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Determination of the utility of ultraviolet-C irradiation for dried bay leaves microbial decontamination through safety and quality evaluations
LWT - Food Science and Technology 117 (2020) 108634
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Determination of the utility of ultraviolet-C irradiation for dried bay leaves microbial decontamination through safety and quality evaluations
T
Alonzo A. Gabriel∗, Katrina Moira D. Melo, Juan Carlos D. Michelena Laboratory of Food Microbiology and Hygiene, Department of Food Science and Nutrition, College of Home Economics, Alonso Hall, Antonio Ma. Regidor St., University of the Philippines Diliman, Quezon City, 1101, Philippines
A R T I C LE I N FO
A B S T R A C T
Keywords: Bay leaves Herbs Microbial inactivation Nonthermal processing UV-C irradiation
This study was conducted to determine the efficacy of UV-C irradiation for safety and quality of dried bay leaves. Cocktails of microorganisms of the same species were prepared and inoculated onto the leaves, which were thereafter subjected to UV-C irradiation. Results showed that all tested organisms, which included Escherichia coli O157:H7, Salmonella enterica, Pseudomonas aeruginosa, Listeria monocytogenes, and Staphylococcus aureus, exhibited inactivation behaviors in the irradiated leaves that are characterized by initial log-linear population decline, followed by inactivation tail. The Total Log Reductions in the organisms after exposure to 3942 mJ/cm2 UV-C dose ranged from 2.70 to 3.93 log CFU/g. Furthermore, subjecting the bay leaves to UV-C of as much as 13662 mJ/cm2 did not significantly alter color, and visual sensory properties, and did not result in mercury deposition. Microbiological quality indicators of the bay leaves, including Total Plate Count and Yeast and Molds Count were improved by the UV-C irradiation. These results contribute in further understanding the scope and limitations of using UV-C as a decontamination technique for specific food product such as dried bay leaves.
1. Introduction Spices and herbs have long been valued throughout the world for their various functions in food and medicine. Peter and Babu (2012) recounted that during ancient times, they were prized for their antimicrobial properties that aided in food preservation, and with the passing of time spices have been indispensable in the culinary arts due to their ability to enhance the flavor of food and drink. While they have no nutritional value, they are prized for specific sensory values, and can even exert beneficial effects on digestive processes (Garbowska, Berthold-Pluta, & Stasiak-Rózańska, 2015). Peter and Babu (2012) further explained that spices and herbs are terms that tend to be used interchangeably in daily conversation, but they are defined as two separate entities: spices are defined as the dried parts of aromatic plants other than the leaves, while herbs are defined as the dried leaves of aromatic plants. Despite this definition, the application remains the same. While not a natural growth medium for pathogenic microorganisms, several reports have shown that spices and herbs can serve as a vector for pathogenic microorganisms under the right conditions, usually related to poor adherence to GMP or due to the environment spices are exposed to (Banerjee & Sarkar, 2003; Zweifel & Stephan, 2012). Spices
such as anise seeds, basil, black pepper, cinnamon, chili, curry powder, and oregano were previously identified as causes of illness outbreaks in countries including Canada, Denmark, England and Wales, France, Germany, New Zealand, Norway, Serbia, and the United States (Sagoo et al., 2009; Van Doren et al., 2013; Zweifel & Stephan, 2012). McKee (1995) reported a 6.0 to 8.0 log CFU/g of microbial contamination levels in retail spices in various places in the world. Current methods for herbs and spices decontamination include physical means such as steam sterilization and gamma irradiation, and the use of chemicals such as ethylene oxide or propylene oxide (Song et al., 2014). However, these methods come with different disadvantages. Steam sterilization alters the chemical and organoleptic properties of spices and herbs due to its nature as a high temperature treatment process (Peter & Babu, 2012). Chemical treatments are sometimes associated with carcinogenic byproducts, and the relatively inert methods like gamma irradiation are expensive (Peter & Babu, 2012; Song et al., 2014). Thus, there is a need to find an alternative method to decontaminate spices, one that does not alter chemical and organoleptic properties of the spices, is not carcinogenic, and is relatively inexpensive. UV-C irradiation is one of the possible alternative methods for decontamination as it manages to address these concerns. Koutchma (2009) explained that the biocidal mechanism of UV-C is
∗ Corresponding author. Laboratory of Food Microbiology and Hygiene, Department of Food Science and Nutrition, Teodora Alonso Hall, College of Home Economics, A. Ma. Regidor Street, University of the Philippines, Diliman Campus, 1101, Quezon City, Philippines. E-mail address: [email protected] (A.A. Gabriel).
https://doi.org/10.1016/j.lwt.2019.108634 Received 25 March 2019; Received in revised form 24 July 2019; Accepted 15 September 2019 Available online 18 September 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
LWT - Food Science and Technology 117 (2020) 108634
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sterile nutrient broth (NB, HiMedia) prior to incubation at 35 °C for 18–24 h. A loopful of cells from the resulting culture was thereafter transferred to another 10-ml sterile NB and was incubated at 35 °C for 18–24 h. A loopful was obtained from each of the 2nd culture passage and streaked onto NA slant prior to incubation at 35 °C for 18–24 h. The culture slants were finally stored at 4 °C until used in the challenge studies. Each of the test strains was subjected to the 2-passage culture maintenance every 14 d.
distinct from those of chemical disinfectants or thermal processes that involve damages to cellular structures. Ultraviolet irradiation inactivates cells by altering the physicochemical nature of the DNA, which eventually affects downstream expression of essential proteins involved in biochemical processes (Donahue, Canitez, & Bushway, 2004). While usually applied to liquid food matrices (Ferrario, Alzamora, & Guerrero, 2015; Gabriel & Marquez, 2017; Gabriel & Nakano, 2010; Kasahara, Carrasco, & Aguilar, 2015; Koutchma, 2008), studies have also been done to inactivate microorganisms artificially inoculated on solid surfaces (Skåra & Rosnes, 2016; Gabriel et al., 2016; 2018) and solid food samples (Chun, Kim, Lee, Yu, & Song, 2010; Fonseca & Rushing, 2006; Gabriel, Tongco, & Barnes, 2017; Ha, Kang, Back, & Kim, 2015). This study was conducted in order to assess the utility of UV-C as a decontamination technology for herbs such as dried bay leaves artificially inoculated with selected foodborne bacteria. The results obtained in this study shall be useful in further identifying the scope and limitation of the utility of this emerging technology in a specific food commodity.
2.4. Cocktail inoculum preparation and bay leaf inoculation In order to prepare cocktails of bacteria from the same species, each working strain was subjected to the previously described 2-passage culture technique. Equal aliquots were thereafter obtained from the 2nd culture passage and pooled in a sterile Erlenmeyer flask, which was vortex mixed to homogenize the cell suspensions. Cells were harvested by obtaining 1 ml of the suspension and transferring it to a sterile Eppendorf tube and spinning at 2419 x g for 15 min using a bench top centrifuge (Cole Parmer, Illinois, USA). The supernatant was decanted after centrifugation, and cell pellet was resuspended in 0.5 ml 0.85% NaCl solution and was homogenized by vortex mixing until the cell pellet was resuspended. The cells were allowed to acclimatize for 5 min prior to inoculation. Sample inoculation procedure was adapted from Gabriel et al. (2017). The cocktailed test species was inoculated by spraying 3 ml of the cocktailed bacterial strains onto 10 g portions of gamma-irradiated bay leaf samples in a biosafety cabinet. Inoculum was sprayed on to the samples at a distance 5–8 cm, ensuring that only one side of the samples was covered. Inoculated samples were allowed to dry for 30 min in a biosafety cabinet before UV-C treatment.
2. Materials & methods 2.1. Bay leaves sample preparation Five kilograms of dried bay leaves obtained from a wet market in Angeles City, Pampanga, Philippines were subjected to gamma irradiation at a dose of 25 kGy, in order to remove background microflora from the sample. Prior to irradiation, the samples were previously sealed in nylon LDPE bags (Afdell Packaging Solutions Inc., Manila, Philippines) in 10 g portions, and were stored at 25 °C after treatment until used in the experimentation. Irradiation was done in the Irradiation Facility of the Nuclear Services Division of the Philippine Nuclear Research Institute of the Department of Science and Technology using a60Co package irradiator (Comprad Communications, Hungary). Post gamma-irradiation microbial enumeration showed that the aerobic plate count and yeast and molds count of the bay leaves were below detection limit (< 1.0 log CFU/g). The effects of the irradiation process to remove the background microflora on the physicochemical quality of the bay leaves were not measured in this study.
2.5. UV-C inactivation and survivor enumeration Five grams of inoculated sample per replicate were subjected to UVC irradiation, which was done for as long as 90 min, at a lamp-to-surface distance of 15 cm. The test exposure time was based on the results of initial studies (data not presented) showing that subjecting the inoculated food sample to UV-C treatments longer than 90 min did not result in any further population change. A fabricated box with a 15W UV-C lamp (Sankyo Denki, Japan) was used as a source of UV-C radiation. The dominant radiation emitted by the UV-C lamp in the fabricated box was confirmed by subjecting the lamp to optical emission spectroscopy at the treatment distance. The emission measurement was conducted using a spectrometer (Ocean Optics, Inc. FL., USA) with a dispersion of 0.2467 nm per pixel, and an optical resolution of 1.0855 nm in the range of 200–1100 nm. Fig. 1 shows the emission spectra obtained during testing. Irradiance measurements from the UV lamp were also obtained during UV irradiation using a UV radiometer (UVX Radiometer, UVP, Upland, California, USA). The treated samples were immediately homogenized with 45 ml 0.1% peptone water (HiMedia, Mumbai, India). Aliquots of 1 ml further diluted in 9 ml 0.1% peptone water, and appropriate dilutions were spread plated onto NA. Plates were incubated at 35 °C for 18–24 h, except for samples containing L. monocytogenes, which were incubated for 48 h prior to survivor enumeration.
2.2. Test organisms The study used a total of five bacterial species as test organisms for the study. Each species had 2-7 strains, and a mixture of strains of the same species was prepared prior to inoculation. Seven strains of Salmonella enterica were used, including serovars Typhimurium (American Type Culture Collection, ATCC 14028), Diarizonae (ATCC 12325 and 29934), Abortus-Equi (ATCC 9842), Enteritidis (Laboratory of Food Microbiology and Hygiene, LFMH S1-10), Infantis (LFMH S210), and Montevideo (LFMH S3-10) were used as test organisms. Five strains of Escherichia coli O157:H7 (LFMH EMY-10, EDT-10, EMN-10, ECR-10, EHC-10) maintained in the LFMH were also used in the study, along with two strains of Listeria monocytogenes, namely 1/2 c (LFMH L1-10) and 4b (LFMH L2-10). A 4-strain mix of Pseudomonas aeruginosa from the Institute of Molecular Biology and Biotechnology, University of the Philippines, at Los Baños, Laguna, Philippines (BIOTECH-1919, -1313, and −1314) and a strain from ATCC (27853) was also challenged. Lastly, a cocktail of Staphylococcus aureus strains LFMH SA-16, SA-17-1 and SA-17-2 were also used as test organisms.
2.6. Inactivation behavior and parameter determination Emerging colonies were enumerated and survivor populations were expressed as log CFU/g. For each test organism, the inactivation behavior and inactivation kinetic parameters were determined using the online freeware Dynamic Model Fit (online DMFit) available at ComBase (2019). In the online DMFit inactivation behavior and inactivation kinetic parameters were determined by fitting the survivor populations per treatment time into inactivation models, which included (1) the Baranyi and Roberts model (no lag), (2) the Biphasic Inactivation Model (no lag), and (3) the linear model. Model fit were
2.3. Microbial propagation and culture maintenance Each test strain was subjected to propagation and maintenance to ensure uniform culture age and physiological state prior to inactivation studies. A loopful of cells were obtained from refrigerated nutrient agar (NA, HiMedia, Mumbai, India) stock cultures and transferred to 10-ml 2
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Fig. 1. Emission spectra of the 15 W UV-C lamp source showing predominant emission wavelength at 254 nm, at 15.0 cm source-to-detector distance.
quantified using the coefficient of determination (R2), standard error of fit (SE) and maximum inactivation rate (Kmax). The total log reduction (TLR) after 90 min UV-C exposure were determined per test microorganism.
were determined following the 997.12 Official Methods of Analysis of AOAC International (AOAC, 2012), which employed a Cold-Vapor Atomic Absorption Spectrophotometer AA-7000 (Shimadzu, Kyoto, Japan). The Same/Different tests, also referred to as difference paired comparisons, or simple difference tests (Lawless and Heymann, 2010), was conducted to determine if consumers could detect significant difference in the control and UV-C-treated samples solely through visual inspection. In order to investigate differences between any of the 3 treatments, 3 combinations were tested, i.e. 5-lamp vs. 1-lamp (denoted A vs. B), 1-lamp vs. control (denoted B vs. C), and control vs. 5-lamp (denoted C vs. A). Samples placed in white plastic cups were presented to 50 untrained consumer-type panelists in pairs, and a random 3-digit code was assigned to each of these pairs. To offset the effect that sample order has on panelists’ perceptions, all four possible samples orders were presented during evaluation ([AA, AB, BA, BB], [BB, BC, CB, CC], [CC, CA, AC, AA]). Panelists were served all 4 sample orders simultaneously, and were instructed to evaluate the pairs one-at-a-time. A total of 200 responses were obtained and analyzed.
2.7. Quality evaluations of UV-C-irradiated bay leaves The study also subjected non-gamma irradiated and uninoculated bay leaves to UV-C processing to determine the effects of irradiation on various quality attributes. Five-g bay leaf samples were placed in separate sterile dishes and subjected to 90-min UV-C irradiation using 1 lamp with an average surface irradiance of 0.43 ± 0.12 mW/cm2 or 5 lamps with irradiance equal to 1.70 ± 0.42 mW/cm2. Samples were thereafter subjected to quality determinations. The microbiology of the treated samples, specifically, the Total Aerobic Plate Count (APC) and the Yeast and Molds Count (YMC) were enumerated before and after UV-C treatment. For the APC, samples were subjected to serial 10-fold dilution in 0.1% PW, after which appropriate dilutions were surfaceplated onto pre-solidified Plate Count Agar (HiMedia), and incubated at 35 °C for 24 h. The YMC were determined by surface plating on presolidified, Potato Dextrose Agar (HiMedia) previously acidified with 10% tartaric acid (RTC Laboratory Services and Supply House, Quezon City, Philippines), and incubated at 30 °C f0r 24–120 h. Changes in the Commission Internationale de L'eclairage (CIE) color space coordinates of the bay leaves were also determined after UV-C treatments. A Konica Minolta Chroma Meter with CR-A33e light projection tube and Granular Attachment CR-A50 was used to measure the coordinates that corresponded to CIE L* (lightness-darkness), a* (redness-greenness), and b* (yellowness-blueness) . Since the study utilized a mercury vapor lamp as UV-C source during prolonged exposure times, the deposition of heavy metal on the treated samples was also determined. Although the risk of mercury contamination has only been associated with the breakage and overall integrity of devices containing the heavy metal (Roda, Giamperti, Vecchio, Apostoli, & Coccini, 2016), the study explored the possibility of mercury deposition after prolonged exposure to UV-C irradiation produced from mercury vapor lamps. Samples were brought to the Philippine Institute of Pure and Applied Chemistry (PIPAC), Ateneo de Manila University, Quezon City, Philippines where mercury contents
2.8. Statistical analyses Individual sets of data obtained from independently replicated experiments were subjected to single factor analysis of variance (ANOVA) using the general linear model procedure (PROC GLM) of SAS Studio Version 3.7 University Edition (Cary, North Carolina, USA). For posthoc determinations of significant differences at 95% level of significance, the Duncan's Multiple Range Test (DMRT) was used. For the Same/Different test, the Chi-square (χ2) test was used to determine the test panel detected differences in the compared bay leaf sample pairs. 3. Results and discussion 3.1. Microbial inactivation patterns in UV-C-irradiated dried bay leaves The inactivation curves shown in Fig. 2a–e illustrate that all test organisms exhibited an inactivation behavior composed of an initial fast, logarithmic-linear population decay followed by a tail where limited or no additional inactivation takes place even with prolonged 3
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Fig. 2. Inactivation behaviors of (a.) Escherichia coli O157:H7, (b.) Salmonella enterica, (c.) Pseudomonas aeruginosa, (d.) Listeria monocytogenes, and (e.) Staphylococcus aureus in UV-C-irradiated (3942 mJ/cm2) bay leaves fitted in the Baranyi and Roberts model using DMFit Freeware of ComBase (2019).
subjected to UV-C inactivation (Sastry, Datta, & Worobo, 2000). When fitted in the Baranyi and Roberts, and the Biphasic Models, the Kmax values presented in Table 1 are those determined from the initial fast logarithmic linear population decline as shown in Fig. 1. This initial population change was followed by a tail where no significant population change were determined for all test organisms, hence the Ktail values of about zero CFU/min were no longer reported. Previous studies similarly reported such an inactivation behavior for test organisms such as S. enterica, L. monocytogenes, and innate microflora of UV-irradiated solid food commodities such as herbs, seafood, and desiccated coconut meat flakes (Cheigh, Hwang, & Chung, 2013; Dogu-Baykut, Gunes, & Decker, 2014; Gabriel et al., 2017). Furthermore, majority of the organisms tested by Gabriel and Marquez (2017), which included E. coli O157:H7, P. aeruginosa, L. monocytogenes, and S.
exposure to the inactivating agent (Cerf, 1977). These curves agree with the initial inactivation model fitting summarized in Table 1, where the least values for model fit parameter were those of the Linear Model. Fitting into the Biphasic Model also resulted in slightly better fit parameters. Hence these curves were generated by fitting into the Baranyi and Roberts Model using the online freeware available in ComBase (2019). Furthermore, in Table 1, it can be observed that the inactivation data for the Gram-negative bacteria, E. coli O157:H7, S. enterica and P. aeruginosa had similar model fit parameters for both the Baranyi and Roberts and the Biphasic Model, as indicated by the R2 and SE of Fit values. On the other hand, for the Gram-positive bacteria, which included L. monocytogenes and S. aureus, better fit parameters were measured in the Biphasic Model. The observed 2-phase inactivation behavior was previously explained to be typical among organisms 4
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3.60 ± 0.83ab
4.05 ± 0.45a
4.13 ± 0.25a
3.57 ± 0.15b
3.45 ± 0.80a
2.70 ± 0.38b
2.84 ± 0.41ab
3.86 ± 0.36a 0.82 ± 0.06 0.72 ± 0.14 0.56 ± 0.13
0.42 ± 0.07
0.81 ± 0.10 0.45 ± 0.11 0.76 ± 0.10
0.23 ± 0.08
0.95 ± 0.21 0.64 ± 0.11 0.60 ± 0.09
0.19 ± 0.14
0.95 ± 0.13 0.41 ± 0.19 0.73 ± 0.11 0.65 ± 0.08
0.43 ± 0.19 Staphylococcus aureus
0.81 ± 0.16
0.69 ± 0.25 Listeria monocytogenes
0.49 ± 0.17
0.60 ± 0.09 Pseudomonas aeruginosa
0.64 ± 0.11
0.65 ± 0.08 Salmonella enterica
0.73 ± 0.11
−0.10 ± 0.06 −0.15 ± 0.15 −0.27 ± 0.10 −0.26 ± 0.16 −0.11 ± 0.18 0.66 ± 0.16 0.65 ± 0.06 Escherichia coli O157:H7
1 Model fit and inactivation kinetic parameters are reported as averages ± SD from at least 8 values obtained from at least 4 independent experiment runs. When fitted in the Baranyi and Roberts, and Biphasic Models, the Kmax values were determined from the initial logarithmic linear population decline. In all test organisms, this initial population decline was followed by inactivation tail where no additional significant inactivation was determined by the models (Ktail = 0). When fitted in the linear model, the Kmax values are determined as the slope of the straight line that best-fitted all enumerated populations. The Total Log Reduction (TLR) values represent the fraction of the population that are susceptible to the inactivating agent. The Surviving Population (SP) values represent the faction of the population that are resistant towards UV-C.
3.07 ± 0.39b 3.93 ± 0.72a
−0.03 ± 0.01 −0.03 ± 0.01 −0.02 ± 0.00 −0.02 ± 0.00 −0.03 ± 0.00 0.52 ± 0.14
−0.10 ± 0.06 −0.16 ± 0.14 −0.26 ± 0.10 −0.31 ± 0.13 −0.26 ± 0.18 0.65 ± 0.16 0.65 ± 0.07
0.74 ± 0.12
Kmax(log CFU/ min) SE Fit R2 Kmax(log CFU/ min) SE Fit R Kmax(log CFU/ min) SE Fit R
Linear Biphasic
2 2
Baranyi and Roberts
Model Fit Parameters per Inactivation Model Test Organisms
Table 1 Inactivation model fit parameters1 and inactivation kinetic parameters1 of microorganisms in UV-C irradiated (3942 mJ/cm2) dried bay leaves.
Total Log Reduction TLR (log CFU/g)
Surviving Population SP(log CFU/g)
A.A. Gabriel, et al.
aureus exhibited this inactivation pattern in UV-C irradiated human milk. When allowed to adhere onto different stainless steel surfaces, P. aeruginosa (Gabriel et al., 2016) and S. enterica (Gabriel et al., 2018) exhibited similar inactivation behavior upon UV-C treatment. This non-linear inactivation behavior may be attributed to the nonhomogenous susceptibility of the organisms inoculated in the test food matrix, which may be due to food- or microorganism-related factors (Cerf, 1977; Gabriel & Marquez, 2017; Gabriel et al., 2017; Kamau, Doores, & Pruitt, 1990; Whiting & Buchanan, 1992). All tested organisms were a cocktail of strains which can have variable UV-C resistance. Furthermore, the characteristic topography of the food product, which possesses cracks, and crevices may provide shadow zones where microorganisms can establish and be protected from the UV-C irradiation (Gabriel et al., 2017). 3.2. Microbial population reduction in UV-C-irradiated dried bay leaves The total logarithmic reduction (TLR) in and Surviving Populations (SP) of the test organism populations after exposure to 90 min of UV-C with surface irradiance of 0.73 mW/cm2 are presented in Table 1. These TLR values were achieved after a possible maximum UV-C energy exposure of 3942 mJ/cm2. Results showed that the TLR values of the test organisms ranged from 2.63 to 3.93 log CFU/g, while the SP values ranged from 3.07 to 4.13 log CFU/g. The tested cocktail of E. coli O157:H7 was demonstrated to have the least resistance towards UV-C, while the cocktail of P. aeruginosa had the greatest resistance to the inactivating agent. However, the observed resistance of E. coli O157:H7 was not significantly different (p > 0.05) from that of the tested S. aureus (TLR = 3.86 log CFU), S. enterica (TLR = 3.45 log CFU), and L. monocytogenes (TLR = 2.84 log CFU). These values are far from the 5log pathogen reduction rates that are commonly recommended to ensure food safety; and hence may indicate that UV-C may not be applicable in decontaminating dried bay leaves that are heavily contaminated with the test organisms. Except for S. aureus, the same set of organisms were challenged by Gabriel and Marquez (2017) in human breast milk, who established a different trend in UV-C resistance. The observed disparity in UV-C resistance may be attributed to the differences in the intrinsic properties of food into which the organisms were introduced. 3.3. Quality attributes of UV-C-irradiated dried bay leaves In order to have a more holistic evaluation of the scope and utility of UV-C irradiation as an antimicrobial technology for dried bay leaves, quality attributes of UV-C-processed samples were also determined. The study also attempted to increase the treatment surface irradiance to which the bay leaves were exposed prior to quality determinations because preliminary studies demonstrated that increasing UV-C intensity also increases the antimicrobial effects of the biocide (data not presented). Hence aside from the previously tested single-lamp set up with a surface irradiance of 0.73 mW/cm2 that resulted in a 90-min UVC energy dose of 3942 mJ/cm2, the study also used a 5-lamp set up with a surface irradiance of 2.53 mW/cm2 that resulted in a 90-min UV-C energy dose of 13662 mJ/cm2 to treat bay leaves samples. Results showed (Table 2) that the CIE color coordinate L* increased with increasing UV-C dose, denoting lightening of the treated samples. The b* values also increase, denoting a shift from less yellow to more yellow. On the other hand, a definitive trend was not observed for the a* value which denoted that the sample neither become redder or greener. Despite these measured changes in the CIE color space coordinates, none of them was statistically significant (p > 0.05). These results agree with those determined in the sensory evaluations conducted in the various sample pairs. Results of the Chi-square tests showed that the 50-member sensory evaluation panel was not able to detect differences between non-processed and processed samples, and between processed samples (data not presented). Gabriel, Ballesteros, 5
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Table 2 Quality attributes of UV-C irradiated dried bay leaves. Quality attributes
CIE Color space coordinates L* (lightness-darkness) a* (redness-greenness) b* (yellowness-blueness) Graphical representation of colorsb Residual mercury (ppm) Microbiological quality Total Aerobic Plate Count (log CFU/g) Yeast and Molds Count (log CFU/g)
UV-C energy dose delivered to dried bay leavesa Untreated
3942 mJ/cm2
13662 mJ/cm2
72.10 ± 1.46a −3.85 ± 0.02a 11.23 ± 1.46a
72.60 ± 0.93a −4.01 ± 0.62a 12.29 ± 1.48a
72.90 ± 1.64a −3.97 ± 0.49a 12.38 ± 1.30a
< 0.03 ± 0.00a
< 0.03 ± 0.00a
< 0.03 ± 0.00a
4.95 ± 0.24a 2.83 ± 0.12a
3.60 ± 0.44b 2.49 ± 0.22a
3.28 ± 0.42b 1.70 ± 0.81b
a, b
Values are reported as averages of at least 3 readings ± SD. Values followed by the same letters in the same row are not significantly different (p > 0.05). UV-C energy dose were delivered by exposing dried bay leaves to UV-C for 90 min using 1 UV-C lamp (surface irradiance = 0.43 mW/cm2, dose = 2322 mJ/cm2) and 5 UV-C lamps (surface irradiance = 1.70 mW/cmb, dose = 9180 mJ/cmb). b Graphical representations of colors were generated using the Nix Free Color Converter available at https://www.nixsensor.com/free-color-converter/. a
UV-C dose. These information contribute to the knowledge necessary in further evaluating the scope and limitation of the utility of UV-C irradiation as a decontamination technology for a specific food commodity such as dried bay leaves.
et al. (2018) and Gabriel, Manalo, et al. (2018) similarly established a UV-C-based process for ready-to-drink calamansi juice and reported the non-significant difference in the sensory properties of processed and non-processed commodities. Furthermore, since prolonged exposure to UV-C irradiation produced by mercury vapor lamps may potentially result in deposition of mercury in the food sample and compromise safety, the study also determined the mercury levels in all samples. Results showed that mercury levels in all samples were below detection limit of the approved AOAC method (> 3 ppm). Mercury (Hg) in foods can elicit a variety of toxic effects in humans. It is known that high levels of exposure to inorganic mercury cause kidney and liver failure, while significantly lower levels of exposure to methylmercury (MeHg) are still associated with a variety of long-term neurodevelopmental deficits in children and may eventually impair cardiovascular health in adults (Sunderland & Tumpney, 2013). Finally, the microbial quality indicators of the UV-C processed bay leaves were measured and showed that significant changes in microbial population after processing. Total aerobic plate counts (TPC) of the processed leaves were significantly (p < 0.05) lower than the control sample. However, there was no difference between the TPC of samples subjected to 1 and 5 lamps. On the other hand, UV-C processing only significantly reduced the yeast and molds count (YMC) of the bay leaves after exposure to the more severe 5-lamp process. The difference in the effects of UV-C on TPC and YMC may be attributed to the differences in the ultrastructure of the organisms inactivated by the biocides. Bacterial cells that are measured by the TPC may be more susceptible towards UV-C since the DNA molecules are not enclosed in a nuclear envelope that are found in yeasts and molds.
Acknowledgements This study was partially supported by the Philippine Council for Industry, Energy and Emerging Technology Research and Development, Department of Science and Technology, through its Human Resource Development Program (HRDP). The assistance provided by the Plasma Physics Laboratory of the University of the Philippines at Diliman in the measurement of the optical properties of the UV-C lamp is also being acknowledged. The authors would also like to extend their gratitude to Philippine Institute of Pure and Applied Chemistry (PIPAC, Ateneo de Manila University) in conducting the measurement of mercury levels in the samples. References Association of Official Analytical Chemists (AOAC) International (2012). In G. W. LatimerJr. (Ed.). Official Methods of Analysis of AOAC International (19th ed). Gaithersburg, MD, USA: AOAC International. Banerjee, M., & Sarkar, P. K. (2003). Microbiological quality of some retail spices in India. Food Research International, 36(5), 469–474. https://doi.org/10.1016/S09639969(02)00194-1. Cerf, O. (1977). A review: Tailing of survival curves of bacterial spores. Journal of Applied Bacteriology, 42, 1–19. https://doi.org/10.1111/j.1365-2672.1977.tb00665.x. Cheigh, C. I., Hwang, H. J., & Chung, M. S. (2013). Intense pulsed light (IPL) and UV-C treatments for inactivating Listeria monocytogenes on solid medium and seafoods. Food Research International, 54, 745–752. https://doi.org/10.1016/j.foodres.2013.08. 025. Chun, H. H., Kim, J. Y., Lee, B. D., Yu, D. J., & Song, K. B. (2010). Effect of UV-C irradiation on the inactivation of inoculated pathogens and quality of chicken breasts during storage. Food Control, 21(3), 276–280. https://doi.org/10.1016/j.foodcont. 2009.06.006. ComBase (2019). Online DMFit. Available at: https://browser.combase.cc/DMFit.aspx. Dogu-Baykut, E., Gunes, G., & Decker, E. A. (2014). Impact of shortwave ultraviolet (UVC) radiation on the antioxidant activity of thyme (Thymus vulgaris L.). Food Chemistry, 157, 167–173. https://doi.org/10.1016/j.foodchem.2014.02.027. Donahue, D., Canitez, N., & Bushway, A. (2004). UV inactivation of E. coli O157:H7 in apple cider: Quality, sensory and shelf-life analysis. Journal of Food Processing and Preservation, 28, 268–287. https://doi.org/10.1111/j.1745-4549.2004.23062.x. Ferrario, M., Alzamora, S. M., & Guerrero, S. (2015). Study of the inactivation of spoilage microorganisms in apple juice by pulsed light and ultrasound. Food Microbiology, 46, 635–642. https://doi.org/10.1016/j.fm.2014.06.017. Fonseca, J. M., & Rushing, J. W. (2006). Effect of ultraviolet-C light on quality and microbial population of fresh-cut watermelon. Postharvest Biology and Technology, 40(3), 256–261. https://doi.org/10.1016/j.postharvbio.2006.02.003. Gabriel, A. A., Ballesteros, M. L. P., Rosario, L. M. D., Tumlos, R. B., & Ramos, H. J. (2018). Elimination of Salmonella enterica on common stainless steel food contact surfaces using UV-C and atmospheric pressure plasma jet. Food Control, 86, 90–100. https://doi.org/10.1016/j.foodcont.2017.11.011. Gabriel, A. A., Manalo, M. R., Feliciano, R. J., Garcia, N. K. A., Dollete, U. G. M., Acanto,
4. Conclusion Summing up, this study was able to establish the UV-C inactivation behavior and inactivation kinetic parameters of common foodborne bacteria artificially inoculated onto dried bay leaves. In this study the cocktail of P. aeruginosa was deemed an appropriate reference organism for the establishment of UV-C process schedule for the tested food commodity. This study was also able to demonstrate that UV-C irradiation may not be able to effectively decontaminate heavily contaminated dried bay leaves, which emphasizes the importance of adherence towards food safety systems in the manufacture and distribution of the test commodity. Furthermore, the study also showed that the applied UV-C dose did not result in the alteration of color and visible sensory properties of the leaves, and did not result in the deposition of mercury from the UV-C lamp source. Finally, microbial quality indices measured in the study were improved by the applied 6
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McKee, L. H. (1995). Microbial contamination of spices and herbs: A review. LebensmittelWissenschaft und -Technologie- Food Science and Technology, 28(1), 1–11. https://doi. org/10.1016/S0023-6438(95)80004-2. Peter, K. V., & Babu, K. N. (2012). Introduction to herbs and spices: Medicinal uses and sustainable production. Handbook of Herbs and Spices (2 nd ed, Vol 2) (pp. 1–16). Woodhead Publishing. https://doi.org/10.1533/9780857095688.1. Roda, E., Giamperti, A., Vecchio, S., Apostoli, P., & Coccini, T. (2016). Case report: Mercury vapour long-lasting exposure: Lymphosite muscarinic receptors as neurochemical markers of accidental intoxication. Case Reports in Medicine. Hindawi Publishing Corp, 1–8. Article ID 9783876 https://doi.org/10.1155/2016/9783876. Sagoo, S. K., Little, C. L., Greenwood, M., Mithani, V., Grant, K. A., McLauchlin, J., et al. (2009). Assessment of the microbiological safety of dried spices and herbs from production and retail premises in the United Kingdom. Food Microbiology, 26(1), 39–43. https://doi.org/10.1016/j.fm.2008.07.005. Sastry, S. K., Datta, A. K., & Worobo, R. W. (2000). Ultraviolet light. Journal of Food Science, 65(s8), 90–92. https://doi.org/10.1111/j.1750-3841.2000.tb00623.x. Skåra, T., & Rosnes, J. T. (2016). Emerging methods and principles in food contact surface decontamination/prevention. Innovation and future trends in food manufacturing and supply chain technologies. Elsevier Ltdhttps://doi.org/10.1016/B978-1-78242-447-5. 00006-X. Song, W.-J., Sung, H.-J., Kim, S.-Y., Kim, K.-P., Ryu, S., & Kang, D.-H. (2014). Inactivation of Escherichia coli O157:H7 and Salmonella Typhimurium in black pepper and red pepper by gamma irradiation. International Journal of Food Microbiology, 172, 125–129. https://doi.org/10.1016/j.ijfoodmicro.2013.11.017. Sunderland, E. M., & Tumpney, M. (2013). Mercury in foods. In M. Rose, & A. Fernandes (Eds.). Persistent organic pollutants and toxic metals in foods. Woodhead publishing series in food science, technology and nutrition No. 247 (pp. 392–413). UK: FERA. Van Doren, J. M., Neil, K. P., Parish, M., Gieraltowski, L., Gould, L. H., & Gombas, K. L. (2013). Foodborne illness outbreaks from microbial contaminants in spices, 19732010. Food Microbiology, 36(2), 456–464. https://doi.org/10.1016/j.fm.2013.04. 014. Whiting, R. C., & Buchanan, R. L. (1992). Use of predictive microbial modeling in a HACCP program. Second ASEPT international conference: Predictive microbiology and HACCP. Laval cedex, France: Asept (pp. 125–141). . Zweifel, C., & Stephan, R. (2012). Spices and herbs as source of Salmonella-related foodborne diseases. Food Research International, 45(2), 765–769. https://doi.org/10. 1016/j.foodres.2011.02.024.
C. A., et al. (2018). A Candida parapsilosis inactivation-based UV-C process for calamansi (Citrus microcarpa) juice drink. Lebensmittel-Wissenschaft & Technologie, 90, 157–163. https://doi.org/10.1016/j.lwt.2017.12.020. Gabriel, A. A., & Marquez, G. F. (2017). Inactivation behaviors of selected bacteria in ultraviolet-C treated human breast milk. Innovative Food Science & Emerging Technologies, 41, 216–223. https://doi.org/10.1016/j.ifset.2017.03.010. Gabriel, A. A., & Nakano, H. (2010). Influences of simultaneous physicochemical stress exposure on injury and subsequent responses of E. coli O157:H7 to resuscitative and inactivate challenges. International Journal of Food Microbiology, 139, 182–192. https://doi.org/10.1016/j.ijfoodmicro.2010.02.028. Gabriel, A. A., Tongco, A. M. P., & Barnes, A. A. (2017). Utility of UV-C radiation as antiSalmonella decontamination treatment for desiccated coconut flakes. Food Control, 71, 117–123. https://doi.org/10.1016/j.foodcont.2016.06.026. Gabriel, A. A., Ugay, M. C. C. F., Siringan, M. A. T., Rosario, L. M. D., Tumlos, R. B., & Ramos, H. J. (2016). Atmospheric pressure plasma jet inactivation of Pseudomonas aeruginosa biofilms on stainless steel surfaces. Innovative Food Science and Emerging Technologies, 36, 311–319. https://dx.doi.org/10.1016/j.ifset.2016.07.015. Garbowska, M., Berthold-Pluta, A., & Stasiak-Rózańska, L. (2015). Microbiological quality of selected spices and herbs including the presence of Cronobacter spp. Food Microbiology, 49, 1–5. https://doi.org/10.1016/j.fm.2015.01.004. Ha, J. W., Kang, D. H., Back, K.-H., & Kim, Y.-H. (2015). Enhanced inactivation of foodborne pathogens in ready-to-eat sliced ham by near-infrared heating combined with UV-C irradiation and mechanism of the synergistic bactericidal action. Applied and Environmental Microbiology, 81(1), 2–8. https://doi.org/10.1128/AEM.01862-14. Kamau, D. N., Doores, S., & Pruitt, K. M. (1990). Enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus by the lactoperoxidase system. Applied and Environmental Microbiology, 56, 2711–2716. Kasahara, I., Carrasco, V., & Aguilar, L. (2015). Inactivation of Escherichia coli in goat milk using pulsed ultraviolet light. Journal of Food Engineering, 152, 43–49. https:// doi.org/10.1016/j.jfoodeng.2014.11.012. Koutchma, T. (2008). UV light for processing foods. Ozone: Science & Engineering, 30(1), 93–98. https://doi.org/10.1080/01919510701816346. Koutchma, T. (2009). Advances in ultraviolet light technology for non-thermal processing of liquid foods. Food and Bioprocess Technology, 2(2), 138–155. https://doi.org/10. 1007/s11947-008-0178-3. Lawless, H. T., & Heymann, H. (2010). Sensory Evaluation of Food. Principles and Practices (2nd ed). Springer.
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Determination of the utility of ultraviolet-C irradiation for dried bay leaves microbial decontamination through safety and quality evaluations
T
Alonzo A. Gabriel∗, Katrina Moira D. Melo, Juan Carlos D. Michelena Laboratory of Food Microbiology and Hygiene, Department of Food Science and Nutrition, College of Home Economics, Alonso Hall, Antonio Ma. Regidor St., University of the Philippines Diliman, Quezon City, 1101, Philippines
A R T I C LE I N FO
A B S T R A C T
Keywords: Bay leaves Herbs Microbial inactivation Nonthermal processing UV-C irradiation
This study was conducted to determine the efficacy of UV-C irradiation for safety and quality of dried bay leaves. Cocktails of microorganisms of the same species were prepared and inoculated onto the leaves, which were thereafter subjected to UV-C irradiation. Results showed that all tested organisms, which included Escherichia coli O157:H7, Salmonella enterica, Pseudomonas aeruginosa, Listeria monocytogenes, and Staphylococcus aureus, exhibited inactivation behaviors in the irradiated leaves that are characterized by initial log-linear population decline, followed by inactivation tail. The Total Log Reductions in the organisms after exposure to 3942 mJ/cm2 UV-C dose ranged from 2.70 to 3.93 log CFU/g. Furthermore, subjecting the bay leaves to UV-C of as much as 13662 mJ/cm2 did not significantly alter color, and visual sensory properties, and did not result in mercury deposition. Microbiological quality indicators of the bay leaves, including Total Plate Count and Yeast and Molds Count were improved by the UV-C irradiation. These results contribute in further understanding the scope and limitations of using UV-C as a decontamination technique for specific food product such as dried bay leaves.
1. Introduction Spices and herbs have long been valued throughout the world for their various functions in food and medicine. Peter and Babu (2012) recounted that during ancient times, they were prized for their antimicrobial properties that aided in food preservation, and with the passing of time spices have been indispensable in the culinary arts due to their ability to enhance the flavor of food and drink. While they have no nutritional value, they are prized for specific sensory values, and can even exert beneficial effects on digestive processes (Garbowska, Berthold-Pluta, & Stasiak-Rózańska, 2015). Peter and Babu (2012) further explained that spices and herbs are terms that tend to be used interchangeably in daily conversation, but they are defined as two separate entities: spices are defined as the dried parts of aromatic plants other than the leaves, while herbs are defined as the dried leaves of aromatic plants. Despite this definition, the application remains the same. While not a natural growth medium for pathogenic microorganisms, several reports have shown that spices and herbs can serve as a vector for pathogenic microorganisms under the right conditions, usually related to poor adherence to GMP or due to the environment spices are exposed to (Banerjee & Sarkar, 2003; Zweifel & Stephan, 2012). Spices
such as anise seeds, basil, black pepper, cinnamon, chili, curry powder, and oregano were previously identified as causes of illness outbreaks in countries including Canada, Denmark, England and Wales, France, Germany, New Zealand, Norway, Serbia, and the United States (Sagoo et al., 2009; Van Doren et al., 2013; Zweifel & Stephan, 2012). McKee (1995) reported a 6.0 to 8.0 log CFU/g of microbial contamination levels in retail spices in various places in the world. Current methods for herbs and spices decontamination include physical means such as steam sterilization and gamma irradiation, and the use of chemicals such as ethylene oxide or propylene oxide (Song et al., 2014). However, these methods come with different disadvantages. Steam sterilization alters the chemical and organoleptic properties of spices and herbs due to its nature as a high temperature treatment process (Peter & Babu, 2012). Chemical treatments are sometimes associated with carcinogenic byproducts, and the relatively inert methods like gamma irradiation are expensive (Peter & Babu, 2012; Song et al., 2014). Thus, there is a need to find an alternative method to decontaminate spices, one that does not alter chemical and organoleptic properties of the spices, is not carcinogenic, and is relatively inexpensive. UV-C irradiation is one of the possible alternative methods for decontamination as it manages to address these concerns. Koutchma (2009) explained that the biocidal mechanism of UV-C is
∗ Corresponding author. Laboratory of Food Microbiology and Hygiene, Department of Food Science and Nutrition, Teodora Alonso Hall, College of Home Economics, A. Ma. Regidor Street, University of the Philippines, Diliman Campus, 1101, Quezon City, Philippines. E-mail address: [email protected] (A.A. Gabriel).
https://doi.org/10.1016/j.lwt.2019.108634 Received 25 March 2019; Received in revised form 24 July 2019; Accepted 15 September 2019 Available online 18 September 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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sterile nutrient broth (NB, HiMedia) prior to incubation at 35 °C for 18–24 h. A loopful of cells from the resulting culture was thereafter transferred to another 10-ml sterile NB and was incubated at 35 °C for 18–24 h. A loopful was obtained from each of the 2nd culture passage and streaked onto NA slant prior to incubation at 35 °C for 18–24 h. The culture slants were finally stored at 4 °C until used in the challenge studies. Each of the test strains was subjected to the 2-passage culture maintenance every 14 d.
distinct from those of chemical disinfectants or thermal processes that involve damages to cellular structures. Ultraviolet irradiation inactivates cells by altering the physicochemical nature of the DNA, which eventually affects downstream expression of essential proteins involved in biochemical processes (Donahue, Canitez, & Bushway, 2004). While usually applied to liquid food matrices (Ferrario, Alzamora, & Guerrero, 2015; Gabriel & Marquez, 2017; Gabriel & Nakano, 2010; Kasahara, Carrasco, & Aguilar, 2015; Koutchma, 2008), studies have also been done to inactivate microorganisms artificially inoculated on solid surfaces (Skåra & Rosnes, 2016; Gabriel et al., 2016; 2018) and solid food samples (Chun, Kim, Lee, Yu, & Song, 2010; Fonseca & Rushing, 2006; Gabriel, Tongco, & Barnes, 2017; Ha, Kang, Back, & Kim, 2015). This study was conducted in order to assess the utility of UV-C as a decontamination technology for herbs such as dried bay leaves artificially inoculated with selected foodborne bacteria. The results obtained in this study shall be useful in further identifying the scope and limitation of the utility of this emerging technology in a specific food commodity.
2.4. Cocktail inoculum preparation and bay leaf inoculation In order to prepare cocktails of bacteria from the same species, each working strain was subjected to the previously described 2-passage culture technique. Equal aliquots were thereafter obtained from the 2nd culture passage and pooled in a sterile Erlenmeyer flask, which was vortex mixed to homogenize the cell suspensions. Cells were harvested by obtaining 1 ml of the suspension and transferring it to a sterile Eppendorf tube and spinning at 2419 x g for 15 min using a bench top centrifuge (Cole Parmer, Illinois, USA). The supernatant was decanted after centrifugation, and cell pellet was resuspended in 0.5 ml 0.85% NaCl solution and was homogenized by vortex mixing until the cell pellet was resuspended. The cells were allowed to acclimatize for 5 min prior to inoculation. Sample inoculation procedure was adapted from Gabriel et al. (2017). The cocktailed test species was inoculated by spraying 3 ml of the cocktailed bacterial strains onto 10 g portions of gamma-irradiated bay leaf samples in a biosafety cabinet. Inoculum was sprayed on to the samples at a distance 5–8 cm, ensuring that only one side of the samples was covered. Inoculated samples were allowed to dry for 30 min in a biosafety cabinet before UV-C treatment.
2. Materials & methods 2.1. Bay leaves sample preparation Five kilograms of dried bay leaves obtained from a wet market in Angeles City, Pampanga, Philippines were subjected to gamma irradiation at a dose of 25 kGy, in order to remove background microflora from the sample. Prior to irradiation, the samples were previously sealed in nylon LDPE bags (Afdell Packaging Solutions Inc., Manila, Philippines) in 10 g portions, and were stored at 25 °C after treatment until used in the experimentation. Irradiation was done in the Irradiation Facility of the Nuclear Services Division of the Philippine Nuclear Research Institute of the Department of Science and Technology using a60Co package irradiator (Comprad Communications, Hungary). Post gamma-irradiation microbial enumeration showed that the aerobic plate count and yeast and molds count of the bay leaves were below detection limit (< 1.0 log CFU/g). The effects of the irradiation process to remove the background microflora on the physicochemical quality of the bay leaves were not measured in this study.
2.5. UV-C inactivation and survivor enumeration Five grams of inoculated sample per replicate were subjected to UVC irradiation, which was done for as long as 90 min, at a lamp-to-surface distance of 15 cm. The test exposure time was based on the results of initial studies (data not presented) showing that subjecting the inoculated food sample to UV-C treatments longer than 90 min did not result in any further population change. A fabricated box with a 15W UV-C lamp (Sankyo Denki, Japan) was used as a source of UV-C radiation. The dominant radiation emitted by the UV-C lamp in the fabricated box was confirmed by subjecting the lamp to optical emission spectroscopy at the treatment distance. The emission measurement was conducted using a spectrometer (Ocean Optics, Inc. FL., USA) with a dispersion of 0.2467 nm per pixel, and an optical resolution of 1.0855 nm in the range of 200–1100 nm. Fig. 1 shows the emission spectra obtained during testing. Irradiance measurements from the UV lamp were also obtained during UV irradiation using a UV radiometer (UVX Radiometer, UVP, Upland, California, USA). The treated samples were immediately homogenized with 45 ml 0.1% peptone water (HiMedia, Mumbai, India). Aliquots of 1 ml further diluted in 9 ml 0.1% peptone water, and appropriate dilutions were spread plated onto NA. Plates were incubated at 35 °C for 18–24 h, except for samples containing L. monocytogenes, which were incubated for 48 h prior to survivor enumeration.
2.2. Test organisms The study used a total of five bacterial species as test organisms for the study. Each species had 2-7 strains, and a mixture of strains of the same species was prepared prior to inoculation. Seven strains of Salmonella enterica were used, including serovars Typhimurium (American Type Culture Collection, ATCC 14028), Diarizonae (ATCC 12325 and 29934), Abortus-Equi (ATCC 9842), Enteritidis (Laboratory of Food Microbiology and Hygiene, LFMH S1-10), Infantis (LFMH S210), and Montevideo (LFMH S3-10) were used as test organisms. Five strains of Escherichia coli O157:H7 (LFMH EMY-10, EDT-10, EMN-10, ECR-10, EHC-10) maintained in the LFMH were also used in the study, along with two strains of Listeria monocytogenes, namely 1/2 c (LFMH L1-10) and 4b (LFMH L2-10). A 4-strain mix of Pseudomonas aeruginosa from the Institute of Molecular Biology and Biotechnology, University of the Philippines, at Los Baños, Laguna, Philippines (BIOTECH-1919, -1313, and −1314) and a strain from ATCC (27853) was also challenged. Lastly, a cocktail of Staphylococcus aureus strains LFMH SA-16, SA-17-1 and SA-17-2 were also used as test organisms.
2.6. Inactivation behavior and parameter determination Emerging colonies were enumerated and survivor populations were expressed as log CFU/g. For each test organism, the inactivation behavior and inactivation kinetic parameters were determined using the online freeware Dynamic Model Fit (online DMFit) available at ComBase (2019). In the online DMFit inactivation behavior and inactivation kinetic parameters were determined by fitting the survivor populations per treatment time into inactivation models, which included (1) the Baranyi and Roberts model (no lag), (2) the Biphasic Inactivation Model (no lag), and (3) the linear model. Model fit were
2.3. Microbial propagation and culture maintenance Each test strain was subjected to propagation and maintenance to ensure uniform culture age and physiological state prior to inactivation studies. A loopful of cells were obtained from refrigerated nutrient agar (NA, HiMedia, Mumbai, India) stock cultures and transferred to 10-ml 2
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Fig. 1. Emission spectra of the 15 W UV-C lamp source showing predominant emission wavelength at 254 nm, at 15.0 cm source-to-detector distance.
quantified using the coefficient of determination (R2), standard error of fit (SE) and maximum inactivation rate (Kmax). The total log reduction (TLR) after 90 min UV-C exposure were determined per test microorganism.
were determined following the 997.12 Official Methods of Analysis of AOAC International (AOAC, 2012), which employed a Cold-Vapor Atomic Absorption Spectrophotometer AA-7000 (Shimadzu, Kyoto, Japan). The Same/Different tests, also referred to as difference paired comparisons, or simple difference tests (Lawless and Heymann, 2010), was conducted to determine if consumers could detect significant difference in the control and UV-C-treated samples solely through visual inspection. In order to investigate differences between any of the 3 treatments, 3 combinations were tested, i.e. 5-lamp vs. 1-lamp (denoted A vs. B), 1-lamp vs. control (denoted B vs. C), and control vs. 5-lamp (denoted C vs. A). Samples placed in white plastic cups were presented to 50 untrained consumer-type panelists in pairs, and a random 3-digit code was assigned to each of these pairs. To offset the effect that sample order has on panelists’ perceptions, all four possible samples orders were presented during evaluation ([AA, AB, BA, BB], [BB, BC, CB, CC], [CC, CA, AC, AA]). Panelists were served all 4 sample orders simultaneously, and were instructed to evaluate the pairs one-at-a-time. A total of 200 responses were obtained and analyzed.
2.7. Quality evaluations of UV-C-irradiated bay leaves The study also subjected non-gamma irradiated and uninoculated bay leaves to UV-C processing to determine the effects of irradiation on various quality attributes. Five-g bay leaf samples were placed in separate sterile dishes and subjected to 90-min UV-C irradiation using 1 lamp with an average surface irradiance of 0.43 ± 0.12 mW/cm2 or 5 lamps with irradiance equal to 1.70 ± 0.42 mW/cm2. Samples were thereafter subjected to quality determinations. The microbiology of the treated samples, specifically, the Total Aerobic Plate Count (APC) and the Yeast and Molds Count (YMC) were enumerated before and after UV-C treatment. For the APC, samples were subjected to serial 10-fold dilution in 0.1% PW, after which appropriate dilutions were surfaceplated onto pre-solidified Plate Count Agar (HiMedia), and incubated at 35 °C for 24 h. The YMC were determined by surface plating on presolidified, Potato Dextrose Agar (HiMedia) previously acidified with 10% tartaric acid (RTC Laboratory Services and Supply House, Quezon City, Philippines), and incubated at 30 °C f0r 24–120 h. Changes in the Commission Internationale de L'eclairage (CIE) color space coordinates of the bay leaves were also determined after UV-C treatments. A Konica Minolta Chroma Meter with CR-A33e light projection tube and Granular Attachment CR-A50 was used to measure the coordinates that corresponded to CIE L* (lightness-darkness), a* (redness-greenness), and b* (yellowness-blueness) . Since the study utilized a mercury vapor lamp as UV-C source during prolonged exposure times, the deposition of heavy metal on the treated samples was also determined. Although the risk of mercury contamination has only been associated with the breakage and overall integrity of devices containing the heavy metal (Roda, Giamperti, Vecchio, Apostoli, & Coccini, 2016), the study explored the possibility of mercury deposition after prolonged exposure to UV-C irradiation produced from mercury vapor lamps. Samples were brought to the Philippine Institute of Pure and Applied Chemistry (PIPAC), Ateneo de Manila University, Quezon City, Philippines where mercury contents
2.8. Statistical analyses Individual sets of data obtained from independently replicated experiments were subjected to single factor analysis of variance (ANOVA) using the general linear model procedure (PROC GLM) of SAS Studio Version 3.7 University Edition (Cary, North Carolina, USA). For posthoc determinations of significant differences at 95% level of significance, the Duncan's Multiple Range Test (DMRT) was used. For the Same/Different test, the Chi-square (χ2) test was used to determine the test panel detected differences in the compared bay leaf sample pairs. 3. Results and discussion 3.1. Microbial inactivation patterns in UV-C-irradiated dried bay leaves The inactivation curves shown in Fig. 2a–e illustrate that all test organisms exhibited an inactivation behavior composed of an initial fast, logarithmic-linear population decay followed by a tail where limited or no additional inactivation takes place even with prolonged 3
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Fig. 2. Inactivation behaviors of (a.) Escherichia coli O157:H7, (b.) Salmonella enterica, (c.) Pseudomonas aeruginosa, (d.) Listeria monocytogenes, and (e.) Staphylococcus aureus in UV-C-irradiated (3942 mJ/cm2) bay leaves fitted in the Baranyi and Roberts model using DMFit Freeware of ComBase (2019).
subjected to UV-C inactivation (Sastry, Datta, & Worobo, 2000). When fitted in the Baranyi and Roberts, and the Biphasic Models, the Kmax values presented in Table 1 are those determined from the initial fast logarithmic linear population decline as shown in Fig. 1. This initial population change was followed by a tail where no significant population change were determined for all test organisms, hence the Ktail values of about zero CFU/min were no longer reported. Previous studies similarly reported such an inactivation behavior for test organisms such as S. enterica, L. monocytogenes, and innate microflora of UV-irradiated solid food commodities such as herbs, seafood, and desiccated coconut meat flakes (Cheigh, Hwang, & Chung, 2013; Dogu-Baykut, Gunes, & Decker, 2014; Gabriel et al., 2017). Furthermore, majority of the organisms tested by Gabriel and Marquez (2017), which included E. coli O157:H7, P. aeruginosa, L. monocytogenes, and S.
exposure to the inactivating agent (Cerf, 1977). These curves agree with the initial inactivation model fitting summarized in Table 1, where the least values for model fit parameter were those of the Linear Model. Fitting into the Biphasic Model also resulted in slightly better fit parameters. Hence these curves were generated by fitting into the Baranyi and Roberts Model using the online freeware available in ComBase (2019). Furthermore, in Table 1, it can be observed that the inactivation data for the Gram-negative bacteria, E. coli O157:H7, S. enterica and P. aeruginosa had similar model fit parameters for both the Baranyi and Roberts and the Biphasic Model, as indicated by the R2 and SE of Fit values. On the other hand, for the Gram-positive bacteria, which included L. monocytogenes and S. aureus, better fit parameters were measured in the Biphasic Model. The observed 2-phase inactivation behavior was previously explained to be typical among organisms 4
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3.60 ± 0.83ab
4.05 ± 0.45a
4.13 ± 0.25a
3.57 ± 0.15b
3.45 ± 0.80a
2.70 ± 0.38b
2.84 ± 0.41ab
3.86 ± 0.36a 0.82 ± 0.06 0.72 ± 0.14 0.56 ± 0.13
0.42 ± 0.07
0.81 ± 0.10 0.45 ± 0.11 0.76 ± 0.10
0.23 ± 0.08
0.95 ± 0.21 0.64 ± 0.11 0.60 ± 0.09
0.19 ± 0.14
0.95 ± 0.13 0.41 ± 0.19 0.73 ± 0.11 0.65 ± 0.08
0.43 ± 0.19 Staphylococcus aureus
0.81 ± 0.16
0.69 ± 0.25 Listeria monocytogenes
0.49 ± 0.17
0.60 ± 0.09 Pseudomonas aeruginosa
0.64 ± 0.11
0.65 ± 0.08 Salmonella enterica
0.73 ± 0.11
−0.10 ± 0.06 −0.15 ± 0.15 −0.27 ± 0.10 −0.26 ± 0.16 −0.11 ± 0.18 0.66 ± 0.16 0.65 ± 0.06 Escherichia coli O157:H7
1 Model fit and inactivation kinetic parameters are reported as averages ± SD from at least 8 values obtained from at least 4 independent experiment runs. When fitted in the Baranyi and Roberts, and Biphasic Models, the Kmax values were determined from the initial logarithmic linear population decline. In all test organisms, this initial population decline was followed by inactivation tail where no additional significant inactivation was determined by the models (Ktail = 0). When fitted in the linear model, the Kmax values are determined as the slope of the straight line that best-fitted all enumerated populations. The Total Log Reduction (TLR) values represent the fraction of the population that are susceptible to the inactivating agent. The Surviving Population (SP) values represent the faction of the population that are resistant towards UV-C.
3.07 ± 0.39b 3.93 ± 0.72a
−0.03 ± 0.01 −0.03 ± 0.01 −0.02 ± 0.00 −0.02 ± 0.00 −0.03 ± 0.00 0.52 ± 0.14
−0.10 ± 0.06 −0.16 ± 0.14 −0.26 ± 0.10 −0.31 ± 0.13 −0.26 ± 0.18 0.65 ± 0.16 0.65 ± 0.07
0.74 ± 0.12
Kmax(log CFU/ min) SE Fit R2 Kmax(log CFU/ min) SE Fit R Kmax(log CFU/ min) SE Fit R
Linear Biphasic
2 2
Baranyi and Roberts
Model Fit Parameters per Inactivation Model Test Organisms
Table 1 Inactivation model fit parameters1 and inactivation kinetic parameters1 of microorganisms in UV-C irradiated (3942 mJ/cm2) dried bay leaves.
Total Log Reduction TLR (log CFU/g)
Surviving Population SP(log CFU/g)
A.A. Gabriel, et al.
aureus exhibited this inactivation pattern in UV-C irradiated human milk. When allowed to adhere onto different stainless steel surfaces, P. aeruginosa (Gabriel et al., 2016) and S. enterica (Gabriel et al., 2018) exhibited similar inactivation behavior upon UV-C treatment. This non-linear inactivation behavior may be attributed to the nonhomogenous susceptibility of the organisms inoculated in the test food matrix, which may be due to food- or microorganism-related factors (Cerf, 1977; Gabriel & Marquez, 2017; Gabriel et al., 2017; Kamau, Doores, & Pruitt, 1990; Whiting & Buchanan, 1992). All tested organisms were a cocktail of strains which can have variable UV-C resistance. Furthermore, the characteristic topography of the food product, which possesses cracks, and crevices may provide shadow zones where microorganisms can establish and be protected from the UV-C irradiation (Gabriel et al., 2017). 3.2. Microbial population reduction in UV-C-irradiated dried bay leaves The total logarithmic reduction (TLR) in and Surviving Populations (SP) of the test organism populations after exposure to 90 min of UV-C with surface irradiance of 0.73 mW/cm2 are presented in Table 1. These TLR values were achieved after a possible maximum UV-C energy exposure of 3942 mJ/cm2. Results showed that the TLR values of the test organisms ranged from 2.63 to 3.93 log CFU/g, while the SP values ranged from 3.07 to 4.13 log CFU/g. The tested cocktail of E. coli O157:H7 was demonstrated to have the least resistance towards UV-C, while the cocktail of P. aeruginosa had the greatest resistance to the inactivating agent. However, the observed resistance of E. coli O157:H7 was not significantly different (p > 0.05) from that of the tested S. aureus (TLR = 3.86 log CFU), S. enterica (TLR = 3.45 log CFU), and L. monocytogenes (TLR = 2.84 log CFU). These values are far from the 5log pathogen reduction rates that are commonly recommended to ensure food safety; and hence may indicate that UV-C may not be applicable in decontaminating dried bay leaves that are heavily contaminated with the test organisms. Except for S. aureus, the same set of organisms were challenged by Gabriel and Marquez (2017) in human breast milk, who established a different trend in UV-C resistance. The observed disparity in UV-C resistance may be attributed to the differences in the intrinsic properties of food into which the organisms were introduced. 3.3. Quality attributes of UV-C-irradiated dried bay leaves In order to have a more holistic evaluation of the scope and utility of UV-C irradiation as an antimicrobial technology for dried bay leaves, quality attributes of UV-C-processed samples were also determined. The study also attempted to increase the treatment surface irradiance to which the bay leaves were exposed prior to quality determinations because preliminary studies demonstrated that increasing UV-C intensity also increases the antimicrobial effects of the biocide (data not presented). Hence aside from the previously tested single-lamp set up with a surface irradiance of 0.73 mW/cm2 that resulted in a 90-min UVC energy dose of 3942 mJ/cm2, the study also used a 5-lamp set up with a surface irradiance of 2.53 mW/cm2 that resulted in a 90-min UV-C energy dose of 13662 mJ/cm2 to treat bay leaves samples. Results showed (Table 2) that the CIE color coordinate L* increased with increasing UV-C dose, denoting lightening of the treated samples. The b* values also increase, denoting a shift from less yellow to more yellow. On the other hand, a definitive trend was not observed for the a* value which denoted that the sample neither become redder or greener. Despite these measured changes in the CIE color space coordinates, none of them was statistically significant (p > 0.05). These results agree with those determined in the sensory evaluations conducted in the various sample pairs. Results of the Chi-square tests showed that the 50-member sensory evaluation panel was not able to detect differences between non-processed and processed samples, and between processed samples (data not presented). Gabriel, Ballesteros, 5
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Table 2 Quality attributes of UV-C irradiated dried bay leaves. Quality attributes
CIE Color space coordinates L* (lightness-darkness) a* (redness-greenness) b* (yellowness-blueness) Graphical representation of colorsb Residual mercury (ppm) Microbiological quality Total Aerobic Plate Count (log CFU/g) Yeast and Molds Count (log CFU/g)
UV-C energy dose delivered to dried bay leavesa Untreated
3942 mJ/cm2
13662 mJ/cm2
72.10 ± 1.46a −3.85 ± 0.02a 11.23 ± 1.46a
72.60 ± 0.93a −4.01 ± 0.62a 12.29 ± 1.48a
72.90 ± 1.64a −3.97 ± 0.49a 12.38 ± 1.30a
< 0.03 ± 0.00a
< 0.03 ± 0.00a
< 0.03 ± 0.00a
4.95 ± 0.24a 2.83 ± 0.12a
3.60 ± 0.44b 2.49 ± 0.22a
3.28 ± 0.42b 1.70 ± 0.81b
a, b
Values are reported as averages of at least 3 readings ± SD. Values followed by the same letters in the same row are not significantly different (p > 0.05). UV-C energy dose were delivered by exposing dried bay leaves to UV-C for 90 min using 1 UV-C lamp (surface irradiance = 0.43 mW/cm2, dose = 2322 mJ/cm2) and 5 UV-C lamps (surface irradiance = 1.70 mW/cmb, dose = 9180 mJ/cmb). b Graphical representations of colors were generated using the Nix Free Color Converter available at https://www.nixsensor.com/free-color-converter/. a
UV-C dose. These information contribute to the knowledge necessary in further evaluating the scope and limitation of the utility of UV-C irradiation as a decontamination technology for a specific food commodity such as dried bay leaves.
et al. (2018) and Gabriel, Manalo, et al. (2018) similarly established a UV-C-based process for ready-to-drink calamansi juice and reported the non-significant difference in the sensory properties of processed and non-processed commodities. Furthermore, since prolonged exposure to UV-C irradiation produced by mercury vapor lamps may potentially result in deposition of mercury in the food sample and compromise safety, the study also determined the mercury levels in all samples. Results showed that mercury levels in all samples were below detection limit of the approved AOAC method (> 3 ppm). Mercury (Hg) in foods can elicit a variety of toxic effects in humans. It is known that high levels of exposure to inorganic mercury cause kidney and liver failure, while significantly lower levels of exposure to methylmercury (MeHg) are still associated with a variety of long-term neurodevelopmental deficits in children and may eventually impair cardiovascular health in adults (Sunderland & Tumpney, 2013). Finally, the microbial quality indicators of the UV-C processed bay leaves were measured and showed that significant changes in microbial population after processing. Total aerobic plate counts (TPC) of the processed leaves were significantly (p < 0.05) lower than the control sample. However, there was no difference between the TPC of samples subjected to 1 and 5 lamps. On the other hand, UV-C processing only significantly reduced the yeast and molds count (YMC) of the bay leaves after exposure to the more severe 5-lamp process. The difference in the effects of UV-C on TPC and YMC may be attributed to the differences in the ultrastructure of the organisms inactivated by the biocides. Bacterial cells that are measured by the TPC may be more susceptible towards UV-C since the DNA molecules are not enclosed in a nuclear envelope that are found in yeasts and molds.
Acknowledgements This study was partially supported by the Philippine Council for Industry, Energy and Emerging Technology Research and Development, Department of Science and Technology, through its Human Resource Development Program (HRDP). The assistance provided by the Plasma Physics Laboratory of the University of the Philippines at Diliman in the measurement of the optical properties of the UV-C lamp is also being acknowledged. The authors would also like to extend their gratitude to Philippine Institute of Pure and Applied Chemistry (PIPAC, Ateneo de Manila University) in conducting the measurement of mercury levels in the samples. References Association of Official Analytical Chemists (AOAC) International (2012). In G. W. LatimerJr. (Ed.). Official Methods of Analysis of AOAC International (19th ed). Gaithersburg, MD, USA: AOAC International. Banerjee, M., & Sarkar, P. K. (2003). Microbiological quality of some retail spices in India. Food Research International, 36(5), 469–474. https://doi.org/10.1016/S09639969(02)00194-1. Cerf, O. (1977). A review: Tailing of survival curves of bacterial spores. Journal of Applied Bacteriology, 42, 1–19. https://doi.org/10.1111/j.1365-2672.1977.tb00665.x. Cheigh, C. I., Hwang, H. J., & Chung, M. S. (2013). Intense pulsed light (IPL) and UV-C treatments for inactivating Listeria monocytogenes on solid medium and seafoods. Food Research International, 54, 745–752. https://doi.org/10.1016/j.foodres.2013.08. 025. Chun, H. H., Kim, J. Y., Lee, B. D., Yu, D. J., & Song, K. B. (2010). Effect of UV-C irradiation on the inactivation of inoculated pathogens and quality of chicken breasts during storage. Food Control, 21(3), 276–280. https://doi.org/10.1016/j.foodcont. 2009.06.006. ComBase (2019). Online DMFit. Available at: https://browser.combase.cc/DMFit.aspx. Dogu-Baykut, E., Gunes, G., & Decker, E. A. (2014). Impact of shortwave ultraviolet (UVC) radiation on the antioxidant activity of thyme (Thymus vulgaris L.). Food Chemistry, 157, 167–173. https://doi.org/10.1016/j.foodchem.2014.02.027. Donahue, D., Canitez, N., & Bushway, A. (2004). UV inactivation of E. coli O157:H7 in apple cider: Quality, sensory and shelf-life analysis. Journal of Food Processing and Preservation, 28, 268–287. https://doi.org/10.1111/j.1745-4549.2004.23062.x. Ferrario, M., Alzamora, S. M., & Guerrero, S. (2015). Study of the inactivation of spoilage microorganisms in apple juice by pulsed light and ultrasound. Food Microbiology, 46, 635–642. https://doi.org/10.1016/j.fm.2014.06.017. Fonseca, J. M., & Rushing, J. W. (2006). Effect of ultraviolet-C light on quality and microbial population of fresh-cut watermelon. Postharvest Biology and Technology, 40(3), 256–261. https://doi.org/10.1016/j.postharvbio.2006.02.003. Gabriel, A. A., Ballesteros, M. L. P., Rosario, L. M. D., Tumlos, R. B., & Ramos, H. J. (2018). Elimination of Salmonella enterica on common stainless steel food contact surfaces using UV-C and atmospheric pressure plasma jet. Food Control, 86, 90–100. https://doi.org/10.1016/j.foodcont.2017.11.011. Gabriel, A. A., Manalo, M. R., Feliciano, R. J., Garcia, N. K. A., Dollete, U. G. M., Acanto,
4. Conclusion Summing up, this study was able to establish the UV-C inactivation behavior and inactivation kinetic parameters of common foodborne bacteria artificially inoculated onto dried bay leaves. In this study the cocktail of P. aeruginosa was deemed an appropriate reference organism for the establishment of UV-C process schedule for the tested food commodity. This study was also able to demonstrate that UV-C irradiation may not be able to effectively decontaminate heavily contaminated dried bay leaves, which emphasizes the importance of adherence towards food safety systems in the manufacture and distribution of the test commodity. Furthermore, the study also showed that the applied UV-C dose did not result in the alteration of color and visible sensory properties of the leaves, and did not result in the deposition of mercury from the UV-C lamp source. Finally, microbial quality indices measured in the study were improved by the applied 6
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