Ultrasound Improves Antimicrobial Effect of Sodium Hypochlorite and Instrumental Texture on Fresh-Cut Yellow Melon

Post Graduate Program in Food Science, Institute of Chemistry, Federal University of Rio de Janeiro, Av. Athos da Silveira Ramos, 149, Cidade Universitária, 21941-909 Rio de Janeiro, Brazil Analytical and Molecular Laboratory Center, Faculty of Veterinary Medicine, Fluminense Federal University, Vital Brazil Filho, 64, Niterói, 24230-340 Rio de Janeiro, Brazil Department of Food Engineer, Federal University of Espı́rito Santo, Alto Universitário, s/n, Alegre, 29500-000 Espı́rito Santo, Brazil Department of Food Technology, Federal University of Viçosa, Av, PH Rolfs, s/n, Campus Universitário, Viçosa, 36570-000 MG, Brazil National Institute of Health Quality Control, Oswaldo Cruz Foundation, Manguinhos, 21040-900 Rio de Janeiro, Brazil


Introduction
Sanitation is the critical stage to reduce microbiological contaminants in fruits and vegetables.Several chemical compounds have been used with high e ciency at this stage.Sodium hypochlorite is the most commonly used sanitizer in the food industry [1,2].Its antimicrobial e ect is due to penetration into the target cell followed by its dissociation into hypochlorous acid [3,4].is compound results in the occurrence of oxidation reactions and reduction of intracellular pH (hypochlorite ion (OCl − ) and proton (H + )) that alter cellular metabolism.Essential cell compounds such as proteins, lipids, and mainly enzymes are damaged/inactivated and generate cell death [5].To improve the antimicrobial e ect of sanitizers, other preservation methods have been combined following the hurdle theory [6].Among the nonthermal preservation technologies such as atmospheric cold plasma [7], high hydrostatic pressure [8], UV-C radiation [9], and ultrasound (US) [10] can be e ciently applied in the decontamination process with high potential to maintain food quality.
US is considered a green technology for not generating waste and being environmentally friendly [10,11].e antimicrobial effect of US is due cavitation generated by sound waves in the liquid medium [11].During this process, the formation of bubbles occurs.Sound waves cause compression and expansion of the bubbles [12].On the critical point, the bubbles implode and form microjets. e shear force generated by microjets can break or form pores in the membrane of microorganisms and lead to cell death [11,13].In addition, during bubble implosion, hot spots and free radicals are formed causing DNA fragmentation [13].In this way, US has been applied to reduce pathogenic and spoilage microorganisms of fruits and vegetables.
Several fruits and vegetables have a high risk of causing diseases by being consumed fresh.Among the vegetables, melon has great global economic importance and good consumer acceptance [14,15].Melon is often consumed raw, and due to this, were recorded several outbreaks.In the USA, three deaths and 94 hospitalizations occurred due to consumption of cantaloupe contaminated with Salmonella serotype Typhimurium and Salmonella serotype Newport [16].In addition, 33 deaths and 143 hospitalizations occurred due to contamination by Listeria monocytogenes [17].Spoilage bacteria are also very important for fruits.
e spoilage microbiota causes important physicochemical changes [10], and texture is one of the main physical factors that determine fruit quality [14,18].
US combined with sanitizers is promising in the decontamination process.However, a long application period may cause undesirable physicochemical changes.In this way, the US effect on the physicochemical characteristics of fruits and vegetables remains to be elucidated.erefore, the aim of this study was to evaluate the effects of US isolated and combined with sodium hypochlorite in natural microbiota reduction and physicochemical changes of freshcut yellow melon.

Materials and Methods
2.1.Sample Preparation.Melons (Cucumis melo L.) were obtained at the local supermarket and transported in isothermal boxes to the laboratory for analysis.Melons were washed in tap water.After this step, the melons were sliced in 5 × 5 × 20 cm (length × width × height).A total of 300 g of melon were submitted for each sanitization treatment.e experiment was conducted with three experimental and analytical replicates.

Sanitization Procedures.
Treatments were applied using sterilized water (control, SW), sodium hypochlorite (NaOCl) at 100 mg/L (Alphatec, Rio de Janeiro, Brazil), and ultrasound (US) (a capacity of 12 L) (Model Soniclean 15, Sanders Medical ® , Minas Gerais, Brazil) at 40 kHz and 500 W for 5 min according to Rosário et al. [10] and Duarte et al. [19].e US power (P) dissipated into the liquid was calculated using equation (1), and 100.32 W (0.33 ± 0.01 W/ g) was obtained.Acoustic energy density (AED) dissipated in the US bath was 0.10 KJ/g (equation ( 2)). e combination of ultrasound and sodium hypochlorite (US + NaOCl) was performed in the same conditions previously described.A total of 2 L of the sanitizer solution (0.15 g/mL, amount sufficient to avoid overlapping of the fresh cut in the US bath) at 7 °C were used for each treatment.e temperature was maintained using ice cubes: where C p is the water specific heat (4.18 J/g•K), m is the mass of water on the US bath (g), zT denotes the increase in the T ( °C), and zt is the sonication time (s).
Appropriate serial dilutions were performed, and aliquots were added in plate count agar (PCA) (HiMedia ® , Mumbai, India) and incubated at 35 ± 0.1 °C for 48 h [20].e results were expressed in log 10 of the colony-forming units per gram of melon (log•cfu/g).e decimal reduction (DR) was calculated (equation ( 3)). e value of N corresponds to the MAB (cfu/g) count after the sanitization treatment and the value of N 0 to the control (SW) treatment: (3)

pH.
e pH values of the melons were measured using the digital pH meter (Model mPA-210, Tecnopon, São Paulo, Brazil).e electrode was immersed in 10 g of melon pulp diluted in 100 mL of distilled water [21].

Total Titratable Acidity (TTA)
. TTA was performed using the titrimetric method.A total of 10 g of sample pulp were homogenized with 100 mL of distilled water.Titration was performed using 0.1 N NaOH and 1% phenolphthalein indicator [21].e results were expressed in grams of citric acid per 100 g of melon (percentage, %).

Texture Profile Analysis.
Texture profile analysis (TPA) was performed using Texture Analyzer (Brookfield ® , Model CT3, São Paulo, Brazil).e analyzer was equipped with a 10 kg load cell, and the TA44 probe was used.e analysis was performed following the conditions of a pretest speed of 5.0 mm/s, a test speed of 1.0 mm/s, a posttest speed of 8.0 mm/s, and a penetration distance of 4 mm [22].e parameters of firmness, adhesiveness, springiness, cohesiveness, gumminess, and chewiness were obtained.

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Journal of Food Quality

Statistical Analysis.
To evaluate the signi cant e ect of treatments on the reduction of bacterial microbiota and physicochemical characteristics, one-way ANOVA was performed using Software Assistant ® [23].To verify the di erence between the treatments, Tukey's test was performed.e signi cance level used was 0.05.

E ect of Treatments on the Bacterial Microbiota.
Sanitization is the critical step for reducing microbiological contaminants and the risk of food-borne diseases [24,25].In the present study, the initial contamination of MAB in the melon was 2.9 log•cfu/g (Figure 1).US + NaOCl reduced the microbial count to 2.5 log•cfu/g (0.4 log•cfu/g).A synergistic e ect between US and NaOCl in natural microbiota reduction of the melon was obtained (Figure 1).São José et al. [26] found an improvement in the e ect of lactic acid (1%) and acetic acid (1%) when combined with US (40 kHz) for 2 min in Salmonella serotype Enteritidis reduction on the melon surface.Park et al. [27] also found a synergistic e ect between NaOCl at 100 mg/L and US (35 kHz, 380 W) applied for 5 min on lettuce.e occurrence of the synergistic e ect in the present study is due to the cavitation process formed during the US application (Figure 2).e intense pressure generated by this phenomenon can propel the active principle of the sanitizer into the microbial cell [12,13].US and NaOCl reduced the microbial count to 2.8 and 2.7 log•cfu/g (0.1 and 0.2 log•cfu/g), respectively, and no signi cant di erence (p > 0.05) was obtained.In addition, both treatments did not di er (p > 0.05) from the SW. is fact demonstrates the low e ciency of these isolated methods.e low chlorine e ciency in the microorganisms inactivation has been reported [13,28].Although some countries in Europe (Belgium, Denmark, Germany, and the Netherlands) have prohibited the use of chlorine, this sanitizer is still widely used by the food industry [28,29].erefore, due to the chlorination's ability to cause selection of chlorine-resistant bacteria [30], the high use of this sanitizer may be causing its ine ciency in the inactivation of bacteria.Likewise, low ultrasonic e ciency was also observed in the natural microbiota inactivation of fruits and vegetables [19,31,32]. is e ect is con rmed by the abundant presence of microbiological contaminants in the sanitizing solution after sanitization [10].ese phenomena state that US has the e ect of removing surface microorganisms [33] and improving the antimicrobial e ect of sanitizers.

E ect of Treatments on Physicochemical Characteristics.
Sanitation is an important step for the reduction of microbiological contaminants.However, physicochemical alterations of fruits and vegetables may be a limiting factor for the application of this procedure.For pH and TTA, no signi cant di erence (p > 0.05) between treatments (Table 1) and overall mean 6.46 and 0.13%, respectively, were obtained.Palekar et al. [34] found alteration of melon pH using a nonthermal preservation technology (electron beam irradiation, 1.5 kGy).pH and TTA unchanged at the present study are important results indicating that the oxidative e ect of NaOCl (100 mg/L) and chemical as well as thermal e ects of US are not able to change these values when applied for 5 min.
is fact demonstrates that the US-assisted chemical sanitation is a promising combined preservation method of melons.
Texture pro le has great importance mainly due to the acceptance and shelf life of foods.Firmness can be described as cell wall resistance and intracellular adhesions [35,36].Signi cant di erence of rmness (p < 0.05) between treatments was obtained and US increased this parameter (Table 1).Conversely, São José and Vanetti [32] evaluated rmness immediately after sanitization, and this parameter reduced with the US application (45 kHz) in isolated or combined with sodium dichloroisocyanurate (50 and 200 mg/L) or chlorine dioxide (10 mg/L) on strawberries for 10 min.In the present study, the time of 5 min was used. is fact demonstrates that the long period of US application can decrease rmness of fruits.Firmness reduction can be attributed to the mechanical e ects of cavitation that break down plant tissues [37].However, sanitization of 5 min improved rmness on melons.is e ect is possible because the US has the ability to increase the di usion of substances [38]. is process may have caused compounds homogenization in the melon such as water together with water soluble substances.In this way, US increased rmness without damage to the fruit cells.However, this phenomenon as well as its mechanisms of action need to be better understood.
Adhesiveness is the force required to remove the specimen from the sample [39] which indicates the adhesion properties of the sample.For this parameter, no signi cant di erence (p > 0.05) between treatments and overall mean of 2685.09g/mm were obtained.is result is important for the reduction of microbiological contaminants without physical alterations.Springiness indicates the elastic capacity of the matrix [35], and no signi cant di erence (p > 0.05) between treatments and overall mean of 25.08 g/mm was achieved.Garcia-Loredo et al. [40] found springiness reduction using ascorbic acid 1% and calcium chloride 0.1% in fresh-cut pear.e present study demonstrates that US application does not alter the elastic structure and adhesiveness of melon.
Cohesiveness is a parameter used to refer to the strength of the internal bonds of fruits [41].Gumminess is a term used to represent the disintegration ability of the sample to be swallowed by the consumer [42].In this study, greater values (p < 0.05) of cohesiveness and gumminess in melons treated with US when compared to the control were obtained (Table 1).e US has an ability to inactivate enzymes, remove oxygen trapped inside the fruits [13], and redistribution of water through cell substructures [43], causing this observed e ect.Moreover, it is known that these parameters are naturally reduced with the start of spoilage [22].erefore, the US can maintain these texture parameters and the integrity of the fruit for a longer period after the sanitization.
Chewiness can be de ned as the energy for chewing food until swallowing [44].US used alone did not di er from control (p > 0.05) and caused an increase on chewiness (Table 1).In fruits, this parameter decreases with storage time [42].erefore, this fact makes it increase an important factor.e NaOCl treatments did not di er (p > 0.05) from the control.erefore, it is possible to explain that the oxidative e ect of this sanitizer does not cause undesirable changes in melon chewiness applied for 5 min.

Conclusion
US improved the sodium hypochlorite e ect on reducing the bacterial microbiota of melon.A synergistic e ect in USassisted chemical sanitation was achieved.is hurdle e ect did not a ect physicochemical characteristics.Furthermore, texture parameters such as rmness, cohesiveness, gumminess, and chewiness have been improved with the US application.erefore, to sanitize melon, US (40 kHz, 500 W) for 5 min (0.10 KJ/g) is appropriate.Finally, US has a potential of fruit industrial application combined to sodium hypochlorite in the decontamination process of fresh-cut yellow melon.

Data Availability
e data used to support the ndings of this study are available from the corresponding author upon request.

Figure 2 :
Figure 2: (a) Hypochlorous acid penetrates the bacteria and dissociates.(b) e bubble implosion generated by the cavitation process forms a microjet that impulses hypochlorous acid into the bacterial cell.erefore, a greater amount of this compound is obtained in the cellular cytoplasm resulting in a greater antibacterial e ect of sodium hypochlorite.
e di erence is signi cant at p < 0.05 by Tukey's test. *

Table 1 :
Mean values of texture pro le analysis and physicochemical parameters of melon., sterilized water; US, ultrasound (40 kHz, 500 W); NaOCl, sodium hypochlorite (100 mg/L); TTA, total titratable acidity (%).Means followed by the same letter in the same column do not di er from each other (p > 0.05) by Tukey's test. SW