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Inactivation of Salmonella Typhimurium in fresh vegetables using water-assisted microwave heating
Food Control 26 (2012) 19e22
Contents lists available at SciVerse ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Short communication
Inactivation of Salmonella Typhimurium in fresh vegetables using water-assisted microwave heating B. De La Vega-Miranda a, N.A. Santiesteban-López b, A. López-Malo a, M.E. Sosa-Morales a, * a b
Departamento de Ingeniería Química, Alimentos y Ambiental, Universidad de las Américas Puebla, Ex-Hacienda Santa Catarina Mártir, Cholula 72810, Puebla, Mexico Facultad de Administración (Gastronomía), Benemérita Universidad Autónoma de Puebla, Av. San Claudio y 20 Sur, Edificio J, Ciudad Universitaria, 72420 Puebla, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 September 2011 Received in revised form 21 December 2011 Accepted 3 January 2012
A water-assisted microwave treatment was studied against the pathogenic bacterium Salmonella Typhimurium in fresh jalapeño peppers and coriander foliage. Vegetables were immersed in water and treated in a microwave oven at 950 W to reach up 63 C: jalapeño pepper for 25 s and coriander foliage for 10 s. After the microwave heating, samples were cooled in water at 4 C. Samples were observed with confocal microscope before and after treatment. The proposed protocols resulted in a reduction of 4e5 log cycles on the Salmonella population, which is the main issue from the microbiological viewpoint. Even color of the vegetables was affected by the treatments (p < 0.05) mainly by darkening in both vegetables and loss of greenness in jalapeño pepper, no changes in firmness were observed. Sensory acceptance of a salsa formulated with the treated vegetables had high scores (7.21 in a 9-points hedonic scale). Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Salmonella Microwave treatment Jalapeño pepper Coriander foliage
1. Introduction Jalapeño pepper (Capsicum annuum L.) is a spicy green vegetable used widely in Mexican food. Coriander leaves or cilantro (Coriandrum sativum L.) is other green vegetable widely used in Mexican cuisine, also in some Italian and Oriental recipes. On June 2008, the FDA (2008) reported a Salmonella outbreak in the U.S. caused by a Mexican salsa containing fresh tomato, coriander leaves and jalapeño pepper. Enteric bacteria may contaminate vegetables, depending on water quality for irrigation, post-harvest handling and conditions during food preparation (Abougrain, Nahaisi, Madi, Saied, & Ghenghesh, 2010), and coriander leaves may have substantial amounts of microorganisms, such as thermophiles, enterobacteriaceae, Pseudomonas spp. and molds (Mitkowska, Hickey, Alonso-Gomez, & Wilkinson, 2011). Salmonella is a Gramnegative and facultative anaerobic bacterium, which contaminates many foods. In the industry, Salmonella is inactivated by applying thermal treatments during food processing. For raw products, some disinfectant treatments in domestic or industrial kitchens are carried out, including application of ozone, chlorine or iodine solutions
* Corresponding author. Tel.: þ52 222 229 2126; fax: þ52 222 229 2727. E-mail address: [email protected] (M.E. Sosa-Morales). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.01.002
(Mossel & Garcia, 2003). However, and due to the reported outbreaks, it is possible that current disinfecting procedures are not being properly used or are not enough to inhibit the bacteria. Other proposed treatment for disinfesting coriander leaves was reported by Kamat, Pingulkar, Bhushan, Gholap, and Thomas (2003), throughout application of low dose gamma irradiation from 1 to 3 kGy; the treatment was effective against pathogenic bacteria (Listeria spp. and Yersinia spp.). In the case of jalapeño peppers, immersion in solutions such as 200 ppm of sodium hypochlorite, acidified sodium chlorite, or peroxy acetic acid for 10 min, could reduce the population of Salmonella Saintpaul in 1.5e1.7 log units for stem/calyx by and 2.1 to 2.4 log units for the flesh of the peppers (Liao, Cooke, & Niemira, 2010). An easy thermal treatment could be applied to these vegetables in many facilities using a domestic microwave oven, since this kind of ovens are very popular. Microwaves have been used as a blanching method in herbs like marjoram and rosemary resulting in higher quality herbs compared with to water and steam blanching methods (Singh, Raghavan, & Abraham, 1996). Microwaves have not been used for disinfecting purposes in coriander foliage, just for drying of the herb (Sarimeseli, 2011; Shaw, Meda, Tabil, & Opoku, 2007), neither applied to jalapeño peppers. Thus, the objective of this study was to develop microwave treatments in order to have safe products without affecting their physical and sensory characteristics.
20
B. De La Vega-Miranda et al. / Food Control 26 (2012) 19e22
2. Materials and methods
2.5. Physical-chemical and sensory analyses
2.1. Vegetables
Color for both jalapeño pepper and coriander foliage was measured by triplicate with a colorimeter using the CIELab scale (Sarimeseli, 2011). Our colorimeter (BYK Gardner, System/05, Reston VA, USA) was previously calibrated with the black and white tiles, and expressed through the L* (lightness), a* (green-red contribution) and b* (blue-yellow contribution) parameters. For jalapeño pepper firmness, an aluminum 60 cone moving at 1 mm/ s was used to penetrate 5 mm the skin in a whole piece. For coriander, foliage (leaves and stem) were stacked and rolled to form a 2.5 cm thick roll. Samples were cut 50% of their thickness (Bourne, 1982) using a stainless steel cutting wire moving at 2 mm/s. A Texture Analyzer TA.XT2 (Stable Micro Systems, Haslemere, England) was used to measure the maximum force (N) for both vegetables. Texture is a complicated measurement, due to the high variation between samples, even coming from the same batch. To avoid high deviations in firmness determination in vegetables, some authors recommend to analyze around 20 samples (Horak et al., 2006). 16 samples of each vegetable (jalapeño pepper or coriander) were used in our study. Samples of Mexican salsa were prepared with 60% tomato, 20% white onion, 10% jalapeño pepper and 10% coriander leaves. One sample contained both jalapeño pepper and coriander treated with microwaves (non-inoculated with Salmonella) and other, a control, was formulated with non-treated vegetables. An affective test with hedonic scale (Horak et al., 2006; Larmond, 1976; Tochampa, Jittrepotch, Kongbangkerd, Kraboun, & Rojsuntornkitti, 2011) was applied to 20 non-trained judges, tasting the two salsas, to evaluate their general acceptance.
Medium-size jalapeño peppers (C. annuum L.) between 19 and 25 g and coriander foliage (C. sativum L.) were acquired in a local market (Puebla, Mexico). Similar and desirable color, texture and overall appearance features were found in both raw products. Jalapeño pepper had initial color 30.4 0.6, 8.8 0.2, 9.1 0.5 in L*, a*, b* scale, and firmness of 10.9 1.3 N; coriander foliage had initial color 25.9 0.1, 5.6 0.1, 7.1 0.2 in L*, a*, b* scale, and firmness of 6.1 1.5 N. 2.2. Salmonella inoculum preparation Inoculum preparation was made by triplicate, prior to the microwave treatment. For that, one loopful of Salmonella Typhimurium ATCC 14028 strain was inoculated in 100 mL of triptycase soy broth, TSB (Becton Dickinson de México, Cuatitlán Izcalli, Estado de México) for 24 h at 35 2 C. A medium size jalapeño pepper or 9 g of coriander foliage were immersed in a liter of distilled water with 1 mL of the TSB inoculated with Salmonella and it was let stand for 15 min to reach a high population of the bacteria. The vegetables were drained under sterile conditions. In order to verify the initial inoculum in the vegetables, as well as to count the viable cells of Salmonella after treatments, 10 g of vegetable was homogenized with 90 mL of peptone water (1% p/v); serial dilutions were made and plated on SalmonellaeShigella agar (Becton Dickinson de México, Cuatitlán Izcalli, Estado de México). Plates were incubated (24 h at 35 C) and counted. Sampling, plating and counting was made by duplicate.
2.6. Data analysis 2.3. Confocal image analysis Cuts of about 20 mm of the peduncle and the surface of the jalapeño pepper and coriander leaves were obtained. 500 mL of a 1:5000 solution of propidium iodide was employed as a dye. After 1 min, the samples were washed 3 times with distilled water and transferred to a slide. A drop of mounting medium for fluorescence (Vectashield, H-1000, Vector Laboratories, USA) was added to the samples; then, they were covered with a slide and observed with a confocal microscope (LSM5 Pascal, Axioscope 2MOT, Zeiss, Germany) at wavelengths of 532 and 543 nm. Images were built in two dimensions. 2.4. Microwave treatments Output power of the microwave oven used for the study (1200 W, 2450 MHz, Panasonic, China) was determined following the method 60705 IEC (Martin, 2008). Temperatures were monitored with optical fiber sensors and FISO Commander software (FISO Technologies, Quebec, Canada). The temperature was measured by introducing the sensor just under the skin, parallel to the surface of the jalapeño body or the coriander stem. One jalapeño pepper (19e25 g) was immersed in distilled water in a 1:1 pepper:water proportion; for cilantro, 9 g were immersed in 1:1.5 coriander: water proportion. A Pyrex glass cylindrical container was used for the treatments, which was previously sterilized. Temperature was monitored till reach up 63 C in the samples. This value has been reported as the target temperature for Salmonella inactivation (FDA, 2008). In order to have a short treatment time, the maximum power was applied. After heat treatment, samples were cooled in cold water at 4 C for 10 min. Protocols, which include microwave heat treatment to reach 63 C and cooling for every studied vegetable, were made by triplicate.
Results were analyzed through an analysis of variance (ANOVA) using a statistical software Minitab Release 14.0 software (Minitab Inc., State College, PA, USA); differences were established at the significant level of P ¼ 0.05. 3. Results and discussion After inoculation, average Salmonella population was 3 108 CFU/g in both vegetables, reaching up a level to simulate a severe microbial contamination. Fig. 1 shows the temperature profiles in the sample, for jalapeño pepper and coriander, while they were treated at 950 W (the
Fig. 1. Average temperature profiles during the water-assisted microwave treatment for jalapeño pepper and coriander. Temperatures were acquired by triplicate.
B. De La Vega-Miranda et al. / Food Control 26 (2012) 19e22
21
Table 1 Color and firmness of jalapeño pepper and coriander foliage before and after the proposed protocol (water-assisted microwave treatment followed by cooling). Vegetable
Jalapeño pepper Coriander
L*
a*
b*
Firmness (N)
Before protocol
After protocol
Before protocol
After protocol
Before protocol
After protocol
Before protocol
After protocol
30.48 0.65 25.98 0.19
24.35 0.64 19.61 0.06
8.51 0.57 5.66 0.18
5.67 0.47 5.99 0.09
8.28 0.51 7.18 0.19
5.75 0.60 5.68 0.09
10.93 1.33 6.17 1.58
9.29 1.92 8.21 1.58
L*, a* and b* were obtained by triplicates. Firmness was measured in 16 samples.
calculated output power). Time to reach up 63 C in the sample was 25 s for jalapeño pepper and 10 s for coriander foilage. Larger microbial logarithmic cycle reductions were obtained for jalapeño pepper (5.12) compared with coriander samples (4.45) after the applied protocol (microwave heating þ cooling). This inactivation is enough to consider the applied protocol as a disinfection treatment for jalapeño pepper, as 5 log cycles reduction was achieved. This means that a microbiological safe product, respect to Salmonella presence, can be obtained by a simple microwave treatment. Application of microwave heating in other foods has been reported, resulting in important reduction of pathogenic bacteria, such as Listeria monocytogenes in shrimps (Gundacarapu, Hung, Brackett, & Mallikarjunan, 1995) or in fresh cheeses (Diaz-Escobar, SosaMorales, & Lopez-Malo, 2008). For coriander foliage, other conditions should be tested to reach larger inactivation, for example, the
addition of some drops of colloidal silver solutions during cooling, commercially available for vegetable disinfesting purposes. Color was significantly affected by the treatment (p < 0.05), as it is shown in Table 1. Lightness (L*) decreased from 30.38 before the treatment to 24.35 after microwave heating for jalapeño pepper, indicating a darker aspect. a* parameter increased (from 8.51 to 5.67), which implicates a reduction in the green component. For coriander, color became darker too (L* decreased from 25.98 to 19.61), but greenness was not affected by the microwave treatment remaining in similar values (5.66 and 5.99, before and after the treatment, respectively). Firmness of both jalapeño pepper and coriander remained without significant changes (p > 0.05), which means an advantage of the proposed treatment (Table 1). Firmness of jalapeño pepper ranged between 9.29 and 10.93 N, with lower values after treatment. On the other hand, before the treatment of
Fig. 2. Two-dimensional confocal microscopy images of fresh jalapeño pepper (a), inoculated with Salmonella and microwave-treated pepper (b); fresh coriander leaves (c) and inoculated with Salmonella and microwave-treated coriander leaves (d). Magnification: 63.
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B. De La Vega-Miranda et al. / Food Control 26 (2012) 19e22
coriander, the firmness was evaluated as 6.17 N, and a slight higher value after treatment was measured, being 8.21 N, even it was not significant as it was commented previously. This apparent increase could be related to a turgency loss, which results in higher force to cut the roll of coriander foliage. Fig. 2 shows different images from confocal microscopy. For jalapeño pepper, the aspect of fresh cells at 532 nm is illustrated in Fig. 2a; Salmonella aggregates remained adhered at the cell wall of the vegetable after the protocol (microwave heating þ cooling), identified as red spots in Fig. 2b (532 nm). Pepper cells morphology did not change in shape after the treatment, but a thin separation between them was observed, seeming smaller in size. Regarding coriander leaves, fresh tissues had irregular shapes, some of them with oval appearance, and showed a native fluorescence at 532 nm (Fig. 2c), with some reddish/oranges lines or spots. When inoculated and treated coriander leaves cells were observed at 543 nm (Fig. 2d), the Salmonella colonies were more fluorescent and located in the peripheral region of the coriander cells, like the observed in the oval shapes. These images confirm the adherence and presence of Salmonella in the treated vegetables, thus, bacteria really infested the vegetables. Adhesion and penetration are typical mechanisms of pathogenic microorganisms. Three basic steps are observed during the infection process of Salmonella: ability to enter the cell, establishment of an adequate habitat/niche in the cell, and expansion of bacteria out of the cell; entry and survival within the cell are critical stages for pathogens (Knodler et al., 2010). Liao et al. (2010) also reported microscopy, stereofluorescence imaging or scanning electron microscopy, as useful techniques to detect the presence of Salmonella in jalapeño peppers, located mostly in stem and calyx. From the sensory evaluation, Mexican salsa containing nontreated vegetables obtained high scores for overall acceptance (7.27 1.51), and salsa containing both jalapeño pepper and coriander treated with microwaves received scores of 7.21 1.18, without difference between the salsas (p > 0.05). Thus, the microwave treatment did not affect the sensory perception of the vegetables cut and integrated to the salsa, perhaps as they were combined with other ingredients and the judge is unable to appreciate the individual characteristics of the fresh vegetables. A panel with a bigger number of judges would help to elucidate possible differences in specific sensory attributes, such as odor, flavor or texture. 4. Conclusions A short thermal treatment for jalapeño pepper and coriander foliage using a domestic microwave oven could be used in kitchens and restaurants by consumers or chefs to prevent bacterial infections, reducing the Salmonella population in 4e5 log cycles. Treated vegetables had similar firmness and sensory acceptance than those non-treated items. Further studies are recommended to study the
effect of the treatment in chlorophyll or individual aroma/flavor compounds in the vegetables. But, the reduction of Salmonella in the vegetables is even more important from the microbiological safety point, and that objective was reached. Acknowledgments Authors thank the financial support from CONACyT (Consejo Nacional de Ciencia y Tecnología, Mexico), Project 084859. References Abougrain, A. K., Nahaisi, M. H., Madi, N. S., Saied, M. M., & Ghenghesh, K. S. (2010). Parasitological contamination in salad vegetables in Tripoli-Libya. Food Control, 21, 760e762. Bourne, M. C. (1982). Food texture and viscosity: Concept and measurement. New York: Academic Press. Diaz-Escobar, G.E., Sosa-Morales, M.E., & Lopez-Malo, A. (2008). Listeria innocua inhibition during microwave heating on fresh cheeses from cow’s milk and from goat’s milk. Proceedings of the IMPI 42nd Symposium. New Orleans, LO. FDA Food and Drug Administration US. (2008). Salmonella Saintpaul outbreak. http://www.fda.gov/oc/opacom/hottopics/tomatoes.html Available at. Gundacarapu, S., Hung, Y., Brackett, R., & Mallikarjunan, P. (1995). Evaluation of microbiological safety of shrimp cooked in a microwave oven. Journal of Food Protection, 58(7), 742e747. Horak, C. I., Pietranera, M. A., Malvicini, M., Narvaiz, P., Gonzalez, M., & Kairiyama, E. (2006). Improvement of hygienic quality of fresh, pre-cut, ready-to-eat vegetables using gamma irradiation. In Proceedings of Nuclear techniques in food and Agriculture. Islamabad, Pakistan, 22e30 July 2005 (pp. 23e40). Vienna, Austria: International Atomic Energy Agency. Kamat, A., Pingulkar, K., Bhushan, B., Gholap, A., & Thomas, P. (2003). Potential application of low dose gamma irradiation to improve the microbiological safety of fresh coriander leaves. Food Control, 12, 529e537. Knodler, L. A., Vallance, B. A., Celli, J., Winfree, S., Hansen, B., Montero, M., et al. (2010). Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia. Proceedings of the National Academy of Sciences of the United States of America, 107(41), 17733e17738. Larmond, E. (1976). Laboratory methods for sensory evaluation of food. Publication 1637. Ottawa: Department of Agriculture of Canada. Liao, C.-H., Cooke, P. H., & Niemira, B. A. (2010). Localization, growth, and inactivation of Salmonella Saintpaul on jalapeño peppers. Journal of Food Science, 75(6), M377eM382. Martin, G. (2008). Factors affecting microwave power. Proceedings of the IMPI 42nd Symposium. New Orleans, LO. Mitkowska, A. M., Hickey, D. K., Alonso-Gomez, M., & Wilkinson, M. G. (2011). The microbiological quality of commercial herb and spice preparations used in the formulation of a chicken supreme ready meal and microbial survival following a simulated industrial heating process. Food Control, 22, 616e625. Mossel, D. A. A., & Garcia, B. (2003). Microbiología de los Alimentos (3rd ed.). Zaragoza, Spain: Acribia. (In Spanish). Sarimeseli, A. (2011). Microwave drying characteristics of coriander (Coriandrum sativum L.) leaves. Energy Conversion and Management, 52, 1449e1453. Shaw, M., Meda, V., Tabil, L., Jr., & Opoku, A. (2007). Drying and color characteristics of coriander foliage using convective thin-layer and microwave drying. Journal of Microwave and Electromagnetic Energy, 41(2), 59e68. Singh, M., Raghavan, B., & Abraham, K. O. (1996). Processing of marjoram (Majorana hortensis Moench.) and rosemary (Rosmarinus officinalis L.). Effect on blanching methods on quality. Nahrung/Food, 40(5), 264e266. Tochampa, W., Jittrepotch, N., Kongbangkerd, T., Kraboun, K., & Rojsuntornkitti, K. (2011). The study of microwave heating time on chemical and microbiological properties and sensory evaluation in sweet fermented glutinous rice (KhaoMark). International Food Research Journal, 18, 239e248.
Contents lists available at SciVerse ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Short communication
Inactivation of Salmonella Typhimurium in fresh vegetables using water-assisted microwave heating B. De La Vega-Miranda a, N.A. Santiesteban-López b, A. López-Malo a, M.E. Sosa-Morales a, * a b
Departamento de Ingeniería Química, Alimentos y Ambiental, Universidad de las Américas Puebla, Ex-Hacienda Santa Catarina Mártir, Cholula 72810, Puebla, Mexico Facultad de Administración (Gastronomía), Benemérita Universidad Autónoma de Puebla, Av. San Claudio y 20 Sur, Edificio J, Ciudad Universitaria, 72420 Puebla, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 September 2011 Received in revised form 21 December 2011 Accepted 3 January 2012
A water-assisted microwave treatment was studied against the pathogenic bacterium Salmonella Typhimurium in fresh jalapeño peppers and coriander foliage. Vegetables were immersed in water and treated in a microwave oven at 950 W to reach up 63 C: jalapeño pepper for 25 s and coriander foliage for 10 s. After the microwave heating, samples were cooled in water at 4 C. Samples were observed with confocal microscope before and after treatment. The proposed protocols resulted in a reduction of 4e5 log cycles on the Salmonella population, which is the main issue from the microbiological viewpoint. Even color of the vegetables was affected by the treatments (p < 0.05) mainly by darkening in both vegetables and loss of greenness in jalapeño pepper, no changes in firmness were observed. Sensory acceptance of a salsa formulated with the treated vegetables had high scores (7.21 in a 9-points hedonic scale). Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Salmonella Microwave treatment Jalapeño pepper Coriander foliage
1. Introduction Jalapeño pepper (Capsicum annuum L.) is a spicy green vegetable used widely in Mexican food. Coriander leaves or cilantro (Coriandrum sativum L.) is other green vegetable widely used in Mexican cuisine, also in some Italian and Oriental recipes. On June 2008, the FDA (2008) reported a Salmonella outbreak in the U.S. caused by a Mexican salsa containing fresh tomato, coriander leaves and jalapeño pepper. Enteric bacteria may contaminate vegetables, depending on water quality for irrigation, post-harvest handling and conditions during food preparation (Abougrain, Nahaisi, Madi, Saied, & Ghenghesh, 2010), and coriander leaves may have substantial amounts of microorganisms, such as thermophiles, enterobacteriaceae, Pseudomonas spp. and molds (Mitkowska, Hickey, Alonso-Gomez, & Wilkinson, 2011). Salmonella is a Gramnegative and facultative anaerobic bacterium, which contaminates many foods. In the industry, Salmonella is inactivated by applying thermal treatments during food processing. For raw products, some disinfectant treatments in domestic or industrial kitchens are carried out, including application of ozone, chlorine or iodine solutions
* Corresponding author. Tel.: þ52 222 229 2126; fax: þ52 222 229 2727. E-mail address: [email protected] (M.E. Sosa-Morales). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.01.002
(Mossel & Garcia, 2003). However, and due to the reported outbreaks, it is possible that current disinfecting procedures are not being properly used or are not enough to inhibit the bacteria. Other proposed treatment for disinfesting coriander leaves was reported by Kamat, Pingulkar, Bhushan, Gholap, and Thomas (2003), throughout application of low dose gamma irradiation from 1 to 3 kGy; the treatment was effective against pathogenic bacteria (Listeria spp. and Yersinia spp.). In the case of jalapeño peppers, immersion in solutions such as 200 ppm of sodium hypochlorite, acidified sodium chlorite, or peroxy acetic acid for 10 min, could reduce the population of Salmonella Saintpaul in 1.5e1.7 log units for stem/calyx by and 2.1 to 2.4 log units for the flesh of the peppers (Liao, Cooke, & Niemira, 2010). An easy thermal treatment could be applied to these vegetables in many facilities using a domestic microwave oven, since this kind of ovens are very popular. Microwaves have been used as a blanching method in herbs like marjoram and rosemary resulting in higher quality herbs compared with to water and steam blanching methods (Singh, Raghavan, & Abraham, 1996). Microwaves have not been used for disinfecting purposes in coriander foliage, just for drying of the herb (Sarimeseli, 2011; Shaw, Meda, Tabil, & Opoku, 2007), neither applied to jalapeño peppers. Thus, the objective of this study was to develop microwave treatments in order to have safe products without affecting their physical and sensory characteristics.
20
B. De La Vega-Miranda et al. / Food Control 26 (2012) 19e22
2. Materials and methods
2.5. Physical-chemical and sensory analyses
2.1. Vegetables
Color for both jalapeño pepper and coriander foliage was measured by triplicate with a colorimeter using the CIELab scale (Sarimeseli, 2011). Our colorimeter (BYK Gardner, System/05, Reston VA, USA) was previously calibrated with the black and white tiles, and expressed through the L* (lightness), a* (green-red contribution) and b* (blue-yellow contribution) parameters. For jalapeño pepper firmness, an aluminum 60 cone moving at 1 mm/ s was used to penetrate 5 mm the skin in a whole piece. For coriander, foliage (leaves and stem) were stacked and rolled to form a 2.5 cm thick roll. Samples were cut 50% of their thickness (Bourne, 1982) using a stainless steel cutting wire moving at 2 mm/s. A Texture Analyzer TA.XT2 (Stable Micro Systems, Haslemere, England) was used to measure the maximum force (N) for both vegetables. Texture is a complicated measurement, due to the high variation between samples, even coming from the same batch. To avoid high deviations in firmness determination in vegetables, some authors recommend to analyze around 20 samples (Horak et al., 2006). 16 samples of each vegetable (jalapeño pepper or coriander) were used in our study. Samples of Mexican salsa were prepared with 60% tomato, 20% white onion, 10% jalapeño pepper and 10% coriander leaves. One sample contained both jalapeño pepper and coriander treated with microwaves (non-inoculated with Salmonella) and other, a control, was formulated with non-treated vegetables. An affective test with hedonic scale (Horak et al., 2006; Larmond, 1976; Tochampa, Jittrepotch, Kongbangkerd, Kraboun, & Rojsuntornkitti, 2011) was applied to 20 non-trained judges, tasting the two salsas, to evaluate their general acceptance.
Medium-size jalapeño peppers (C. annuum L.) between 19 and 25 g and coriander foliage (C. sativum L.) were acquired in a local market (Puebla, Mexico). Similar and desirable color, texture and overall appearance features were found in both raw products. Jalapeño pepper had initial color 30.4 0.6, 8.8 0.2, 9.1 0.5 in L*, a*, b* scale, and firmness of 10.9 1.3 N; coriander foliage had initial color 25.9 0.1, 5.6 0.1, 7.1 0.2 in L*, a*, b* scale, and firmness of 6.1 1.5 N. 2.2. Salmonella inoculum preparation Inoculum preparation was made by triplicate, prior to the microwave treatment. For that, one loopful of Salmonella Typhimurium ATCC 14028 strain was inoculated in 100 mL of triptycase soy broth, TSB (Becton Dickinson de México, Cuatitlán Izcalli, Estado de México) for 24 h at 35 2 C. A medium size jalapeño pepper or 9 g of coriander foliage were immersed in a liter of distilled water with 1 mL of the TSB inoculated with Salmonella and it was let stand for 15 min to reach a high population of the bacteria. The vegetables were drained under sterile conditions. In order to verify the initial inoculum in the vegetables, as well as to count the viable cells of Salmonella after treatments, 10 g of vegetable was homogenized with 90 mL of peptone water (1% p/v); serial dilutions were made and plated on SalmonellaeShigella agar (Becton Dickinson de México, Cuatitlán Izcalli, Estado de México). Plates were incubated (24 h at 35 C) and counted. Sampling, plating and counting was made by duplicate.
2.6. Data analysis 2.3. Confocal image analysis Cuts of about 20 mm of the peduncle and the surface of the jalapeño pepper and coriander leaves were obtained. 500 mL of a 1:5000 solution of propidium iodide was employed as a dye. After 1 min, the samples were washed 3 times with distilled water and transferred to a slide. A drop of mounting medium for fluorescence (Vectashield, H-1000, Vector Laboratories, USA) was added to the samples; then, they were covered with a slide and observed with a confocal microscope (LSM5 Pascal, Axioscope 2MOT, Zeiss, Germany) at wavelengths of 532 and 543 nm. Images were built in two dimensions. 2.4. Microwave treatments Output power of the microwave oven used for the study (1200 W, 2450 MHz, Panasonic, China) was determined following the method 60705 IEC (Martin, 2008). Temperatures were monitored with optical fiber sensors and FISO Commander software (FISO Technologies, Quebec, Canada). The temperature was measured by introducing the sensor just under the skin, parallel to the surface of the jalapeño body or the coriander stem. One jalapeño pepper (19e25 g) was immersed in distilled water in a 1:1 pepper:water proportion; for cilantro, 9 g were immersed in 1:1.5 coriander: water proportion. A Pyrex glass cylindrical container was used for the treatments, which was previously sterilized. Temperature was monitored till reach up 63 C in the samples. This value has been reported as the target temperature for Salmonella inactivation (FDA, 2008). In order to have a short treatment time, the maximum power was applied. After heat treatment, samples were cooled in cold water at 4 C for 10 min. Protocols, which include microwave heat treatment to reach 63 C and cooling for every studied vegetable, were made by triplicate.
Results were analyzed through an analysis of variance (ANOVA) using a statistical software Minitab Release 14.0 software (Minitab Inc., State College, PA, USA); differences were established at the significant level of P ¼ 0.05. 3. Results and discussion After inoculation, average Salmonella population was 3 108 CFU/g in both vegetables, reaching up a level to simulate a severe microbial contamination. Fig. 1 shows the temperature profiles in the sample, for jalapeño pepper and coriander, while they were treated at 950 W (the
Fig. 1. Average temperature profiles during the water-assisted microwave treatment for jalapeño pepper and coriander. Temperatures were acquired by triplicate.
B. De La Vega-Miranda et al. / Food Control 26 (2012) 19e22
21
Table 1 Color and firmness of jalapeño pepper and coriander foliage before and after the proposed protocol (water-assisted microwave treatment followed by cooling). Vegetable
Jalapeño pepper Coriander
L*
a*
b*
Firmness (N)
Before protocol
After protocol
Before protocol
After protocol
Before protocol
After protocol
Before protocol
After protocol
30.48 0.65 25.98 0.19
24.35 0.64 19.61 0.06
8.51 0.57 5.66 0.18
5.67 0.47 5.99 0.09
8.28 0.51 7.18 0.19
5.75 0.60 5.68 0.09
10.93 1.33 6.17 1.58
9.29 1.92 8.21 1.58
L*, a* and b* were obtained by triplicates. Firmness was measured in 16 samples.
calculated output power). Time to reach up 63 C in the sample was 25 s for jalapeño pepper and 10 s for coriander foilage. Larger microbial logarithmic cycle reductions were obtained for jalapeño pepper (5.12) compared with coriander samples (4.45) after the applied protocol (microwave heating þ cooling). This inactivation is enough to consider the applied protocol as a disinfection treatment for jalapeño pepper, as 5 log cycles reduction was achieved. This means that a microbiological safe product, respect to Salmonella presence, can be obtained by a simple microwave treatment. Application of microwave heating in other foods has been reported, resulting in important reduction of pathogenic bacteria, such as Listeria monocytogenes in shrimps (Gundacarapu, Hung, Brackett, & Mallikarjunan, 1995) or in fresh cheeses (Diaz-Escobar, SosaMorales, & Lopez-Malo, 2008). For coriander foliage, other conditions should be tested to reach larger inactivation, for example, the
addition of some drops of colloidal silver solutions during cooling, commercially available for vegetable disinfesting purposes. Color was significantly affected by the treatment (p < 0.05), as it is shown in Table 1. Lightness (L*) decreased from 30.38 before the treatment to 24.35 after microwave heating for jalapeño pepper, indicating a darker aspect. a* parameter increased (from 8.51 to 5.67), which implicates a reduction in the green component. For coriander, color became darker too (L* decreased from 25.98 to 19.61), but greenness was not affected by the microwave treatment remaining in similar values (5.66 and 5.99, before and after the treatment, respectively). Firmness of both jalapeño pepper and coriander remained without significant changes (p > 0.05), which means an advantage of the proposed treatment (Table 1). Firmness of jalapeño pepper ranged between 9.29 and 10.93 N, with lower values after treatment. On the other hand, before the treatment of
Fig. 2. Two-dimensional confocal microscopy images of fresh jalapeño pepper (a), inoculated with Salmonella and microwave-treated pepper (b); fresh coriander leaves (c) and inoculated with Salmonella and microwave-treated coriander leaves (d). Magnification: 63.
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coriander, the firmness was evaluated as 6.17 N, and a slight higher value after treatment was measured, being 8.21 N, even it was not significant as it was commented previously. This apparent increase could be related to a turgency loss, which results in higher force to cut the roll of coriander foliage. Fig. 2 shows different images from confocal microscopy. For jalapeño pepper, the aspect of fresh cells at 532 nm is illustrated in Fig. 2a; Salmonella aggregates remained adhered at the cell wall of the vegetable after the protocol (microwave heating þ cooling), identified as red spots in Fig. 2b (532 nm). Pepper cells morphology did not change in shape after the treatment, but a thin separation between them was observed, seeming smaller in size. Regarding coriander leaves, fresh tissues had irregular shapes, some of them with oval appearance, and showed a native fluorescence at 532 nm (Fig. 2c), with some reddish/oranges lines or spots. When inoculated and treated coriander leaves cells were observed at 543 nm (Fig. 2d), the Salmonella colonies were more fluorescent and located in the peripheral region of the coriander cells, like the observed in the oval shapes. These images confirm the adherence and presence of Salmonella in the treated vegetables, thus, bacteria really infested the vegetables. Adhesion and penetration are typical mechanisms of pathogenic microorganisms. Three basic steps are observed during the infection process of Salmonella: ability to enter the cell, establishment of an adequate habitat/niche in the cell, and expansion of bacteria out of the cell; entry and survival within the cell are critical stages for pathogens (Knodler et al., 2010). Liao et al. (2010) also reported microscopy, stereofluorescence imaging or scanning electron microscopy, as useful techniques to detect the presence of Salmonella in jalapeño peppers, located mostly in stem and calyx. From the sensory evaluation, Mexican salsa containing nontreated vegetables obtained high scores for overall acceptance (7.27 1.51), and salsa containing both jalapeño pepper and coriander treated with microwaves received scores of 7.21 1.18, without difference between the salsas (p > 0.05). Thus, the microwave treatment did not affect the sensory perception of the vegetables cut and integrated to the salsa, perhaps as they were combined with other ingredients and the judge is unable to appreciate the individual characteristics of the fresh vegetables. A panel with a bigger number of judges would help to elucidate possible differences in specific sensory attributes, such as odor, flavor or texture. 4. Conclusions A short thermal treatment for jalapeño pepper and coriander foliage using a domestic microwave oven could be used in kitchens and restaurants by consumers or chefs to prevent bacterial infections, reducing the Salmonella population in 4e5 log cycles. Treated vegetables had similar firmness and sensory acceptance than those non-treated items. Further studies are recommended to study the
effect of the treatment in chlorophyll or individual aroma/flavor compounds in the vegetables. But, the reduction of Salmonella in the vegetables is even more important from the microbiological safety point, and that objective was reached. Acknowledgments Authors thank the financial support from CONACyT (Consejo Nacional de Ciencia y Tecnología, Mexico), Project 084859. References Abougrain, A. K., Nahaisi, M. H., Madi, N. S., Saied, M. M., & Ghenghesh, K. S. (2010). Parasitological contamination in salad vegetables in Tripoli-Libya. Food Control, 21, 760e762. Bourne, M. C. (1982). Food texture and viscosity: Concept and measurement. New York: Academic Press. Diaz-Escobar, G.E., Sosa-Morales, M.E., & Lopez-Malo, A. (2008). Listeria innocua inhibition during microwave heating on fresh cheeses from cow’s milk and from goat’s milk. Proceedings of the IMPI 42nd Symposium. New Orleans, LO. FDA Food and Drug Administration US. (2008). Salmonella Saintpaul outbreak. http://www.fda.gov/oc/opacom/hottopics/tomatoes.html Available at. Gundacarapu, S., Hung, Y., Brackett, R., & Mallikarjunan, P. (1995). Evaluation of microbiological safety of shrimp cooked in a microwave oven. Journal of Food Protection, 58(7), 742e747. Horak, C. I., Pietranera, M. A., Malvicini, M., Narvaiz, P., Gonzalez, M., & Kairiyama, E. (2006). Improvement of hygienic quality of fresh, pre-cut, ready-to-eat vegetables using gamma irradiation. In Proceedings of Nuclear techniques in food and Agriculture. Islamabad, Pakistan, 22e30 July 2005 (pp. 23e40). Vienna, Austria: International Atomic Energy Agency. Kamat, A., Pingulkar, K., Bhushan, B., Gholap, A., & Thomas, P. (2003). Potential application of low dose gamma irradiation to improve the microbiological safety of fresh coriander leaves. Food Control, 12, 529e537. Knodler, L. A., Vallance, B. A., Celli, J., Winfree, S., Hansen, B., Montero, M., et al. (2010). Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia. Proceedings of the National Academy of Sciences of the United States of America, 107(41), 17733e17738. Larmond, E. (1976). Laboratory methods for sensory evaluation of food. Publication 1637. Ottawa: Department of Agriculture of Canada. Liao, C.-H., Cooke, P. H., & Niemira, B. A. (2010). Localization, growth, and inactivation of Salmonella Saintpaul on jalapeño peppers. Journal of Food Science, 75(6), M377eM382. Martin, G. (2008). Factors affecting microwave power. Proceedings of the IMPI 42nd Symposium. New Orleans, LO. Mitkowska, A. M., Hickey, D. K., Alonso-Gomez, M., & Wilkinson, M. G. (2011). The microbiological quality of commercial herb and spice preparations used in the formulation of a chicken supreme ready meal and microbial survival following a simulated industrial heating process. Food Control, 22, 616e625. Mossel, D. A. A., & Garcia, B. (2003). Microbiología de los Alimentos (3rd ed.). Zaragoza, Spain: Acribia. (In Spanish). Sarimeseli, A. (2011). Microwave drying characteristics of coriander (Coriandrum sativum L.) leaves. Energy Conversion and Management, 52, 1449e1453. Shaw, M., Meda, V., Tabil, L., Jr., & Opoku, A. (2007). Drying and color characteristics of coriander foliage using convective thin-layer and microwave drying. Journal of Microwave and Electromagnetic Energy, 41(2), 59e68. Singh, M., Raghavan, B., & Abraham, K. O. (1996). Processing of marjoram (Majorana hortensis Moench.) and rosemary (Rosmarinus officinalis L.). Effect on blanching methods on quality. Nahrung/Food, 40(5), 264e266. Tochampa, W., Jittrepotch, N., Kongbangkerd, T., Kraboun, K., & Rojsuntornkitti, K. (2011). The study of microwave heating time on chemical and microbiological properties and sensory evaluation in sweet fermented glutinous rice (KhaoMark). International Food Research Journal, 18, 239e248.