PAPER 198 Ital. J. Food Sci., vol. 27 - 2015 - Keywords: food safety, Listeria monocytogenes, fresh produce, food borne illnesses, tomatoes, food borne pathogens - INACTIVATION OF LISTERIA MONOCYTOGENES ATCC 7644 ON TOMATOES USING SODIUM DODECYL SULPHATE, LEVULINIC ACID AND SODIUM HYPOCHLORITE SOLUTION E. MNYANDU*, O.A. IJABADENIYI and S. SINGH Department of Biotechnology and Food Technology, Durban University of Technology, Durban 4001, South Africa *Corresponding author: Tel. 002778 0248617 email: mketiwae@yahoo.com ABSTRACT The human pathogen Listeria monocytogenes poses a serious threat to public health. A study was carried out to evaluate the effectiveness of four sanitizers, used individually or combined, against L. monocytogenes ATCC 7644. The contact times for bacteria and sanitizer were varied to 1, 3 and 5 minutes. Levulinic acid, sodium dodecyl sulphate (SDS), sodium hypochlorite solution (chlorine) and a combination of SDS and levulinic acid (mixture) were tested. Results revealed that 0.5% levulinic acid, when used individually, is capable of reducing the surviving colonies by 3.63 log CFU/mL, 4.05 log CFU/mL, 6.71 log CFU/mL after exposure for 1, 3 and 5 minutes re- spectively. SDS resulted in an 8 log CFU/mL reduction after 1, 3 and 5 minutes. A combination of 0.5% levulinic acid and 0.05% SDS caused a 3.69 log CFU /mL reduction, 4.4 log CFU/mL re- duction, 7.97 log CFU/mL reduction for 1, 3 and 5 minutes respectively. Chlorine was the least effective with 2.93 log CFU/mL reduction, 3.16 log CFU/ mL reduction and 4.53 log CFU/ mL reduction respectively. When stored for up to 72 hours at 4°C, the surviving colonies remained viable and decreased in number significantly P < 0.05 = 0.001. The titratable acidity of samples treated with levulinic acid and samples treated with SDS/Lev mixture was lowered significantly compared to the control sample. No significant differences were noted in these same parameters for samples treated with chlorine or SDS. The application of SDS in the fresh produce industry as a sanitizing agent may be successful in eradicating or reducing the viability of L. monocytogenes on fresh produce, thereby replacing the routine chlorine washing. mailto:mketiwae%40yahoo.com?subject= Ital. J. Food Sci., vol. 27 - 2015 199 INTRODUCTION The increase in fresh produce consumption has caused a rapid evolution in the fresh pro- duce industry (JOHNSTON et al., 2005). This, coupled with recommendations to eat minimal- ly processed foods, has led to an increase in the consumption of fresh fruits and vegetables among consumers (BERGER et al., 2010). The consumption of minimally processed foods and fresh produce has also been encouraged among the immune compromised populations, such as those affected by HIV/AIDS, children and preg- nant women (BERGER et al., 2010; GANDHI and CHIKINDAS, 2007). Consumer demands and habits have also shifted, with many consumers in the busy world preferring to eat ready-to-eat foods and eating from salad bars (OMS-OLIU et al., 2010; BERDEGUÉ et al., 2005). A variety of fresh produce such as lettuce can- taloupes, peppers, tomatoes, herbs and green leafy vegetables, among others, have been linked to food borne illnesses associated with either Salmonella, Escherichia coli O157:H7 or Listeria monocytogenes contamination (TAUXE et al., 2010). Contamination of fresh produce by these pathogens occurs by various means. IJABADENI- YI et al. (2011a) cited irrigation water as major pre-harvest source of contamination of fresh pro- duce. Other factors as cited by JOHNSTON et al. (2005) include use of biocides as fertilizer, poor worker hygiene and poor sanitation. L. monocytogenes among other food borne pathogens have been implicated as a public health threat (VELUSAMY et al., 2010) and are es- timated to cause about 1,600 incidents of illness, more than 1400 hospitalisations and about 250 deaths per year in United states (KYLE, 2012). These pathogens are responsible for food borne Listeriosis. They can grow in the soil, drains and on food preparation surfaces (GÁLVEZ et al., 2010; PAN et al., 2006; DJORDJEVIC et al., 2002). They have been largely associated with dairy products, but recent research has also shown their increasing association with fresh produce (GANDHI and CHIKINDAS, 2007) includ- ing tomatoes. Tomatoes are widely consumed and can be eaten raw, partially cooked or can be processed into other products. They are a very rich source of carotenoids, folate, vitamin C, mineral ele- ments and phenolic compounds (FRUSCIANTE et al., 2007). Of major importance are the anti- oxidants (carotenoids). Epidemiological research has shown that the antioxidants are capable of preventing chances of cancers and cardio vas- cular diseases (LEONARDI et al., 2000). Toma- toes also provide a dietary source of soluble and insoluble fibres such as pectin, hemicellulose, and cellulose. Due to their nutritional value, they form an important part of the human diet. The elimination of food borne pathogens that can contaminate tomatoes is essential for prevent- ing food borne illnesses that may be associated with the consumption of tomatoes. Many methods are being used to try and elim- inate the food borne pathogens. Use of phage or phage products in food production has been con- sidered as a novel method for bio-control of path- ogens in fresh and ready-to-eat food products (HAGENS and LOESSNER, 2010), but the cost as- sociated with their use is very high. Other meth- ods include bacteriocin-activated films high-hy- drostatic pressure, high-pressure homogeniza- tion, in-package pasteurization, food irradia- tion, pulsed electric fields, or pulsed light and electrolyzed water (GÁLVEZ et al., 2010). Sani- tizers such as carvacrol, vanillin, peroxyacetic acid, hydrogen peroxide, N-acetyl-l-cysteine and citrox among others have also been tried (ABA- DIAS et al., 2011). Sanitizers affect cell compo- nents, for example proteins, DNA, RNA and cell wall constituents through physicochemical in- teractions or chemical reactions. They cause ir- reversible damage to these structures and a loss of cell contents, thereby rendering the bacteria inactive or dead (CERF et al., 2010). The action of sanitizers is governed by con- tact time (exposure time), pH and temperature, among other factors. Some researchers conclude that sanitizers are not effective in eradicating food borne pathogens when used individually, although a combination of agents increases the sanitizer ability (SAGONG et al., 2011; ZHAO et al., 2009). Recent studies have also shown that if not used properly, sanitizers can be detrimen- tal to the quality of fresh produce (SALGADO et al., 2013; GUAN et al., 2010). With regard to to- matoes, pH and acidity are the most important determinants of tomato quality (ANTHON et al., 2011), hence the interaction of tomatoes with sanitizers during washing should be monitored. The study was performed to evaluate the effec- tiveness of SDS, chlorine and levulinic acid in reducing the viability of L. monocytogenes on to- matoes and the effect of these sanitizers on pH, titratable acidity and total soluble solids. MATERIALS AND METHODS Fresh produce Tomatoes were purchased from a local super- market on three separate occasions in Durban, South Africa. On the day of purchase the toma- toes were washed in running water. The toma- toes were then washed in 70% alcohol (IJABAD- ENIYI et al., 2011a). Prior to subjection to differ- ent sanitizer treatments, the tomatoes were test- ed for the presence of L. monocytogenes. Bacterial strains Listeria monocytogenes ATCC 7644 (Mer - ck, South Africa) was used for this study. The 200 Ital. J. Food Sci., vol. 27 - 2015 strain was cultured in Fraser broth for 24 hours at 37ºC and stored at 4ºC (IJABADENIYI et al., 2011a). Prior to each experiment, a fresh cul- ture was prepared from the stock culture by sub-culturing in Fraser broth for 24 hours at 37ºC, an 8 log cfu/mL culture of L. monocy- togenes, using McFarland Standards (JI et al., 2010). Chemicals and chemical treatments Sodium dodecyl sulphate (SDS), levulinic acid, sodium hypochlorite solution, all purchased from Merck, South Africa, were tested, individ- ually or combined with varying contact times (1, 3 and 5 minutes) for their effect on L. monocy- togenes ATCC 7644 in tomatoes. The chemicals were used as follows; 1% SDS individually 0.5% Levulinic acid individually 200 ppm Sodium hypochlorite solution in- dividually and 0.5% levulinic acid/0.05% SDS combined and termed mixture. Inoculation of bacterial strains into tomatoes The method of ZHAO et al. (2009) was followed. A 25 g sample of tomatoes was cut into approxi- mately 5 cm long pieces in the lamina flow hood. The samples were submerged into bacterial sus- pension (108 cfu/mL, 50 mL of bacterial solu- tion into 950 mL of distilled water) for 60 sec- onds and then air dried for 20 minutes in the lamina flow hood. The samples were then sus- pended into 500 mL test solution and agitated by a magnetic stirrer at 100 rpm for 1, 3 and 5 minutes. Following treatment, the individual samples were placed in double zipper bags con- taining 25 mL of phosphate buffered saline and pummelled for one minute. The suspension was serially diluted (1:10) in 0.1% buffered peptone water and enumerated for L. monocytogenes ATCC 7644. Enumeration of L. monocytogenes A method by Taormina and BEUCHAT (2001) was followed. Populations of L. monocytogenes ATCC 7644 were determined by surface plating serially diluted samples; 0.1 mL in duplicates on Listeria Selective Agar (Oxford formulation; Oxoid Ltd, Wade Road, Basingstoke, Hants UK). Plates were incubated for 24 hours at 37ºC, af- ter which colonies were counted. Preparation of samples for Scanning Electron Microscopy Untreated samples and samples subjected to chlorine, levulinic and SDS/Lev were used for SEM viewing. A method used by Ijabadeniyi et al. (2011b) was followed with a few modifica- tions, according to the requirements of Univer- sity of KwaZulu Natal microscopy unit. Pieces of tomatoes inoculated with L. monocytogenes ATCC 7644 and subjected to different treat- ments were cut into small pieces of 2 x 2 mm using a sterile blade. Primary fixation was car- ried out in 2.5% glutaraldehyde for 12 hours, followed by rinsing three times in phosphate buffer (0.1 M, pH 7.0). Post fixation was done using 0.5% Osmium tetroxide for one hour. Fixed samples were dehydrated in graded etha- nol (30%, 50%, 75% and 100%) each for 5 min- utes. The samples were then dried in a critical point dryer with carbon dioxide as a transition gas. The samples were mounted on specimen stubs and coated with gold palladium. The sam- ples were then analysed using Desmond Clar- ence scanning electron microscopy. Analysis of tomato physicochemical properties Preparation of samples: The method of ZHAO et al. (2009) was followed for sample preparation, except that the tomato was further homogenised into slurry. A 25 g sample of tomatoes was cut into approximately 5 cm long pieces. The sam- ples were then suspended into 500 mL test so- lutions as follows: 25 grams of tomatoes + 500 mL de-ionised wa- ter (control) 25 grams of tomatoes + 500 mL 1% SDS 25 grams of tomatoes + 500 mL of 0.5% levulin- ic acid 25 grams of tomatoes + 500 mL of 200 ppm so- dium hypochlorite solution 25 grams of tomatoes + 500 mL of 0.5% levulin- ic acid/0.05% SDS (mixture) The samples were agitated by a magnet- ic stirrer at 100 rpm for 1, 3 and 5 minutes (contact times). After each contact time was achieved, samples were immediately drained and the tomatoes were homogenized to form a slurry using Waring Commercial Laborato- ry blender. The slurry was used to test for pH, titratable acidity and total soluble solids im- mediately. Determination of pH The determination of pH was done on fresh- ly made tomato paste using the Thermo Scien- tific Orion 2star pH meter. The electrodes were rinsed with distilled water in between samples. Determination of Titratable Acidity For estimating titratable acidity, the slurry was filtered using Whatman syringe filters. A 100 mL of the filtrate was titrated by adding 0.1N so- dium hydroxide until a pH of 8.1 was attained. The volume of the sodium hydroxide added to the solution was multiplied by a correction fac- tor of 0.064 to estimate titratable acidity as a Ital. J. Food Sci., vol. 27 - 2015 201 percentage of citric acid (CHEEMA et al., 2014; TURHAN and SENIZ, 2009). Determination of Soluble Solids Content TSS is an index of soluble solids concentra- tion in fruit. For an estimation of soluble solids content, 1.5 mL tomato slurry was centrifuged at 10,000 rpm (15 min, 25°C), and the super- natant was filtered through Whatman nonster- ile syringe filters (0.45 μm). The filtered toma- to serum (40 μL) was measured using a dig- ital refractometer ATAGO (ATAGO, USA Inc. Kirkland, WA, USA). Measurements were tak- en once for each sample, and 70% ethanol was used to clean in between samples. The refrac- tion index was expressed as percent soluble sol- ids in°Brix (Wilkerson et al., 2013; Javanmar- di and Kubota, 2006). Data analysis Three trials were conducted for each exper- iment. Analysis of the data was performed us- ing SPSS version 21 (IBM Statistics). Analysis of variance was conducted with repeated measures and Greenhouse Geisser correction to study the effect of contact time on the survival of L. mono- cytogenes, ATCC 7644 and the effect of each san- itizer on the survival of L. monocytogenes ATCC 7644 at varied time intervals (0, 24, 48 and 72 hours). The number of surviving colonies was plotted against contact time (1, 3 and 5 min- utes) and also against time interval (0, 24, 48 and 72 hours). Log reductions for each contact time and sanitizer were also calculated and is presented in a table. Pair wise comparison with Bonferroni adjustment was used to determine any significant difference between subjects. To analyse results for physicochemical properties, ANOVA was used to assess if there was a signif- icant difference in pH, total soluble solids and titratable acidity of treated and untreated to- mato samples. RESULTS Effect of storage time, sanitizer treatments and contact time on the survival of L. monocytogenes ATCC 7644 The treatment of L. monocytogenes with sani- tizers resulted in a decrease in the populations of bacteria. All the sanitizers tested had the abil- ity to reduce the surviving colonies, with vary- ing degree of effectiveness. Among the sanitiz- ers tested, sodium hypochlorite solution was the least effective, with the highest counts of surviving colonies. The next in the list is lev- ulinic acid, then a mixture of SDS and levulin- ic (termed mixture), with SDS the most effec- tive of them all. The results of repeated meas- ures (CASTRO et al.) with Greenhouse-Geisser correction showed that there was a significant difference at 5% level between effectiveness of sanitizers used, [F(3, 9) = 63.00; P< 0.05 = 0.01]. The surviving colonies were reduced progres- sively as storage time increased from 0 hours to 72 hours. The means of surviving colonies are shown in Table 1. Marginal means for each sanitizer’s contact time were also plotted in Fig. 1 for 1, 3 and 5 minutes. As shown in the figure, sodium hy- Table 1 - Mean 1 count of L. monocytogenes ATCC 7644 after treatment with different sanitizers at different contact times and storage times. Contact times Time intervals 0 Hours 24 Hours 48 Hours 72 Hours 1 minute a 5.36±0.02 a 5.14±0.03 a 5.02±0.03 a 4.75±0.04 Chlorine 3 minutes a 5.06±0.03 a 5.06±0.03 a 4.78±0.05 a 4.45±0.04 5 minutes b 4.17±0.09 b 3.77±0.09 b 3.33±0.10 b 2.60±0.09 1 minute c 4.60±0.01 c 4.59 ±0.02 c 4.27±0.08 c 4.01±0.06 SDS/Lev 3 minutes c 4.35±0.05 c 4.24 ±0.06 c 3.39±0.36 c 2.53±0.08 5 minutes d 1.33±0.15 d 1.40±0.03 d 0.56±0.09 d 0.00 1 minute e 4.68±0.03 e 4.60±0.02 e 4.15±0.14 e 4.06±0.11 Levulinic 3 minutes e 4.68±0.03 e 4.34±0.09 e 4.12±0.10 e 2.60±0.30 5 minutes f 3.17±0.07 f 2.06±0.04 f 1.50±0.10 f 0.43±0.20 1 minute g 0.00 g 0.00 g 0.00 g 0.00 SDS 3 minutes g 0.00 g 0.00 g 0.00 g 0.00 5 minutes g 0.00 g 0.00 g 0.00 g 0.00 Mean counts ±Standard Deviation (Log 10 CFU /mL). 1Means followed by different letters in the same column are significantly different. 202 Ital. J. Food Sci., vol. 27 - 2015 Fig. 1 - Means of surviving colonies of L. monocytogenes ATCC 7644; based on marginal means. The highest means associated with chlorine show that it was least effective. pochlorite has the highest mean values, mean- ing that the highest number of surviving colo- nies was observed after exposure to this san- itizer compared to other solutions. Sodium chloride was thus not very efficient in reduc- ing survival of the pathogen in this particu- lar study. Increasing the contact time (1, 3 and 5 min- utes) significantly reduced the surviving colo- nies for all sanitizers tested at 5% level; [F (3, 180) = 30.70; P< 0.001]. However, the results of ANOVA with Green House Geisser correction showed that the reduction for 1 minute and 3 minutes of treatment were not significantly dif- ferent (P = 0.16). This shows that increasing the contact time of each of the sanitizer to 3 min- utes did not make much difference to the sur- viving colonies. Overall log reductions When exposed for 1 minute to 200 ppm chlo- rine, L. monocytogenes were inactivated by 2.93 log CFU/mL. A log reduction of 3.16 log CFU/ mL and 4.53 log CFU/mL was achieved after in- creasing contact time to 3 minutes and 5 min- utes respectively. A mixture of 0.5% levulin- ic acid and 0.05% SDS (mixture) reduced the surviving colonies to 3.69 log CFU/mL, 4.4 log CFU/mL and log 7.97 CFU/mL after exposure for 1 minute, 3 minutes and 5 minutes, respec- tively. Using 0.5% levulinic acid resulted in log reductions of 3.63 log CFU/mL, 4.05 log CFU/ mL and 6.71 CFU/mL after exposure for 1 min- ute, 3 minutes and 5 minutes. The overall log reductions are presented in Table 2. Observations of specimens using a scanning electron microscope Samples of tomatoes treated with sodium hy- pochlorite solution, levulinic acid and mixture were viewed under SEM to verify the existence of colonies even after exposure to sanitizers. The results for this current work showed that there were surviving colonies after exposure to sodium chlorite solution, levulinic acid and a mixture. However, viewing samples treated with the above sanitizers did not clearly show the remains of surviving colonies. The SEM images did not show the presence of an abun- dance of bacteria on the surfaces. It is possible that the bacteria that were inoculated on the surfaces could have been washed out during the sample preparation procedure. The proce- dure used for sample preparation might not be suitable in this specific case. Bacteria might also have migrated into other hidden sections of the pictures due to the irregularities of the topography. Table 2 - Log reductions (CFU/ mL) for chlorine, mixture, levulinic acid and SDS at 1, 3 and 5 minutes. Overall log reduction Sanitizer 1 minute 3 minutes 5 minutes Chlorine 2.93 3.16 4.53 Mixture 3.69 4.40 7.17 Levulinic 3.63 4.05 6.71 SDS 8.00 8.00 8.00 Ital. J. Food Sci., vol. 27 - 2015 203 Titratable acidity, pH and total soluble solids of tomato samples Table 3 presents the results of the TA, pH and TSS. The TA of tomato samples treated with levulinic acid and SDS /Lev mixture was sig- nificantly different from the control (P< 0.05). The TA for levulinic acid treated tomatoes was 2.78%, 2.81%, and 2.81%; while the TA for mix- ture treated tomatoes was 3.81%, 3.73%, 3.74% for 1, 3 and 5 minutes respectively. The pH for levulinic acid and mixture treated tomato sam- ples was relatively lower than the pH of the con- trol sample, as shown in the table. There was no significant difference between the TA and pH of the mixture and levulinic acid treated samples. The TA for tomato samples treated with SDS was 0.16 and for samples treated with chlorine was 0.15%, 0.14% and 0.14%. These results did not vary significantly from the control. The pH for the SDS and chlorine treated samples were also slightly different from the control sample, as shown in the table. TSS for levulinic acid treat- ed samples were reduced significantly to 3.20% brix for 1, 3 and 5 minutes, while the TSS for mixture treated samples was reduced to 3.24, 3.26, 3.24% brix respectively. Though the TSS for SDS treated and chlorine treated samples were reduced, the effect was not significant ac- cording to the findings of this study. Contact time was varied from 1 minute to 5 minutes; but there were no significant changes in theses pa- rameters from 1 minute to 5 minutes. DISCUSSION The food manufacturing industry depends on the use of sanitizers for reducing the risk asso- ciated with food borne pathogens. Many sanitiz- ers have been tried, but to date food borne path- ogens are still a problem in the food and fresh produce industry. Some researchers have sug- gested that this is due to development of resist- ance by the bacteria with repeated exposure to sanitizers (MANI-LÓPEZ et al., 2012; RIAZI and MATTHEWS, 2011). Most fruits and vegetable units resort to chlo- rine based sanitizers because they are cheaper and have a long standing credibility with reduc- ing surviving bacteria. However, this is proved not to be the case in this current research, as well as other previous research. Findings from this study show that though chlorine has been widely used for washing produce and sanitis- ing food surfaces, it is not really capable of kill- ing all food borne pathogens. This is shown by high mean counts associated with chlorine as presented in the results above. Chlorine washing has also been tried on Escherichia coli O157:H7 and Salmonella, but the reports on that work also shows that chlorine is not effective against food borne pathogens (KESKINEN et al., 2009b). Other researchers also agree that chlorine can- not reduce food borne pathogens effectively (IJABADENIYI et al., 2011b; ALLENDE et al., 2009; MAHMOUD et al., 2007). Several research projects are under way to try Table 3 - Effects of levulinic acid, chlorine, SDS/Lev mixture and SDS on physicochemical properties of tomatoes. Tomato treatment Contact pH of sample Titratable acidity Total soluble times (% citric acid) solids (%Brix) 1 minute 4.77 a 0.16 a 4.90 a Distilled water 3 minutes 4.78 a 0.14 a 4.90 a 5 minutes 4.78 a 0.16 a 4.90 a Levulinic acid 1 minute 3.61 b 2.78 b 3.20 b 3 minutes 3.67 b 2.81 b 3.20 b 5 minutes 3.69 b 2.81 b 3.20 b Mixture (SDS/Lev) 1 minute 3.81 b 2.76 b 3.24 b 3 minutes 3.73 b 2.78 b 3.26 b 5 minutes 3.74 b 2.78 b 3.24 b Chlorine 1 minute 5.09 a 0.15 a 4.60 a 3 minutes 5.17 a 0.14 a 4.63 a 5 minutes 5.20 a 0.14 a 4.61 a SDS 1 minute 4.68 a 0.16 b 4.65 b 3 minutes 4.88 a 0.16 b 4.61 b 5 minutes 4.87 a 0.16 b 4.63 b Each value represents the mean of three trials. For each parameter, the values significantly different at P ≤ 0.05 are indicated by different letters. Samples treat- ed in distilled water were used as control. Chlorine = Sodium hypochlorite solution. 204 Ital. J. Food Sci., vol. 27 - 2015 to find other alternative sanitizers because of the challenges that are associated with chlorine (KE- SKINEN et al., 2009a). Some researchers point out that its pH sensitivity affects its effectiveness (ZHAO et al., 2009). Another challenge is that it diminishes quickly upon contact with organ- ic matter and hence leads to reduced effective- ness (NEAL et al., 2012). Other concerns raised include the environmental and health risks as- sociated with the formation of carcinogenic hal- ogenated disinfection by-products such as trih- alomethanes (GIL et al., 2009; KIM et al., 2009). For these reasons chlorine has not been gain- fully useful in the fresh produce industry in re- cent years. Though it has been a long standing sanitizer in the food industry, other sanitizers that have been shown to be more effective than chlorine; through this research and previous re- search can be employed for the betterment of mi- crobiologically quality of fresh produce. Levulinic acid is applied in the food manufac- turing industry as a food additive. It has been designated as a generally safe additive to food by the Food and Drug Administration (FDA)(ZHAO et al., 2009). Levulinic acid disrupts the mem- brane structure of bacteria due to its polarity, thereby exposing cell constituencies and lethali- ty (Thompson, 2007). Levulinic acid can be used over a wide pH and temperature range (SAGONG et al., 2011). In this particular study, levulinic acid showed mean counts that were much low- er than those of chlorine. With these findings, it can be concluded that levulinic acid at 0.5% can perform better than a 200ppm sodium hy- pochlorite solution against L. monocytogenes ATCC 7644. Other researchers also tried lev- ulinic acid in their work with related findings. THOMPSON et al. (2008) concluded that it was effective in inhibiting outgrowth of L. monocy- togenes in ready-to-eat meat products. Other studies using lactic acid, acetic acid and levulin- ic acid on meat revealed that though levulinic acid is effective, it does not provide as effective decontamination as lactic acid, nor as much re- sidual protection as acetic acid (CARPENTER et al., 2011). Levulinic acid shows potential in the fresh produce industry, and therefore further re- search can be pursued on the most usable con- centrations and most applicable pathogens. Its detrimental effects on quality should be taken into consideration as well. Sodium dodecyl sulphate is generally regard- ed as a safe (GRAS) food additive (LU and WU, 2012). In this study, using 1% SDS alone re- sulted in 8 log CFU/mL reduction of L. monocy- togenes. SDS has amphilic properties (12 car- bon chain attached to sulphate group) and its anti-microbial effectiveness increases when pH is decreased, it has the ability to denature cell proteins and damage cells membranes irrevers- ibly (ZHAO et al., 2009). The action of SDS was much better than that of levulinic acid in this particular study when they were used individu- ally. This is because levulinic acid has a shorter carbon chain (5 carbons and a hydroxyl group), which makes it a weak acid, therefore its effec- tiveness is less than SDS. Extra care must be taken if SDS is employed in fresh produce as it was established during this study that very low concentrations of 1% can have a very large im- pact on survival of pathogens. A combination of 0.05% SDS and 0.5% lev- ulinic acid was also used in this study. Find- ings show that this mixture achieved better re- sults as compared to levulinic acid alone. Many researchers have reported on the advantages of mixing SDS and levulinic acid. The findings of ZHAO et al. (2009) show an increased antimicro- bial activity by the combination of SDS and lev- ulinic acid against Salmonella and E. coli O157: H7. Gurtler and Jin (2012) found that a combi- nation of 2% acetic acid, lactic acid and levulin- ic acid reduce Salmonella on tomatoes. ORTEGA et al. (2011) reported that a combination of lev- ulinic acid and SDS was highly effective against E. coli when exposure times were increased to 30 and 60 minutes. On the contrary, GUAN et al. (2010) reported that a combination of these had no commercial value as they have detrimental effects on the quality of fresh produce. Combin- ing sanitizers has shown to have a positive con- tribution in the food market. This has potential for implementation in the fresh produce indus- try. The implementation of a combination of san- itizers can be tried together with an assessment of their effects on sensory qualities. Increasing exposure time significantly de- creased the surviving colonies of L. monocy- togenes. In this particular study, a greater fall in surviving colonies was achieved at 5 minutes exposure time. This is evidence that the longer the bacteria are exposed to chemicals, the great- er the chances of reducing their survival. PARK et al. (2011) also reported that log reductions increase with increasing contact times. Other writers indicate that an exposure time of 3 min- utes is effective against food pathogens (MATT- SON et al., 2011). DING et al. (2011) and Møretrø et al. (2012) also reported that the effectiveness of a sanitizer depends on treatment time. Other writers also note that a significant decrease in the bacterial counts occurs in the first minute and the subsequent decrease after one minute is not significant (STEBBINS et al., 2011; TIRPANA- LAN et al., 2011). From the reports written by other writers and from this research, it can be said that contact time is one of the factors that should be monitored when using sanitizers. In- sufficient contact time will lead to high surviv- al after treatment, while extended contact time may lead to damage in the sensory qualities of fresh produce. The bacteria where further stored for a peri- od of 72 hours at 4 0 C. During this storage pe- riod L. monocytogenes survived up to 72 hours after being treated with sanitizers, except in Ital. J. Food Sci., vol. 27 - 2015 205 SDS. Survival of pathogens after a storage peri- od of 72 hours is also reported by IJABADENIYI et al. (2011b). Sufficient exposure of pathogens to sanitizers is paramount to reduce surviving colonies, as some have the ability to recover even after being treated with sanitizer. For sanitizers to be effectively used on toma- toes, they should cause negligible changes to pH and titratable acidity of the tomatoes, the ma- jor determinants of tomato quality (ANTHON et al., 2011). In this research it was revealed that sanitizers can alter the physicochemical attrib- utes of fresh tomatoes if the sanitizers come into contact with the sub surfaces, thereby affect- ing the final sensory quality of tomatoes. Oth- er recent studies also point out that sanitizers can affect sensory qualities of fresh produce to some extent (PÉREZ-GREGORIO et al., 2011). In this study, major effects were noted on the pH, TA and TSS with levulinic acid and mixture. Changes effected by SDS and chlorine were not significant. These findings pronounce SDS as a better sanitizer to replace the routine chlorine washing as it causes minimal changes to quality. In previous studies, SDS was tested togeth- er with organic acids and hydrogen peroxide on blue berries and no significant difference was detected in pH and total anthocyanin value be- tween untreated and treated blueberries (LI and WU, 2013). In another study using Iceberg and Romane lettuce, chlorine had high quality scores for Romane lettuce, but caused quality deterio- ration on Iceberg lettuce. A combination of SDS and Tsunami did not show any effect on senso- ry attributes of Iceberg lettuce either (SALGADO et al., 2013). Though levulinic acid did not have favourable results in this particular study, previous studies report that using levulinic acid caused no sen- sory changes in turkey meat and pork sausages (VASAVADA et al., 2003). A combination of SDS and levulinic did not give favourable results in this study. Other studies also report that SDS used in combination with other sanitizers such as levulinic acid are of low commercial value com- pared to chlorine, since they cause detrimental effects to sensory attributes (GUAN et al., 2010). Total soluble solids were reduced for all treat- ments with levulinic acid having the highest re- ductions followed by SDS/Lev mixture. This could have been attributed to leaching of con- tents into treatment solutions as a larger surface area of the subsurface area of tomatoes was ex- posed. Leaching of materials is also reported by ALEGRIA et al. (2009). Though previous studies also report that longer contact times result in deterioration of sensory characteristics (Rico et al., 2007), there was no significant difference for all attributes tested in relation to contact time in this particular study. Contact of sanitizers with sub-surfaces of fresh produce should be mini- mised to prevent unnecessary damage to sen- sory quality attributes. ACKNOWLEDGEMENTS The authors would like to extend their sincere gratitude to the Durban University of Technology, Faculty of Applied Sci- ences, for the financial support towards this study. The au- thors also want to thank the University of KwaZulu Natal microscopy unit for their contributions towards this study. CONCLUSION This works confirms that use of sanitizers in food processing at shorter contact time of 1 min- ute may not eradicate food borne pathogens. SDS alone is capable of destroying L. monocy- togenes, causing no detrimental effect to sen- sory attributes of tomatoes. It is also important to consider exposure time to increase the effec- tiveness of sanitizers. Sanitizers can have detri- mental effects on the sensory attributes of fresh produce; hence careful consideration is required when selecting sanitizers for particular produce. 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