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Behavior of Salmonella Rubislaw on ground black pepper (Piper nigrum L.)
Food Control 18 (2007) 268–272 www.elsevier.com/locate/foodcont
Behavior of Salmonella Rubislaw on ground black pepper (Piper nigrum L.) Christiane Asturiano Ristori *, Marco Antonio dos Santos Pereira, Dilma Scala Gelli Food Microbiology Laboratory, Adolfo Lutz Institute, Sa˜o Paulo, SP AV. Dr. Arnaldo No. 355, Cerqueira Ce´sar, CEP 01246-902 Sa˜o Paulo, SP, Brazil Received 18 June 2005; received in revised form 16 October 2005; accepted 20 October 2005
Abstract Among spices, black pepper is highly appreciated and Brazil is one of the largest producers of it in the world. However, spices may reach consumers presenting poor quality, due to the loss of volatile compounds, microbial contamination or even due to insect infestation. Salmonella spp. frequently contaminate ground black pepper and may be recovered from products with low levels of free water, even after they have been submitted to high temperatures. The objective of this trial was to evaluate the behavior of a Salmonella Rubislaw strain on ground black pepper (Piper Nigrum L.), as well as to study the effects of water activity (Aw) and storage temperature (5, 25 and 35 C) for 2 and 15 days. The most probable number technique was used for the quantification of the S. Rubislaw. Data obtained in the present trial indicate that differences in the reduction in counts were observed in relation to Aw (P = 0.006) and storage temperature (P = 0.000). Nevertheless, there was no significance as to the interaction of the two factors (P’s > 0.05). Bonferroni multiple comparisons tests have shown significant differences only when related to the Aw: 0.663, 0.815 and 0.887 (P’s < 0.05). When the product is stored at 5 C, the number of surviving cells is even greater (P = 0.000). Considering the data obtained, we may conclude that, after contamination, S. Rubislaw remains viable in pepper for up to 15 days. 2005 Elsevier Ltd. All rights reserved. Keywords: Ground black pepper; Salmonella Rubislaw; Water activity; Storage temperature
1. Introduction Spices are plants or parts of plants that have been used as flavouring agents in food preparation for thousands of years. Spices may reach consumers presenting poor quality, due to the loss of volatile compounds, microbial contamination (Baxter & Holzapfel, 1982; Pafumi, 1986) or even due to insect infestation (Germano & Germano, 1998; Santos et al., 1999). Black pepper is a highly appreciated spice (Germano & Germano, 1998) and Brazil is one of the largest producers and exporters of it in the world (International
*
Corresponding author. Tel.: +55 011 3068 2932; fax: +55 011 3085 3505. E-mail address: [email protected] (C.A. Ristori). 0956-7135/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2005.10.015
Pepper Community, 2003). In Brazil, Para´ State is the leading producer and exporter, followed by States of Espı´rito Santo and Bahia (Ministe´rio da Agricultura e do Abastecimento, 2001). Spices may contain microbial contaminants as a result of deficiencies in primary production conditions, such as fertilizing and planting, harvesting, drying, storage, transportation, and subsequent grinding and handling. Moreover, in tropical countries such as Brazil, constant high temperature and humidity may favor the development of microorganisms in fruit, seeds and food in general. Salmonella spp. frequently contaminate ground black pepper. This bacterium is a public health problem, with important economic implications, for it affects the international trade of black pepper (D’Aoust, 1994; Gustavsen & Breen, 1984; Santos et al., 1999; Satchell, Bruce, Allen,
C.A. Ristori et al. / Food Control 18 (2007) 268–272
Andrews, & Gerber, 1989). Furthermore, the presence of Salmonella in spices is a risk, since they are frequently added to ready-to-eat foods, without further cooking. Salmonella spp. may be recovered from products with low levels of free water, even after they have been submitted to high temperatures, which may be a result of post-processing contamination or of the ability of Salmonella to survive heat treatment, when the water activity of the product is low (Goepfert, Iskander, & Amundson, 1970; Riemann, 1968). One of the effects of Aw below the optimum for the development of a given bacterium is the increase in the lag phase, a decrease in the rate of multiplication, and a smaller final population. Besides Aw, other factors also affect adaptation and multiplication of bacteria, such as pH, temperature and oxidation–reduction potential (Jay, 1992). In general, Gram-negative bacteria require a higher Aw for their development than Gram-positive bacteria (Jay, 1992). The minimum Aw for Salmonella development is 0.93. However, as already noted above, there may be other factors affecting bacterial behavior (Varnan & Evans, 1991, chapter 4). In addition to Aw, for instance, thermoresistance is another important factor in the study of bacterial survival. Some authors, such as Varnan and Evans (1991, chapter 4), report Salmonella destruction at 74 C, with exposure time ranging from 0.2 to 6.5 min. But several studies on the thermoresistance of this bacterium demonstrate that this value is higher when the Aw of the medium is lower (Archer, Jervis, & Gaze, 1998; Summer, Sandros, Harmon, Scott, & Bernard, 1991). Although the relationship between Aw and thermoresistance is known, the survival of Salmonella in a low Aw product, which would limit the development of the bacterium, has been little studied, as well as its association with different storage temperatures before thermal treatment. Therefore, the contamination of an ingredient with reduced Aw, such as spices, may vary according to storage at different temperatures and heat treatment. Mattick, Jorgensen, Legan, Lappin-Scott, and Humphrey (2000) demonstrated that the previous exposure of Salmonella spp. to reduced Aw levels may reduce the efficiency of the subsequent thermal process, which may vary according to the solute. The objective of this trial was to evaluate the behavior of a Salmonella Rubislaw strain on ground black pepper (Piper Nigrum L.), as well as the functions of water activity (Aw) and storage temperature.
2. Material and methods 2.1. Samples Samples of ground black pepper (Piper Nigrum L.), traded in 50 and 500 g packs, were purchased in local supermarkets in the city of Sa˜o Paulo.
269
2.2. Preparation of the strain A S. Rubislaw strain, isolated from a pepper sample at the Food Microbiology Laboratory at Instituto Adolfo Lutz, was used. This serotype was the most frequent one isolated from this kind of sample at this Laboratory. The strain was stored on nutrient agar, at room temperature. A culture of the strain in the stationary phase was obtained with stock strain transferring to brain heart infusion broth (Merck KGaA 64271 Darmstadt, Germany) and incubated at 35 C for 18–24 h. 2.3. Preparation and storage of the samples The samples were previously analyzed for the presence of Salmonella. Samples were prepared in 500 mL sterile glass flasks, with 40 g of ground black pepper samples. Distilled water was added in the proportion described below (item 2.3.1) in order to adjust Aw. Samples were mixed with a sterile glass rod, for at least 60 s. After being mixed, a buffered peptone water (BPW, Merck KGaA 64271 Darmstadt, Germany) suspension of cells from a stationary phase S. Rubislaw culture (containing an average 109 viable cells/ mL) was added. After adding the culture, the product was mixed again in order to uniformly distribute the contaminant over the sample. Storage temperatures were 5, 25 and 35 C. Each trial (three temperatures for each Aw level) was performed in triplicate. Salmonella were quantified immediately after incorporation in the sample, and after 2 and 15 days of storage at the different temperatures. 2.3.1. Aw determination in the product Aw of each sample (flask) was determined in a water activity analyzer (CX-2 Model, from Aqualab), calibrated for each measurement. Aw levels, after the addition of the liquid to the samples, were: 0.663, 0.772, 0.815, 0.859, 0.887, 0.937. Aw was measured before producing the aliquots (original packaging), after the preparation of each flask, and after 15 days of storage at each temperature range. Contamination and Aw levels in each sample were obtained as described below: Aw 0.663: 40 g pepper + 0.2 mL of the contaminant, Aw 0.772: 40 g pepper + 0.2 mL of the contaminant + 1 mL sterile distilled water, Aw 0.815: 40 g pepper + 0.3 mL of the contaminant + 1 mL sterile distilled water, Aw 0.859: 40 g pepper + 0.2 mL of the contaminant + 3 mL sterile distilled water, Aw 0.887: 40 g pepper + 0.3 mL of the contaminant + 3 mL sterile distilled water, Aw 0.937: 40 g pepper + 0.3 mL of the contaminant + 6 mL sterile distilled water. Note: Contaminant = 10 1 dilution of the stationary phase culture of S. Rubislaw.
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2.4. Quantification of the contaminant (S. Rubislaw)
Temperature 5ºC 5.0
0.663
4.0
0.772
3.0
0.815
2.0
0.859
1.0
0.887
0.0 Initial
2 days Time
6.0 5.0 4.0 3.0 2.0 1.0 0.0
0.663 0.772 0.815 0.859 0.887 Initial
R2: Reduction (Log) of S. Rubislaw in the sample, from the day 0 to day 2 samples. R15: Reduction (Log) of S. Rubislaw in the sample, from the day 0 to day 15 samples. The effects of reduction (variable determined by two dependent measurements: R2 and R15) were tested, along with its interactions with other factors (Aw and T C), as well as the main effects of these factors. The significance level adopted (a) was 0.05. Data analysis was performed with Microsoft Excel 97 and SPSS for Windows 10.
0.937
Temperature 25ºC
3. Statistical analysis
2 days Time
0.937
15 days
Fig. 2. Mean profiles of variable response for the temperature of 25 C.
Temperature 35ºC Log10 MPN/g S. Rubislaw
Microbiological data were converted to Log10 MPN/g before being analyzed. Values for the mean Log10 and standard deviation of each set of S. Rubislaw MPN were calculated. Repeated variance analysis measurements (Neter, Kutner, Nachtsheim, & Wasserman, 1996) and Bonferroni multiple comparisons were the statistical techniques used to compare the mean Log10 reduction of S. Rubislaw values under the different conditions. The variable observed in the trial is the Response: the Log MPN/g value found for S. Rubislaw (used to contaminate the sample) at three distinct times in the storage period (initial, 2 and 15 days). For data analysis, the following variables were created:
15 days
Fig. 1. Mean profiles of variable response for the temperature of 5 C.
Log10 MPN/g S. Rubislaw
The most probable number (MPN) technique was used, according to Peeler, Houghtby, and Rainosek (1992). Culture media for isolation were those recommended by ISO, 2002. For quantification, 10 g of the sample were withdrawn from each flask. Each aliquot was homogenized in 90 mL of BPW. From this homogenous suspension, which corresponded to a 10 1 dilution, other decimal dilutions were obtained, up to 10 6, using BPW as diluent. Dilutions (1 mL of each) were inoculated into a series of three tubes, containing 10 mL of BPW and were incubated at 35 C/ 24 h. After incubation, tubes that were turbid were streaked onto Salmonella-Shigella agar and Brilliant Green agar (Oxoid Ltd., Basingstoke, Hampshire, England), both incubated at 35 C/24 h. Characteristic Salmonella colonies were inoculated in IAL presumptive medium for enterobacteria (Pessoa & Silva, 1972), incubated at 35 C/24 h, for biochemical confirmation.
Log10 MPN/g S. Rubislaw
6.0
7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
0.663 0.772 0.815 0.859 0.887 Initial
2 days Time
0.937
15 days
Fig. 3. Mean profiles of variable response for the temperature of 35 C.
Table 1 Repeated variance analysis measurements Source
Reduction Reduction * Aw Reduction * temperature Reduction * Aw * temperature Error (reduction)
Tests of within-subjects SS
df
MS
F
Sig.
25.163 1.733 0.552 3.255 9.186
1 5 2 10 35
25.16 0.347 0.276 0.325 0.262
95.87 1.321 1.051 1.240
0.000 0.278 0.360 0.301
Tests of between-subjects effects
4. Results The behavior of the variable response (Log10 of the initial S. Rubislaw value), in connection with storage time, storage temperature and water activity are showing in Figs. 1–3. The mean Aw value for the pepper in the original packaging was 0.647.
Aw Temperature Aw * temperature Error SS—sum of squares. MS—mean squares.
df
MS
F
5 2 10 35
4.918 64.921 2.212 1.236
3.980 52.534 1.790
Sig. 0.006 0.000 0.099
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Reduction
Mean
Std. error
95% confidence interval Lower bound
Upper bound
0.663, the least reduction was observed, both at 35 and 25 C; in an Aw equal to 0.887 and storage at these temperatures, the greatest reductions occurred (Table 2). Although Salmonella does not grow in Aw below 0.93 (Varnan & Evans, 1991, chapter 4), it may survive for long periods (Sperber, 1983) under these conditions. Considering the data obtained in this study, we may conclude that after contamination S. Rubislaw remains viable in pepper for up to 15 days. When the product is stored at 5 C, the number of surviving cells is even greater. In this trial, tests were performed with only one strain of S. Rubislaw. The behavior of other strains and serotypes may be different in relation to sensitivity to natural compounds and to the behavior reported here, using ground black pepper and considering the parameters tested (Doesburg, Lamprecht, & Elliott, 1970). We may conclude that there is a significant reduction in counts of the S. Rubislaw strain used to contaminate the samples of ground black pepper and that this reduction is more influenced by storage temperature than by low Aw. Moreover, it should be emphasized that results obtained here demonstrate the importance of the use of good manufacturing practices in the production of ground black pepper, since the products a low Aw alone may not efficiently prevent the survival of the bacterium.
1 2
2.080 3.058
0.117 0.122
1.842 2.812
2.318 3.305
References
Table 2 Estimates for the mean reduction Aw
Mean
Std. error
95% confidence interval Lower bound
Upper bound
0.663 0.772 0.815 0.859 0.887 0.937
1.994 2.611 2.225 2.406 3.517 2.661
0.262 0.262 0.283 0.262 0.262 0.262
1.463 2.079 1.650 1.874 2.985 2.129
2.526 3.143 2.800 2.937 4.049 3.193
Table 3 Estimates for the mean reduction for temperature Temperature (C)
Mean
Std. error
95% confidence interval Lower bound
Upper bound
5 25 35
1.197 2.572 3.938
0.185 0.185 0.193
0.821 2.196 3.546
1.573 2.948 4.329
Table 4 Estimates for the mean reduction for R2 (reduction 1) and R15 (reduction 2)
There was no interaction between the factors (Table 1): reduction · temperature · Aw (P = 0.301), reduction · Aw (P = 0.278), reduction · temperature (P = 0.360), Aw · temperature (P = 0.099). However, the variance analysis demonstrated that the main effects of Aw, temperature and reduction, in relation to the initial Log10 value of S. Rubislaw, are highly significant (P’s < 0.05). Tables 2–4 present the estimates of mean reduction for each level of the factors under study. Bonferroni multiple comparisons tests showed significant differences in relation to the levels of the factors temperature and reduction (P’s = 0.000); regarding the Aw comparisons, only: 0.663 · 0.887; 0.815 · 0.887 presented significant differences (P’s = 0.05). 5. Discussion Juven et al. (1984), when studying the survival of Salmonella spp. in dry products, including animal feed, reported that it was greater at Aw 0.43 and 0.52 than at 0.75. They also found that survival varied among different serotypes. Data obtained in the present trial, however, indicate that the survival of S. Rubislaw in ground black pepper was mainly associated with storage temperature, for the least reduction in counts was observed when pepper was stored at 5 C (Table 3). Differences in the reduction in counts were observed in relation to Aw; however, they were smaller than those observed in relation to temperature. In Aw
Archer, J., Jervis, E. T., & Gaze, J. E. (1998). Heat resistance of Salmonella weltevreden in low-moisture environments. Journal of Food Protection, 61(8), 969–973. Baxter, R., & Holzapfel, W. H. (1982). A microbial investigation of selected spices, herbs, and additives in South Africa. Journal of Food Science, 47, 570–574. D’Aoust, J. Y. (1994). Salmonella and international food trade. International Journal of Food Microbiology, 24, 11–31. Doesburg, J. J., Lamprecht, E. C., & Elliott, M. (1970). Death rates of Salmonellae in fish meals with different water activities. II—During heat treatment. Journal of the Science of Food and Agriculture, 21, 636–640. Germano, P. M. L., & Germano, M. I. S. (1998). Importaˆncia e riscos das especiarias. Higiene Alimentar, 12, 23–26. Goepfert, J. M., Iskander, I. K., & Amundson, C. H. (1970). Relation of the heat resistance of salmonellae to the water activity of the environment. Applied Microbiology, 19, 429–433. Gustavsen, S., & Breen, O. (1984). Investigation of an outbreak of Salmonella Oranienburg infections in Norway, caused by contaminated black pepper. American Journal of Epidemiology, 119(5), 806–812. International Organization for Standardization (2002). Microbiology— General guidance for the detection of Salmonella. International Organization for Standardization, ISO 6579, Geneva, Switzerland. International Pepper Community, IPC (2003). Pepper market review. http://www.peppertrade.com.br/IPC-REPORT2003.htm. Accessed 24.05.05. Jay, J. M. (1992). Modern food microbiology (pp. 44–48). New York: Chapman & Hall. Juven, B. J., Cox, N. A., Bailey, J. S., Thomson, J. E., Charles, O. W., & Shutze, J. V. (1984). Survival of Salmonella in dry food and feed. Journal of Food Protection, 47(6), 445–448. Mattick, K. L., Jorgensen, F., Legan, J. D., Lappin-Scott, H. M., & Humphrey, T. J. (2000). Habituation of Salmonella spp. at reduced water activity and its effect on heat tolerance. Applied and Environmental Microbiology, 66(11), 4921–4925.
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Ministe´rio da Agricultura e do Abastecimento (2001). Brazilian agriculture in figures. http://www.agricultura.gov.br/pls/portal/docs/PAGE/ MAPA/ESTATISTICAS/AGRICULTURA_EM_NUMEROS/3118H. XLS. Accessed 24.05.05. Neter, J., Kutner, M. H., Nachtsheim, C. J., & Wasserman, W. (1996). Applied linear statistical models (4th ed.). Boston: McGraw-Hill. Pafumi, J. (1986). Assessment of the microbiological quality of spices and herbs. Journal of Food Protection, 49(12), 958–963. Peeler, J. T., Houghtby, G. A., & Rainosek, A. P. (1992). The most probable number technique. In C. Vanderzant & D. F. Splittstoesser (Eds.), Compendium of methods for the microbiological examination of foods (3rd, pp. 105–120). Washington, DC: APHA. Pessoa, G. V. A., & Silva, E. A. M. (1972). Meios de Rugai e lisina— motilidade combinados em um so´ tubo para a identificac¸a˜o presuntiva de enterobacte´rias. Revista do Instituto Adolfo Lutz, 32, 97–100. Riemann, H. (1968). Effect of water activity on the heat resistance of Salmonella in ‘‘dry’’ materials. Applied Microbiology, 16, 1621–1622.
Santos, C. C. M., Graciano, R. A. S., Peresi, J. T. M., Ribeiro, A. K., Carvalho, I. S., Quirino, G. K., et al. (1999). Avaliac¸a˜o dos padro˜es de identidade e qualidade da Pimenta do reino comercializada na regia˜o de S.J. do Rio Preto, SP. Higiene Alimentar, 13(61), 101–104. Satchell, F. B., Bruce, V. R., Allen, G., Andrews, W. H., & Gerber, H. R. (1989). Microbiological survey of selected imported spices and associated fecal pellet specimens. Journal of the Association of Official Analytical Chemists, 72(4), 632–637. Sperber, W. H. (1983). Influence of water activity on foodborne bacteria— A review. Journal of Food Protection, 46(2), 142–150. Summer, S. S., Sandros, T. M., Harmon, M. C., Scott, V. N., & Bernard, D. T. (1991). Heat resistance of Salmonella typhimurium and Listeria monocytogenes in sucrose solutions of various water activities. Journal of Food Science, 56(6), 1741–1743. Varnan, A. H., & Evans, M. G. (1991). Foodborne pathogens—an illustrated text (chapter 4, pp. 51–85). St. Louis: Mosby Year Book, London: Wolfe Publishing Ltd.
Behavior of Salmonella Rubislaw on ground black pepper (Piper nigrum L.) Christiane Asturiano Ristori *, Marco Antonio dos Santos Pereira, Dilma Scala Gelli Food Microbiology Laboratory, Adolfo Lutz Institute, Sa˜o Paulo, SP AV. Dr. Arnaldo No. 355, Cerqueira Ce´sar, CEP 01246-902 Sa˜o Paulo, SP, Brazil Received 18 June 2005; received in revised form 16 October 2005; accepted 20 October 2005
Abstract Among spices, black pepper is highly appreciated and Brazil is one of the largest producers of it in the world. However, spices may reach consumers presenting poor quality, due to the loss of volatile compounds, microbial contamination or even due to insect infestation. Salmonella spp. frequently contaminate ground black pepper and may be recovered from products with low levels of free water, even after they have been submitted to high temperatures. The objective of this trial was to evaluate the behavior of a Salmonella Rubislaw strain on ground black pepper (Piper Nigrum L.), as well as to study the effects of water activity (Aw) and storage temperature (5, 25 and 35 C) for 2 and 15 days. The most probable number technique was used for the quantification of the S. Rubislaw. Data obtained in the present trial indicate that differences in the reduction in counts were observed in relation to Aw (P = 0.006) and storage temperature (P = 0.000). Nevertheless, there was no significance as to the interaction of the two factors (P’s > 0.05). Bonferroni multiple comparisons tests have shown significant differences only when related to the Aw: 0.663, 0.815 and 0.887 (P’s < 0.05). When the product is stored at 5 C, the number of surviving cells is even greater (P = 0.000). Considering the data obtained, we may conclude that, after contamination, S. Rubislaw remains viable in pepper for up to 15 days. 2005 Elsevier Ltd. All rights reserved. Keywords: Ground black pepper; Salmonella Rubislaw; Water activity; Storage temperature
1. Introduction Spices are plants or parts of plants that have been used as flavouring agents in food preparation for thousands of years. Spices may reach consumers presenting poor quality, due to the loss of volatile compounds, microbial contamination (Baxter & Holzapfel, 1982; Pafumi, 1986) or even due to insect infestation (Germano & Germano, 1998; Santos et al., 1999). Black pepper is a highly appreciated spice (Germano & Germano, 1998) and Brazil is one of the largest producers and exporters of it in the world (International
*
Corresponding author. Tel.: +55 011 3068 2932; fax: +55 011 3085 3505. E-mail address: [email protected] (C.A. Ristori). 0956-7135/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2005.10.015
Pepper Community, 2003). In Brazil, Para´ State is the leading producer and exporter, followed by States of Espı´rito Santo and Bahia (Ministe´rio da Agricultura e do Abastecimento, 2001). Spices may contain microbial contaminants as a result of deficiencies in primary production conditions, such as fertilizing and planting, harvesting, drying, storage, transportation, and subsequent grinding and handling. Moreover, in tropical countries such as Brazil, constant high temperature and humidity may favor the development of microorganisms in fruit, seeds and food in general. Salmonella spp. frequently contaminate ground black pepper. This bacterium is a public health problem, with important economic implications, for it affects the international trade of black pepper (D’Aoust, 1994; Gustavsen & Breen, 1984; Santos et al., 1999; Satchell, Bruce, Allen,
C.A. Ristori et al. / Food Control 18 (2007) 268–272
Andrews, & Gerber, 1989). Furthermore, the presence of Salmonella in spices is a risk, since they are frequently added to ready-to-eat foods, without further cooking. Salmonella spp. may be recovered from products with low levels of free water, even after they have been submitted to high temperatures, which may be a result of post-processing contamination or of the ability of Salmonella to survive heat treatment, when the water activity of the product is low (Goepfert, Iskander, & Amundson, 1970; Riemann, 1968). One of the effects of Aw below the optimum for the development of a given bacterium is the increase in the lag phase, a decrease in the rate of multiplication, and a smaller final population. Besides Aw, other factors also affect adaptation and multiplication of bacteria, such as pH, temperature and oxidation–reduction potential (Jay, 1992). In general, Gram-negative bacteria require a higher Aw for their development than Gram-positive bacteria (Jay, 1992). The minimum Aw for Salmonella development is 0.93. However, as already noted above, there may be other factors affecting bacterial behavior (Varnan & Evans, 1991, chapter 4). In addition to Aw, for instance, thermoresistance is another important factor in the study of bacterial survival. Some authors, such as Varnan and Evans (1991, chapter 4), report Salmonella destruction at 74 C, with exposure time ranging from 0.2 to 6.5 min. But several studies on the thermoresistance of this bacterium demonstrate that this value is higher when the Aw of the medium is lower (Archer, Jervis, & Gaze, 1998; Summer, Sandros, Harmon, Scott, & Bernard, 1991). Although the relationship between Aw and thermoresistance is known, the survival of Salmonella in a low Aw product, which would limit the development of the bacterium, has been little studied, as well as its association with different storage temperatures before thermal treatment. Therefore, the contamination of an ingredient with reduced Aw, such as spices, may vary according to storage at different temperatures and heat treatment. Mattick, Jorgensen, Legan, Lappin-Scott, and Humphrey (2000) demonstrated that the previous exposure of Salmonella spp. to reduced Aw levels may reduce the efficiency of the subsequent thermal process, which may vary according to the solute. The objective of this trial was to evaluate the behavior of a Salmonella Rubislaw strain on ground black pepper (Piper Nigrum L.), as well as the functions of water activity (Aw) and storage temperature.
2. Material and methods 2.1. Samples Samples of ground black pepper (Piper Nigrum L.), traded in 50 and 500 g packs, were purchased in local supermarkets in the city of Sa˜o Paulo.
269
2.2. Preparation of the strain A S. Rubislaw strain, isolated from a pepper sample at the Food Microbiology Laboratory at Instituto Adolfo Lutz, was used. This serotype was the most frequent one isolated from this kind of sample at this Laboratory. The strain was stored on nutrient agar, at room temperature. A culture of the strain in the stationary phase was obtained with stock strain transferring to brain heart infusion broth (Merck KGaA 64271 Darmstadt, Germany) and incubated at 35 C for 18–24 h. 2.3. Preparation and storage of the samples The samples were previously analyzed for the presence of Salmonella. Samples were prepared in 500 mL sterile glass flasks, with 40 g of ground black pepper samples. Distilled water was added in the proportion described below (item 2.3.1) in order to adjust Aw. Samples were mixed with a sterile glass rod, for at least 60 s. After being mixed, a buffered peptone water (BPW, Merck KGaA 64271 Darmstadt, Germany) suspension of cells from a stationary phase S. Rubislaw culture (containing an average 109 viable cells/ mL) was added. After adding the culture, the product was mixed again in order to uniformly distribute the contaminant over the sample. Storage temperatures were 5, 25 and 35 C. Each trial (three temperatures for each Aw level) was performed in triplicate. Salmonella were quantified immediately after incorporation in the sample, and after 2 and 15 days of storage at the different temperatures. 2.3.1. Aw determination in the product Aw of each sample (flask) was determined in a water activity analyzer (CX-2 Model, from Aqualab), calibrated for each measurement. Aw levels, after the addition of the liquid to the samples, were: 0.663, 0.772, 0.815, 0.859, 0.887, 0.937. Aw was measured before producing the aliquots (original packaging), after the preparation of each flask, and after 15 days of storage at each temperature range. Contamination and Aw levels in each sample were obtained as described below: Aw 0.663: 40 g pepper + 0.2 mL of the contaminant, Aw 0.772: 40 g pepper + 0.2 mL of the contaminant + 1 mL sterile distilled water, Aw 0.815: 40 g pepper + 0.3 mL of the contaminant + 1 mL sterile distilled water, Aw 0.859: 40 g pepper + 0.2 mL of the contaminant + 3 mL sterile distilled water, Aw 0.887: 40 g pepper + 0.3 mL of the contaminant + 3 mL sterile distilled water, Aw 0.937: 40 g pepper + 0.3 mL of the contaminant + 6 mL sterile distilled water. Note: Contaminant = 10 1 dilution of the stationary phase culture of S. Rubislaw.
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2.4. Quantification of the contaminant (S. Rubislaw)
Temperature 5ºC 5.0
0.663
4.0
0.772
3.0
0.815
2.0
0.859
1.0
0.887
0.0 Initial
2 days Time
6.0 5.0 4.0 3.0 2.0 1.0 0.0
0.663 0.772 0.815 0.859 0.887 Initial
R2: Reduction (Log) of S. Rubislaw in the sample, from the day 0 to day 2 samples. R15: Reduction (Log) of S. Rubislaw in the sample, from the day 0 to day 15 samples. The effects of reduction (variable determined by two dependent measurements: R2 and R15) were tested, along with its interactions with other factors (Aw and T C), as well as the main effects of these factors. The significance level adopted (a) was 0.05. Data analysis was performed with Microsoft Excel 97 and SPSS for Windows 10.
0.937
Temperature 25ºC
3. Statistical analysis
2 days Time
0.937
15 days
Fig. 2. Mean profiles of variable response for the temperature of 25 C.
Temperature 35ºC Log10 MPN/g S. Rubislaw
Microbiological data were converted to Log10 MPN/g before being analyzed. Values for the mean Log10 and standard deviation of each set of S. Rubislaw MPN were calculated. Repeated variance analysis measurements (Neter, Kutner, Nachtsheim, & Wasserman, 1996) and Bonferroni multiple comparisons were the statistical techniques used to compare the mean Log10 reduction of S. Rubislaw values under the different conditions. The variable observed in the trial is the Response: the Log MPN/g value found for S. Rubislaw (used to contaminate the sample) at three distinct times in the storage period (initial, 2 and 15 days). For data analysis, the following variables were created:
15 days
Fig. 1. Mean profiles of variable response for the temperature of 5 C.
Log10 MPN/g S. Rubislaw
The most probable number (MPN) technique was used, according to Peeler, Houghtby, and Rainosek (1992). Culture media for isolation were those recommended by ISO, 2002. For quantification, 10 g of the sample were withdrawn from each flask. Each aliquot was homogenized in 90 mL of BPW. From this homogenous suspension, which corresponded to a 10 1 dilution, other decimal dilutions were obtained, up to 10 6, using BPW as diluent. Dilutions (1 mL of each) were inoculated into a series of three tubes, containing 10 mL of BPW and were incubated at 35 C/ 24 h. After incubation, tubes that were turbid were streaked onto Salmonella-Shigella agar and Brilliant Green agar (Oxoid Ltd., Basingstoke, Hampshire, England), both incubated at 35 C/24 h. Characteristic Salmonella colonies were inoculated in IAL presumptive medium for enterobacteria (Pessoa & Silva, 1972), incubated at 35 C/24 h, for biochemical confirmation.
Log10 MPN/g S. Rubislaw
6.0
7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
0.663 0.772 0.815 0.859 0.887 Initial
2 days Time
0.937
15 days
Fig. 3. Mean profiles of variable response for the temperature of 35 C.
Table 1 Repeated variance analysis measurements Source
Reduction Reduction * Aw Reduction * temperature Reduction * Aw * temperature Error (reduction)
Tests of within-subjects SS
df
MS
F
Sig.
25.163 1.733 0.552 3.255 9.186
1 5 2 10 35
25.16 0.347 0.276 0.325 0.262
95.87 1.321 1.051 1.240
0.000 0.278 0.360 0.301
Tests of between-subjects effects
4. Results The behavior of the variable response (Log10 of the initial S. Rubislaw value), in connection with storage time, storage temperature and water activity are showing in Figs. 1–3. The mean Aw value for the pepper in the original packaging was 0.647.
Aw Temperature Aw * temperature Error SS—sum of squares. MS—mean squares.
df
MS
F
5 2 10 35
4.918 64.921 2.212 1.236
3.980 52.534 1.790
Sig. 0.006 0.000 0.099
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Reduction
Mean
Std. error
95% confidence interval Lower bound
Upper bound
0.663, the least reduction was observed, both at 35 and 25 C; in an Aw equal to 0.887 and storage at these temperatures, the greatest reductions occurred (Table 2). Although Salmonella does not grow in Aw below 0.93 (Varnan & Evans, 1991, chapter 4), it may survive for long periods (Sperber, 1983) under these conditions. Considering the data obtained in this study, we may conclude that after contamination S. Rubislaw remains viable in pepper for up to 15 days. When the product is stored at 5 C, the number of surviving cells is even greater. In this trial, tests were performed with only one strain of S. Rubislaw. The behavior of other strains and serotypes may be different in relation to sensitivity to natural compounds and to the behavior reported here, using ground black pepper and considering the parameters tested (Doesburg, Lamprecht, & Elliott, 1970). We may conclude that there is a significant reduction in counts of the S. Rubislaw strain used to contaminate the samples of ground black pepper and that this reduction is more influenced by storage temperature than by low Aw. Moreover, it should be emphasized that results obtained here demonstrate the importance of the use of good manufacturing practices in the production of ground black pepper, since the products a low Aw alone may not efficiently prevent the survival of the bacterium.
1 2
2.080 3.058
0.117 0.122
1.842 2.812
2.318 3.305
References
Table 2 Estimates for the mean reduction Aw
Mean
Std. error
95% confidence interval Lower bound
Upper bound
0.663 0.772 0.815 0.859 0.887 0.937
1.994 2.611 2.225 2.406 3.517 2.661
0.262 0.262 0.283 0.262 0.262 0.262
1.463 2.079 1.650 1.874 2.985 2.129
2.526 3.143 2.800 2.937 4.049 3.193
Table 3 Estimates for the mean reduction for temperature Temperature (C)
Mean
Std. error
95% confidence interval Lower bound
Upper bound
5 25 35
1.197 2.572 3.938
0.185 0.185 0.193
0.821 2.196 3.546
1.573 2.948 4.329
Table 4 Estimates for the mean reduction for R2 (reduction 1) and R15 (reduction 2)
There was no interaction between the factors (Table 1): reduction · temperature · Aw (P = 0.301), reduction · Aw (P = 0.278), reduction · temperature (P = 0.360), Aw · temperature (P = 0.099). However, the variance analysis demonstrated that the main effects of Aw, temperature and reduction, in relation to the initial Log10 value of S. Rubislaw, are highly significant (P’s < 0.05). Tables 2–4 present the estimates of mean reduction for each level of the factors under study. Bonferroni multiple comparisons tests showed significant differences in relation to the levels of the factors temperature and reduction (P’s = 0.000); regarding the Aw comparisons, only: 0.663 · 0.887; 0.815 · 0.887 presented significant differences (P’s = 0.05). 5. Discussion Juven et al. (1984), when studying the survival of Salmonella spp. in dry products, including animal feed, reported that it was greater at Aw 0.43 and 0.52 than at 0.75. They also found that survival varied among different serotypes. Data obtained in the present trial, however, indicate that the survival of S. Rubislaw in ground black pepper was mainly associated with storage temperature, for the least reduction in counts was observed when pepper was stored at 5 C (Table 3). Differences in the reduction in counts were observed in relation to Aw; however, they were smaller than those observed in relation to temperature. In Aw
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