquefaciens was expressed in terms of logarithmic reductions as the difference between counts after the treatments and the initial inoculum, i.e. Log (N/N0). The statistical package Statistica for Windows (v.8, Statsoft Inc., Tulsa, OK, USA) was used for regression analysis. The simplified second order polynomial equation was generated through the backward stepwise procedure and contained only statistically significant terms (p < 0.05) describing HP-induced inactivation of S. liquefaciens. The goodness of fit of the regression model was defined by the adjusted coefficient of determination (R2adj). The statistical significance of the model was evaluated on the basis of the significance of the p-values derived from the Fisher F-test and the significance of the Lack of Fit (LoF) test. 3. Results and discussion 3.1. Inactivation of Serratia liquefaciens on dry-cured ham by HPP Table 1 summarizes the inactivation, expressed as logarithmic reductions, registered for S. liquefaciens CTC1690 inoculated on drycured ham for each combination of pressure, time of treatment and initial fluid temperature, according to the CCD. S. liquefaciens, inoculated on dry-cured ham, was relatively sensitive to HPP. The lowest pressure applied (347 MPa) corresponded to the lowest inactivation obtained (1.40 logs). High inactivation extent, slightly exceeding the 6 logs, was obtained in a total of 10 runs (3, 4, 7, 8, 11, 12, 13, 14, 15, and 17 in Table 1), all performed at 600 MPa or above, for different holding times and at different temperatures. It should be highlighted that the use of a pressure intensity of 750 MPa for 13 min led to levels of S. liquefaciens below the detection limit (<10 ufc/g) irrespectively of the initial fluid temperature (11 or 21 C). At the same pressure level, lowering the holding time to 5 min, allowed some of the S. liquefaciens cells to survive, even after HPP at the highest pressure (852 MPa) was applied. Generally speaking, the inactivation of S. liquefaciens on drycured ham seemed to depend upon the pressure intensity and the holding time; but the influence of temperature seemed to be negligible. For example, at both 450 (runs 1, 2, 5, 6) and 750 MPa (runs 3, 4, 7, 8), the differences between the inactivation at 11 and 21 C were not relevant from a microbiological point of view, being below 0.5 logs. To our knowledge, no published data are available for Serratia spp. inactivation on HP treated meat products. Furthermore, the differences in meat product, processing conditions and/or target microorganisms make it difficult to find references to compare experimental results with. Serratia spp. is a member of the large family of enterobacteria, which is characterized by a relative
36
N. Belletti et al. / Food Microbiology 35 (2013) 34e37
sensitivity to pressurization. Diez et al. (2008) and López-Caballero et al. (1999) reported that the initial number of endogenous enterobacteria, around 2 log (cfu/g), can be reduced below the detection limit in high water activity (i.e. 0.98) meat products like Morcilla de Burgos (i.e. using technological parameters in a range from 300 to 600 MPa, at 15 C, for 10 min). Garriga et al. (2004) reported the reduction of endogenous enterobacteria from an initial presence of 3.88 log (cfu/g) to below the detection limit after pressurization at 600 MPa for 6 min at 16 C on sliced, skin vacuum-packed marinated beef loin (pH 5.88, aw 0.985). Since endogenous enterobacteria are normally present in numbers below 4 logs (cfu/g) in processed dry-cured meat products, only relatively low inactivation extent can be recorded. The family of enterobacteria includes, along with Serratia spp., pathogenic microorganisms of great concern such as Salmonella, and some specific strains of Escherichia coli. E. coli have been shown to have a high sensitivity to pressure. According to Porto-Fett et al. (2010) the combination of 483 MPa at 19 C for 5 min, led to a total reduction of the initial 5 logs (cfu/g) of E. coli O157:H7 inoculated into dry-fermented Genoa salami (characterized by pH values between 4.56 and 4.66 and aw values between 0.884 and 0.940). Our results showed that, similar HPP conditions resulted in the lowest inactivation extent of S. liquefaciens, as can be seen in Table 1, runs 2 (11 C) and 6 (21 C). In the study by Garriga et al. (2002), a mixture of 2 strains of E. coli, inoculated into a meat model (cooked ham homogenized with water, pH 6.5), exhibited a decline of 4.5-logs after HP treatment (400 MPa, 10 min, 17 C); whereas Salmonella London and Salmonella Schwarzengrund were reduced by about 6 logs. However, Bover-Cid et al. (2012) reported that by applying the same experimental conditions assessed in the present study, Salmonella Enterica resulted to be generally more baroresistant when compared to the results discussed here for S. liquefaciens. 3.2. Mathematical modelling
LogðN=N0 Þ ¼ 13:828 4:340 ½P=100 þ 0:266 ½P=1002 0549 ½ti þ 0:22 ½ti 2
Where P is the pressure level in MPa and ti is holding time in min. The high correlation between the experimental and the fitted values (i.e. R2adj ¼ 0.875), the overall significance of the model (indicated by F ¼ 34.24 and the corresponding probability value p < 0.00001), as well as the non-significant Lack of Fit test (p ¼ 0.58) indicated that the equation significantly described the HPP-inactivation of S. liquefaciens within the experimental domain assayed. According to the developed empirical equation, pressure and holding time were the significant factors leading the inactivation of S. liquefaciens on dry-cured ham. The 3D surface plot, shown in Fig. 1, graphically represents the empirical model obtained. The inactivation of S. liquefaciens was expressed as a function of the pressure and time of HPP, while temperature was fixed at the central value of the CCD. The sensitivity of S. liquefaciens to pressure increase was higher compared to the sensitivity to the increase of the holding time, graphically represented by the steepest slope of the pressure axis in comparison with the time axis. The lethality of the process increased linearly for pressures up to 650 MPa and holding times up to 8 min. Above those values, more intense treatments in terms of length and/or intensity would not result in a higher inactivation extent of S. liquefaciens, as graphically represented by the curvature, or tail shape, of the response surface. The factor temperature was not included in the model as it resulted not statistically significant within the range assayed. In HPP, elevated fluid temperatures are reported to promote the inactivation of microorganisms (Chen and Hoover, 2003; Alpas et al., 2000). However, the role of temperature remains unclear when pressurization is performed at room temperature or under refrigeration. In fact, Bover-Cid et al. (2012) reported that in the same experimental conditions used in this study, Salmonella spp.
Results were analysed through the multiple regression analysis to obtain the polynomial equation quantifying the effects of the studied technological factors on the inactivation of S. liquefaciens. Since the magnitude of pressure units (in MPa) was too high in comparison with the magnitude of the factors time (in min) and temperature (in C), unsatisfactory statistical outputs were observed. Therefore, three different approaches, as previously reported (Bover-Cid et al., 2011, 2012), were assessed:
For each approach, the backward stepwise procedure was used and three simplified polynomial equations comprising of only the statistically significant (p < 0.05) terms were obtained and compared on the basis of their statistical performance indexes (R2adj, p-value of the F-test, Lack of Fit test) resulting from the analysis of variance (ANOVA). Approach (i) was discarded (R2adj ¼ 0.819, F ¼ 43.95, p of the Lack of Fit test ¼ 0.34). Approaches (ii) and (iii) were characterized by the same values of the performance indexes. Approach (ii) was chosen to avoid the use of dimensionless factors, resulting in the Equation (1).
0 -1 Inactivation Log(N/N0)
(i) by Log10 transformation of the values for the factor pressure (P), i.e. PLog; (ii) by rescaling the values of the factor pressure (P) with a 1/100 ratio, i.e. P0.01; (iii) by expressing all the considered factors with their dimensionless levels (1.68, 1.00, 0.00, þ1.00, þ1.68), each level was added with 5 to avoid negative and zero values, resulting in 3.32, 4.00, 5.00, 6.00 and 6.68.
(1)
-2 -3 -4 -5 -6 -7 350 450 550 Pressure (MPa)
8
650
10 12
750 850 16
14
6
4
2
Time (minutes)
Fig. 1. Response surface plot describing the effect of pressure and holding time on HPinactivation of Serratia liquefaciens CTC1690 on dry-cured ham according to the developed model (Equation (1)).
N. Belletti et al. / Food Microbiology 35 (2013) 34e37
(also belonging to the Enterobacteriaceae family), was significantly affected by the initial fluid temperature. However, contrary to this, negligible effects of fluid temperature were reported, for the Gram positive Listeria monocytogenes (Bover-Cid et al., 2011). 4. Conclusions Our findings highlight that HPP is an effective post-processing technology for reducing relatively high levels of S. liquefaciens. The sensitivity of Serratia to HPP in sliced dry-cured ham was higher in comparison to that reported for other microorganisms such as pathogenic Salmonella spp. and L. monocytogenes. Setting the optimal HPP technological variables (process criteria) to accomplish safety standards for Salmonella spp. and L. monocytogenes in RTE products, according to previous works (Bover-Cid et al., 2011, 2012), would allow manufacturers to avoid the presence of spoilage microorganisms like Serratia spp. Acknowledgements This work was funded by the Spanish Ministerio de Ciencia e Innovación (INIA, Ref. RTA2007-00032). Nicoletta Belletti acknowledges the fellowship of the “Subprograma DOC-INIA 2010”. References Alpas, H., Kalchayanand, N., Bozoglu, F., Ray, B., 2000. Interactions of high hydrostatic pressure, pressurization temperature and pH on death and injury of pressure-resistant and pressure-sensitive strains of foodborne pathogens. International Journal of Food Microbiology 60, 33e42. Bover-Cid, S., Belletti, N., Garriga, M., Aymerich, T., 2011. Model for Listeria monocytogenes inactivation on dry-cured ham by high hydrostatic pressure processing. Food Microbiology 28, 804e809. Bover-Cid, S., Belletti, N., Garriga, M., Aymerich, T., 2012. Response surface methodology to investigate the effect of high pressure processing on Salmonella inactivation on dry-cured ham. Food Research International 45, 1111e1117.
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Chen, H., Hoover, D.G., 2003. Modeling the combined effect of high hydrostatic pressure and mild heat on the inactivation kinetics of Listeria monocytogenes Scott A in whole milk. Innovative Food Science & Emerging Technologies 4, 25e34. Diez, A.M., Urso, R., Rantsiou, K., Jaime, I., Rovira, J., Cocolin, L., 2008. Spoilage of blood sausages morcilla de Burgos treated with high hydrostatic pressure. International Journal of Food Microbiology 123, 246e253. Erkmen, O., Dogan, C., 2004. Effects of ultra high hydrostatic pressure on Listeria monocytogenes and natural flora in broth, milk and fruit juices. International Journal of Food Science and Technology 39, 91e97. Garriga, M., Aymerich, M.T., Costa, S., Monfort, J.M., Hugas, M., 2002. Bactericidal synergism through bacteriocins and high pressure in a meat model system during storage. Food Microbiology 19, 509e518. Garriga, M., Grèbol, N., Aymerich, M.T., Monfort, J.M., Hugas, M., 2004. Microbial inactivation after high-pressure processing at 600 MPa in commercial meat products over its shelf life. Innovative Food Science & Emerging Technologies 5, 451e457. Hugas, M., Garriga, M., Monfort, J.M., 2002. New mild technologies in meat processing: high pressure as a model technology. Meat Science 62, 359e371. López-Caballero, M.E., Carballo, J., Jiménez-Colmenero, F., 1999. Microbiological changes in pressurized, prepackaged sliced cooked ham. Journal of Food Protection 62, 1411e1415. Losantos, A., Sanabria, C., Cornejo, I., Carrascosa, A.V., 2000. Characterization of Enterobacteriaceae strains isolated from spoiled dry-cured hams. Food Microbiology 17, 505e512. Martín, A., Benito, M.J., Hernández, A., Pérez-Nevado, F., Córdoba, J.J., Córdoba, M.G., 2008. Characterisation of microbial deep spoilage in Iberian dry-cured ham. Meat Science 78, 475e484. Patterson, M.F., 2005. Microbiology of pressure-treated foods. Journal of Applied Microbiology 98, 1400e1409. Patterson, M.F., Linton, M., Doona, C.J., 2007. Introduction to high pressure processing of foods. In: Doona, C.J., Feeherry, F.E. (Eds.), High Pressure Processing of Foods. Blackwell Publishing, pp. 1e14. Porto-Fett, A.C.S., Call, J.E., Shoyer, B.E., Hill, D.E., Pshebniski, C., Cocoma, G.J., Luchansky, J.B., 2010. Evaluation of fermentation, drying, and/or high pressure processing on viability of Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp., and Trichinella spiralis in raw pork and Genoa salami. International Journal of Food Microbiology 140, 61e75. Rendueles, E., Omer, M.K., Alvseike, O., Alonso-Calleja, C., Capita, R., Prieto, M., 2011. Microbiological food safety assessment of high hydrostatic pressure processing: a review. LWT-Food Science and Technology 44, 1251e1260. Silla-Santos, M.H., 1996. Biogenic amines: their importance in foods. International Journal of Food Microbiology 29, 213e231. Stiles, M.E., 1981. Enterobacteriaceae associated with meats and meat handling. Applied Environmental Microbiology 41, 867e872.
Contents lists available at SciVerse ScienceDirect
Food Microbiology journal homepage: www.elsevier.com/locate/fm
Inactivation of Serratia liquefaciens on dry-cured ham by high pressure processing N. Belletti, M. Garriga, T. Aymerich, S. Bover-Cid* IRTA, Food Safety Programme, Finca Camps i Armet, s/n, E-17121 Monells, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 September 2012 Received in revised form 22 January 2013 Accepted 1 March 2013 Available online 14 March 2013
To quantify the inactivation of Serratia liquefaciens exerted by high pressure processing (HPP), slices of dry-cured ham were inoculated and processed combining different levels of technological parameters: pressure (347e852 MPa), time (2.3e15.8 min) and temperature (7.6e24.4 C) according to a central composite design. Bacterial inactivation, as logarithmic reduction, indicated that S. liquefaciens was relatively sensitive to HPP. Six log reductions were achieved in a total of 10 trials combining pressures of 600 MPa or above with different holding times and temperatures. The inactivation of S. liquefaciens was analysed through the multiple regression analysis to generate a second order polynomial equation. Pressure and time were the two factors which significantly determined the inactivation of S. liquefaciens on dry-cured ham. Temperature did not significantly affect the lethality of the process. The response surface methodology was used to determine optimum process conditions to maximize the inactivation of S. liquefaciens in the experimental range tested. The maximum inactivation of S. liquefaciens in dry-cured ham was achieved by combining a pressure of 650 MPa with a holding time of 8 min. Combinations above these values (i.e. 750 MPa for 13 min) would not significantly improve the lethality of the process. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: High pressure processing Process criteria Serratia liquefaciens Dry-cured ham Response surface methodology
1. Introduction High pressure processing (HPP) is gaining more and more acceptance due to its successful commercial applications on jams, fruit juices, sauces, smoothies, guacamole, ready-to-eat meat products, and whole fresh oysters (Patterson et al., 2007). HPP is principally used as a final hygienization measure after production and/or packaging operations to improve the safety and extend the shelf life of food products. One of the main benefits of HPP is that it allows the inactivation of vegetative cells of spoilage and pathogenic microorganisms at a low or moderate temperature, without compromising the sensorial and nutritional food quality attributes (Chen and Hoover, 2003; Hugas et al., 2002). When using HPP, pressure level, holding time and treatment temperature, are the processing factors which determine the inactivation of microorganisms. Bacterial inactivation is also affected by other factors, including those related to the microorganism (type of strain, growth phase, etc) as well as to the food composition (pH, aw, etc) (Rendueles et al., 2011). Thus, to properly inhibit undesirable microorganisms and at the same time retain sensory product attributes, HPP parameters should be tailored on the basis of the type of food to be processed (Erkmen and Dogan, 2004; Patterson, 2005).
* Corresponding author. Tel.: þ34 972 630052x1446, fax: þ34 972 630373. E-mail address: [email protected] (S. Bover-Cid). 0740-0020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fm.2013.03.001
Enterobacteriaceae are considered to be microbial qualityrelated organisms in the development of HACCP systems for drycured ham. Their presence has been related to important causes of spoilage in Iberian and other kinds of dry-cured ham, the socalled ‘‘bone taint’’ or ‘‘deep spoilage’’ (Martín et al., 2008). Species of the genera Serratia, Enterobacter, Proteus, Leclercia, and Hafnia have frequently been isolated from spoiled hams. In particular, strains of Enterobacteriaceae isolated in spoiled Spanish dry-cured hams belonged to the Serratia liquefaciens and Proteus vulgaris species. Both species are reported to be proteolytic and non-pathogenic (Losantos et al., 2000). Furthermore, Serratia, together with Proteus, is the genera most commonly present on working surfaces in the meat processing industry (Stiles, 1981). S. liquefaciens is a major spoilage agent in fresh, cooked or curedmeat. Its proteolytic activity causes the production of off odours and off flavours. Moreover, its ability to produce biogenic amines (e.g. putrescine and cadaverine) in meat products has been reported (Silla-Santos, 1996). Therefore, the control of the presence of Serratia in foods in general and in high added value meat products, such as sliced dry-cured ham, can contribute to guarantee good quality standards for production. Based upon these premises, the aim of this study was to investigate the effect of HPP on the inactivation of a S. liquefaciens strain in a RTE meat product (sliced dry-cured ham). The modelling approach, through a response surface methodology, was applied to quantify the influence of each technological factor of the
N. Belletti et al. / Food Microbiology 35 (2013) 34e37
pressurization process (i.e. pressure, time and temperature) on the logarithmic reductions of S. liquefaciens loads following HPP.
35
Pressure come up rate was on average 220 MPa/min. Pressure release was almost immediate. Samples for each treatment were prepared and sampled at least twice.
2. Materials and methods 2.3. Microbiological determinations 2.1. Bacterial strain and culture preparation S. liquefaciens (CTC1690) was previously isolated from dry-cured ham and was selected as the most baroresistant strain from five S. liquefaciens strains from our own collection (CTC1045, CTC1985, CTC1691, CTC1692). To prepare the inoculum culture, 100 ml of a stock culture (stored in 20% glycerol at 80 C) were transferred to 10 ml Brain Heart Infusion (BHI, from DB, NJ, USA) broth and incubated for 7 h at 37 C. Subsequently, an aliquot was transferred (1/100 dilution) to fresh BHI and incubated overnight (18 h) at 37 C. This culture was centrifuged (model J2-MC, Beckman Instruments S.A. Madrid, Spain) at 10,000 rpm for 10 min at 4 C, the pellet was resuspended in sterile water to achieve a concentration of the cell suspension ca. 109 cfu/ml. 2.2. Sample preparation and experimental design Dry-cured ham (aw ¼ 0.88 and pH ¼ 5.84), was aseptically deboned and sliced (in ca. 1 mm thick slices). Sample preparation was performed under a laminar flow cabin (Telstar BIO-II-A, Telstar S.A., Terrassa, Spain). The S. liquefaciens CTC1690 cell suspension was inoculated at 1% (v/w) onto each slice obtaining a final concentration of ca. 107 cfu/g. The inoculum was uniformly distributed over the surface of the slice using a sterile disposable spreader until it was absorbed (max. 2 min). Inoculated samples were vacuumpackaged and treated under different HPP conditions according to a Central Composite Design (CCD) of three factors (Table 1): pressure (347, 450, 600, 750 and 852 MPa), holding time (2.3, 5.0, 9.0, 13.0 and 15.8 min) and initial fluid temperature (7.6, 11.0, 16.0, 21.0 and 24.4 C). The experimental ranges considered in this work were chosen on the basis of industrial relevance and the technological feasibility, as published in previous works (Bover-Cid et al., 2011, 2012). Wave 6000 (Hyperbaric, Burgos, Spain) and Thiot ingenierie e Hyperbaric (Bretenoux, France e Burgos, Spain) HPP units were used for pressures up to and above 600 MPa, respectively. Table 1 Combinations of intensity of pressure, holding time and fluid temperature according to the CCD and correspondent log-inactivation of Serratia liquefaciens CTC1690 achieved after high pressure processing. Run
Pressure (MPa)
Time (min)
Temperature ( C)
Inactivation (log (N/N0))
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
450 450 750 750 450 450 750 750 600 600 600 600 600 600 600 600 852 347 600 600
5.0 13.0 5.0 13.0 5.0 13.0 5.0 13.0 9.0 9.0 9.0 9.0 9.0 9.0 15.7 2.3 9.0 9.0 9.0 9.0
11.0 11.0 11.0 11.0 21.0 21.0 21.0 21.0 16.0 16.0 16.0 16.0 16.0 16.0 16.0 16.0 16.0 16.0 24.4 7.6
2.55 3.68 6.48 6.78 2.55 3.43 6.79 6.94 4.98 5.39 6.23 6.41 6.56 6.26 6.42 3.01 6.63 1.40 5.65 4.74
a
Mean (standard deviation).
(0.17)a (0.18) (0.63) (0.21) (0.11) (0.46) (0.15) (0.07) (0.50) (0.02) (0.59) (0.22) (0.00) (0.43) (0.98) (0.21) (0.01) (0.16) (0.69) (0.35)
The enumeration of S. liquefaciens cells was carried out on inoculated samples before and after HPP treatment. The dry-cured ham was homogenized (1/10) in 0.1% Bacto Peptone (Difco Laboratories, Detroit, MI, USA) with 0.85% NaCl (Merck, Darmstadt, Germany) in a Masticator Classic (IUL S.A., Barcelona, Spain) for 60 s. The homogenate was serially diluted in the same diluent and pour-plated using violet red bile glucose agar (VRBGA, Difco); plates were incubated at 37 C for 24 h. 2.4. Statistical analysis and mathematical modelling Inactivation of S. liquefaciens was expressed in terms of logarithmic reductions as the difference between counts after the treatments and the initial inoculum, i.e. Log (N/N0). The statistical package Statistica for Windows (v.8, Statsoft Inc., Tulsa, OK, USA) was used for regression analysis. The simplified second order polynomial equation was generated through the backward stepwise procedure and contained only statistically significant terms (p < 0.05) describing HP-induced inactivation of S. liquefaciens. The goodness of fit of the regression model was defined by the adjusted coefficient of determination (R2adj). The statistical significance of the model was evaluated on the basis of the significance of the p-values derived from the Fisher F-test and the significance of the Lack of Fit (LoF) test. 3. Results and discussion 3.1. Inactivation of Serratia liquefaciens on dry-cured ham by HPP Table 1 summarizes the inactivation, expressed as logarithmic reductions, registered for S. liquefaciens CTC1690 inoculated on drycured ham for each combination of pressure, time of treatment and initial fluid temperature, according to the CCD. S. liquefaciens, inoculated on dry-cured ham, was relatively sensitive to HPP. The lowest pressure applied (347 MPa) corresponded to the lowest inactivation obtained (1.40 logs). High inactivation extent, slightly exceeding the 6 logs, was obtained in a total of 10 runs (3, 4, 7, 8, 11, 12, 13, 14, 15, and 17 in Table 1), all performed at 600 MPa or above, for different holding times and at different temperatures. It should be highlighted that the use of a pressure intensity of 750 MPa for 13 min led to levels of S. liquefaciens below the detection limit (<10 ufc/g) irrespectively of the initial fluid temperature (11 or 21 C). At the same pressure level, lowering the holding time to 5 min, allowed some of the S. liquefaciens cells to survive, even after HPP at the highest pressure (852 MPa) was applied. Generally speaking, the inactivation of S. liquefaciens on drycured ham seemed to depend upon the pressure intensity and the holding time; but the influence of temperature seemed to be negligible. For example, at both 450 (runs 1, 2, 5, 6) and 750 MPa (runs 3, 4, 7, 8), the differences between the inactivation at 11 and 21 C were not relevant from a microbiological point of view, being below 0.5 logs. To our knowledge, no published data are available for Serratia spp. inactivation on HP treated meat products. Furthermore, the differences in meat product, processing conditions and/or target microorganisms make it difficult to find references to compare experimental results with. Serratia spp. is a member of the large family of enterobacteria, which is characterized by a relative
36
N. Belletti et al. / Food Microbiology 35 (2013) 34e37
sensitivity to pressurization. Diez et al. (2008) and López-Caballero et al. (1999) reported that the initial number of endogenous enterobacteria, around 2 log (cfu/g), can be reduced below the detection limit in high water activity (i.e. 0.98) meat products like Morcilla de Burgos (i.e. using technological parameters in a range from 300 to 600 MPa, at 15 C, for 10 min). Garriga et al. (2004) reported the reduction of endogenous enterobacteria from an initial presence of 3.88 log (cfu/g) to below the detection limit after pressurization at 600 MPa for 6 min at 16 C on sliced, skin vacuum-packed marinated beef loin (pH 5.88, aw 0.985). Since endogenous enterobacteria are normally present in numbers below 4 logs (cfu/g) in processed dry-cured meat products, only relatively low inactivation extent can be recorded. The family of enterobacteria includes, along with Serratia spp., pathogenic microorganisms of great concern such as Salmonella, and some specific strains of Escherichia coli. E. coli have been shown to have a high sensitivity to pressure. According to Porto-Fett et al. (2010) the combination of 483 MPa at 19 C for 5 min, led to a total reduction of the initial 5 logs (cfu/g) of E. coli O157:H7 inoculated into dry-fermented Genoa salami (characterized by pH values between 4.56 and 4.66 and aw values between 0.884 and 0.940). Our results showed that, similar HPP conditions resulted in the lowest inactivation extent of S. liquefaciens, as can be seen in Table 1, runs 2 (11 C) and 6 (21 C). In the study by Garriga et al. (2002), a mixture of 2 strains of E. coli, inoculated into a meat model (cooked ham homogenized with water, pH 6.5), exhibited a decline of 4.5-logs after HP treatment (400 MPa, 10 min, 17 C); whereas Salmonella London and Salmonella Schwarzengrund were reduced by about 6 logs. However, Bover-Cid et al. (2012) reported that by applying the same experimental conditions assessed in the present study, Salmonella Enterica resulted to be generally more baroresistant when compared to the results discussed here for S. liquefaciens. 3.2. Mathematical modelling
LogðN=N0 Þ ¼ 13:828 4:340 ½P=100 þ 0:266 ½P=1002 0549 ½ti þ 0:22 ½ti 2
Where P is the pressure level in MPa and ti is holding time in min. The high correlation between the experimental and the fitted values (i.e. R2adj ¼ 0.875), the overall significance of the model (indicated by F ¼ 34.24 and the corresponding probability value p < 0.00001), as well as the non-significant Lack of Fit test (p ¼ 0.58) indicated that the equation significantly described the HPP-inactivation of S. liquefaciens within the experimental domain assayed. According to the developed empirical equation, pressure and holding time were the significant factors leading the inactivation of S. liquefaciens on dry-cured ham. The 3D surface plot, shown in Fig. 1, graphically represents the empirical model obtained. The inactivation of S. liquefaciens was expressed as a function of the pressure and time of HPP, while temperature was fixed at the central value of the CCD. The sensitivity of S. liquefaciens to pressure increase was higher compared to the sensitivity to the increase of the holding time, graphically represented by the steepest slope of the pressure axis in comparison with the time axis. The lethality of the process increased linearly for pressures up to 650 MPa and holding times up to 8 min. Above those values, more intense treatments in terms of length and/or intensity would not result in a higher inactivation extent of S. liquefaciens, as graphically represented by the curvature, or tail shape, of the response surface. The factor temperature was not included in the model as it resulted not statistically significant within the range assayed. In HPP, elevated fluid temperatures are reported to promote the inactivation of microorganisms (Chen and Hoover, 2003; Alpas et al., 2000). However, the role of temperature remains unclear when pressurization is performed at room temperature or under refrigeration. In fact, Bover-Cid et al. (2012) reported that in the same experimental conditions used in this study, Salmonella spp.
Results were analysed through the multiple regression analysis to obtain the polynomial equation quantifying the effects of the studied technological factors on the inactivation of S. liquefaciens. Since the magnitude of pressure units (in MPa) was too high in comparison with the magnitude of the factors time (in min) and temperature (in C), unsatisfactory statistical outputs were observed. Therefore, three different approaches, as previously reported (Bover-Cid et al., 2011, 2012), were assessed:
For each approach, the backward stepwise procedure was used and three simplified polynomial equations comprising of only the statistically significant (p < 0.05) terms were obtained and compared on the basis of their statistical performance indexes (R2adj, p-value of the F-test, Lack of Fit test) resulting from the analysis of variance (ANOVA). Approach (i) was discarded (R2adj ¼ 0.819, F ¼ 43.95, p of the Lack of Fit test ¼ 0.34). Approaches (ii) and (iii) were characterized by the same values of the performance indexes. Approach (ii) was chosen to avoid the use of dimensionless factors, resulting in the Equation (1).
0 -1 Inactivation Log(N/N0)
(i) by Log10 transformation of the values for the factor pressure (P), i.e. PLog; (ii) by rescaling the values of the factor pressure (P) with a 1/100 ratio, i.e. P0.01; (iii) by expressing all the considered factors with their dimensionless levels (1.68, 1.00, 0.00, þ1.00, þ1.68), each level was added with 5 to avoid negative and zero values, resulting in 3.32, 4.00, 5.00, 6.00 and 6.68.
(1)
-2 -3 -4 -5 -6 -7 350 450 550 Pressure (MPa)
8
650
10 12
750 850 16
14
6
4
2
Time (minutes)
Fig. 1. Response surface plot describing the effect of pressure and holding time on HPinactivation of Serratia liquefaciens CTC1690 on dry-cured ham according to the developed model (Equation (1)).
N. Belletti et al. / Food Microbiology 35 (2013) 34e37
(also belonging to the Enterobacteriaceae family), was significantly affected by the initial fluid temperature. However, contrary to this, negligible effects of fluid temperature were reported, for the Gram positive Listeria monocytogenes (Bover-Cid et al., 2011). 4. Conclusions Our findings highlight that HPP is an effective post-processing technology for reducing relatively high levels of S. liquefaciens. The sensitivity of Serratia to HPP in sliced dry-cured ham was higher in comparison to that reported for other microorganisms such as pathogenic Salmonella spp. and L. monocytogenes. Setting the optimal HPP technological variables (process criteria) to accomplish safety standards for Salmonella spp. and L. monocytogenes in RTE products, according to previous works (Bover-Cid et al., 2011, 2012), would allow manufacturers to avoid the presence of spoilage microorganisms like Serratia spp. Acknowledgements This work was funded by the Spanish Ministerio de Ciencia e Innovación (INIA, Ref. RTA2007-00032). Nicoletta Belletti acknowledges the fellowship of the “Subprograma DOC-INIA 2010”. References Alpas, H., Kalchayanand, N., Bozoglu, F., Ray, B., 2000. Interactions of high hydrostatic pressure, pressurization temperature and pH on death and injury of pressure-resistant and pressure-sensitive strains of foodborne pathogens. International Journal of Food Microbiology 60, 33e42. Bover-Cid, S., Belletti, N., Garriga, M., Aymerich, T., 2011. Model for Listeria monocytogenes inactivation on dry-cured ham by high hydrostatic pressure processing. Food Microbiology 28, 804e809. Bover-Cid, S., Belletti, N., Garriga, M., Aymerich, T., 2012. Response surface methodology to investigate the effect of high pressure processing on Salmonella inactivation on dry-cured ham. Food Research International 45, 1111e1117.
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