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Chlorination management in commercial fresh produce processing lines
Food Control 106 (2019) 106760
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Chlorination management in commercial fresh produce processing lines Juan A. Tudela, Francisco López-Gálvez, Ana Allende, María I. Gil
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T
Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, Campus Universitario de Espinardo, 30100, Murcia, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Leafy vegetable Antimicrobial treatment Microbial safety Processing wash water Chlorate Disinfection by-product
Despite all the information available and the fact that the fresh produce industry has been active for more than 30 years, chlorination management of process wash water (PWW) still needs to be further optimized. It is necessary to adjust the chlorination management to the demanding goals of avoiding microbial cross-contamination and the presence of disinfection by-products (DBPs). In the present study, two commercial processing plants were examined, and the impact of chlorination management was studied by comparing two chlorine sources (chlorine gas and liquid chlorine) under manual and automatic control procedures. The effect of the reduction of water replenishment rates, product type and cut size, as well as product rinse, were also evaluated. Results showed that there was no presence of chlorate in PWW (< 0.001 mg/L) and in the product (< 0.001 mg/ kg) when chlorine gas alone was used, while chlorate accumulation occurred in water (up to 16 mg/L) and product (> 2 mg/kg) when liquid chlorine was combined to cope with the chlorine demand. Chlorination management using the automatic control system led to better free chlorine and pH regulation. pH levels using the automatic control were in the range 5.5–6.5, while pH was in the range 6.5–8.5 when using manual control systems. Fluctuations in the physicochemical and microbiological characteristics of PWW were linked to changes in product type and cut size. Chemical oxygen demand (COD) of PWW was twice as high when shredded lettuce was washed (1000–1800 mg/L) compared with chopped lettuce (500–900 mg/L). The microbial load in PWW was higher when washing baby leaves (≈4.0 log cfu/100 mL) compared with cut lettuce (≈2.5 log cfu/100 mL), and higher when washing shredded lettuce compared with chopped lettuce. Product type and cut size significantly affected chlorate residues removal by produce rinse. Tap water rinsing led to significant reductions (p < 0.05) in chlorate concentration in the case of chopped lettuce, but not in the case of shredded lettuce and baby leaves. This study highlights the importance of adequate chlorination management for chlorate residue mitigation, including the selection of chlorine source, control system, and proper water rinse. The concept that ‘one size does not fit all’ should be present when establishing the operational procedures for different commodities and product formats.
1. Introduction In the fresh produce industry, disinfection of process wash water (PWW) is necessary to prevent the transfer of pathogenic microorganisms through the water between product lots (Danyluk & Schaffner, 2011; Gil, Selma, López-Gálvez, & Allende, 2009; Gombas et al., 2017; Maffei, Sant'Ana, Franco, & Schaffner, 2017). Fecal bacterial indicators have been detected in PWW at industrial scale when no antimicrobial agents are in place (Hamilton, Mebalds, Aldaoud, & Heat, 2005; Holvoet, Jacxsens, Sampers, & Uyttendaele, 2012). Chlorine-based antimicrobial treatments are widely used by the fresh produce industry to improve the microbiological status of PWW (Fu, Li, Awad, Zhou, & Liu, 2018; Teng et al., 2018). Control systems should maintain a desired free
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chlorine level at a suitable pH for the effective antimicrobial action of chlorine during industrial produce washing (Munther & Wu, 2013; Suslow, 2001). Several lab-scale studies have been performed to assess the antimicrobial efficacy of chlorine in produce washing (GómezLópez, Lannoo, Gil, & Allende, 2014; Luo et al., 2011). In contrast, few studies have addressed the same topic at industrial scale. Luo et al. (2018) assessed the minimum effective dose of free chlorine in industrial leafy greens wash water, and suggested a minimum level of 10 mg/L. López-Gálvez, Tudela, Allende, and Gil (2018), also at industrial scale, observed the presence of microorganisms in produce wash water with free chlorine concentrations > 20 mg/L, probably due to a non-optimal pH regulation. Recent results obtained at lab scale (Tudela et al., 2019) suggest that different products require different
Corresponding author. E-mail address: [email protected] (M.I. Gil).
https://doi.org/10.1016/j.foodcont.2019.106760 Received 16 April 2019; Received in revised form 8 June 2019; Accepted 7 July 2019 Available online 09 July 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
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shredded (6 mm-wide pieces) iceberg lettuce.
free chlorine concentrations in order to maintain the microbial quality of the wash water. Apart from the antimicrobial efficacy, the chemical risk associated with the formation and accumulation of disinfection by-products (DBPs) is considered as a primary concern that the fresh produce industry is facing nowadays (Gil, Marín, Andujar, & Allende, 2016; Shen, Norris, Williams, Hagan, & Li, 2016). Coroneo et al. (2017) and Gil et al. (2016) detected trihalomethanes and chlorates, respectively, in ready to eat vegetables produced in companies that use chlorinated wash water. Chlorate is a DBP resulting from the use of chlorine disinfectants in food and drinking water processing, whose presence in fresh produce poses a potential risk for consumers (EFSA, 2015; EFSA, 2019). The European Food Safety Authority (EFSA), using data collected between 2014 and 2018, indicated that chlorate residues are present at levels that frequently exceed the default maximum residue levels (MRLs) of 0.01 mg/kg, but the general trend towards decreasing levels suggests that manufacturing practices are improving (EFSA, 2019). An optimum chlorination management procedure aims to reduce the microbiological risk without increasing the chemical risk due to the chlorate residues (Tudela et al., 2019). Despite the existing body of knowledge, chlorination management is often poorly performed in many fresh produce processing plants (López-Gálvez et al., 2018). Scientific research seeks advances in the monitoring and control of fresh produce washing operations (Azimi, Munther, Fakoorian, Nguyen, & Simon, 2017; CPS, 2016; Zhou, Luo, Nou, & Millner, 2014). Water reuse is a regular practice by the fresh produce industry to reduce the high-water demand (Casani, Rouhany, & Knøchel, 2005). In particular, the washing process is the processing step that requires the highest water needs (Manzocco et al., 2015). For water saving interventions, reducing water inflow in the washing tank could be a potentially appropriate strategy for the washing operations. The working procedures established for chlorination management have to be flexible enough to be adapted to fluctuating conditions of high and low chlorine demand depending on the product. In general, different types of products are handled in the same production line without considering the specific characteristics of each one (López-Gálvez et al., 2018). It is expected that different products affect the microbiological and physicochemical characteristics of PWW, including DBPs (Fan & Sokorai, 2015) differently. Therefore, proper chlorination management for industrial produce washing is still challenging (Luo et al., 2018; Warriner & Namvar, 2013). The present study aimed to gather relevant data on the chlorination management of PWW at commercial processing conditions to help the fresh produce industry understand the implications of the selection of the operational standards. The obtained data were used to elaborate on some important concepts on chlorination management and chlorate residue mitigation, including the impact of: i) chlorine source, ii) control system, and iii) product type and cut size.
2.2. Processing line design The processing line in PP1 included two tanks (primary wash and secondary wash) (Fig. 1) of 1500 L of cold (≈6 °C) chlorinated tap water. Water was recirculated from the second tank to the first tank. The product was moved along the tanks by the water flow. The product was transferred from the first to the second tank and went out of the second tank via perforated conveyor belts. Between the first and the second tank, the product was rinsed by showers of chlorinated water coming from the second tank. After the secondary wash, the product was rinsed by showers of cold tap water. The washing time from the produce entrance to the showers was 60 s. In PP2, there was only one washing tank with a volume of 3500 L. To reduce the water needs, in samplings 6, 7 and 8 (automatic procedure) water used for the rinsing step was recycled into the washing tank, while in samplings 4 and 5 (manual procedure) the rinsing water was discarded (Table 2). At the exit of the tank, the product was rinsed with cold tap water showers. The washing time from the produce entrance to the showers was 30 s. In both processing plants, there were water catch tanks below the washing tanks to collect the water from the perforated conveyor belts as well as the overflow water from the washing tanks. Except in the case of the manual procedure in PP2, this water was incorporated into the washing tanks at the point where the product was introduced. The flow of the water ensured the homogeneous distribution of disinfectant in the washing tank. 2.3. Chlorination management procedures Sodium hypochlorite (NaClO) (Industrias Gamer, Murcia, Spain) as liquid chlorine and chlorine gas (Cl2) (Air Liquide, Spain) were the sources of free chlorine (FC). Two different chlorination control procedures were compared: i) a manual procedure, which leads to an inaccurate control of free chlorine and pH levels and represents the management performed nowadays in many fresh produce processing plants; and ii) an automatic control system, which was able to maintain specific chlorine and pH levels during the industrial produce washing. This automatic procedure was characterized by the accurate control of FC and pH through the ASAP™ unit (Automated SmartWash Analytic Platform, Smart Wash Solutions, Salinas, USA). In PP1, liquid chlorine was used as a source of FC under both the manual and the automatic chlorination procedures. When the manual procedure was used, liquid chlorine was added in both washing tanks within the range of 40–80 mg/L FC without pH control. In the automatic procedure, the FC goal was set at 10–20 mg/L, and the pH was set at 6.0 by adding phosphoric acid as an acidulant. In PP2, the main difference was that chlorine gas was used as a chlorine source. Chlorinated water was generated by injecting Cl2 into cold water. Chlorinated water (FC = 120 mg/L) was stored in a big container located in an adjacent room before use. As in PP1, two chlorination control procedures were used, manual and automatic procedures. In the manual procedure, the target residual FC was 80 mg/ L, chlorine demand was dealt with by replenishment with chlorinated water (Cl2, FC = 120 mg/L), and no pH regulator was used. In the automatic control procedure, the tank was filled and replenished with chlorinated water from chlorine gas (maximum chlorine 120 mg/L), but when there was a high FC demand, liquid chlorine was dosed to keep the desired FC concentration (10–20 mg/L). Also, under the automatic control procedure, the pH was adjusted to a target value of 6.0 with phosphoric acid.
2. Materials and methods 2.1. Processing plants and plant material Two different processing plants, PP1 and PP2, from the same fresh produce processor were selected for the study. Table 1 shows the commercial conditions of the eight samplings performed in PP1 (1–3) and PP2 (4–8). The eight samplings were distributed in three visits to PP1, and three more visits to PP2. Leafy vegetables such as baby leaves and cut lettuce, including Romaine and Iceberg lettuce, as the main product types for prepared salads, were examined. The products processed in PP1, samplings 1–3, included baby leaves such as lamb's lettuce (Valerianella locusta L.), rocket (Eruca sativa), tatsoi (Brassica rapa subsp. narinosa) and baby spinach (Spinacia oleracea L.), as well as chopped romaine and iceberg lettuce (Lactuca sativa L. var. longifolia and var. capitata) of 30–40 mm-wide pieces. The products processed in PP2, samplings 4–8, included chopped (30-40 mm-wide pieces) and
2.4. Water sampling and analysis Free chlorine (FC), pH, and temperature (T) were assessed each 2
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Table 1 Summary of the conditions of samplings (1–8) at the two processing plants (PP1 and PP2) when washing baby leaves and cut lettuce using liquid chlorine (NaClO) and chlorine gas (Cl2) under manual and automatic chlorination procedures. Sampling
Processing plant (PP)
Plant material
Product size
Chlorine Source
Chlorination Management
Product flow (kg/h)
1 2 3 4 5 6 7 8
1 1 1 2 2 2 2 2
Baby leaves + lettuce Baby leaves + lettuce Baby leaves + lettuce Lettuce Lettuce Lettuce Lettuce Lettuce
Whole + Chopped Whole + Chopped Whole + Shredded Shredded Chopped Shredded Chopped Shredded
Liquid Liquid Liquid Gas Gas Gas + Liquid Gas + Liquid Gas + Liquid
Manual Automatic Automatic Manual Manual Automatic Automatic Automatic
198a+190b + 730c 285a+792c 220a+351d 1,136e 1,765c 1,365e 581c 1,568e
In the context of this study, Baby leaves and whole leaf Batavia were considered whole product. a Baby leaves. b Whole leaf Batavia. c Chopped iceberg lettuce. d Shredded romaine and iceberg lettuce. e Shredded iceberg lettuce.
Fig. 1. Scheme of processing plant 1 (PP1) with two washing tanks (primary wash and secondary wash). Table 2 Water consumption of samplings (4–8) at the processing plant 2 (PP2) when washing shredded and chopped lettuce using chlorine gas (Cl2) combined or not with liquid chlorine (NaClO) under manual and automatic chlorination procedures. Sampling
Product size
Chlorine Source
Chlorination Management
Tank refillingsa (/h)
Chlorinated waterb inflow (L/h)
Rinse waterc flow (L/h)
Total consumption (L/h)
4 5 6 7 8
Shredded Chopped Shredded Chopped Shredded
Gas Gas Gas + Liquid Gas + Liquid Gas + Liquid
Manual Manual Automatic Automatic Automatic
1 0 0 0 0
3000 3000 2000 1200 2000
800 800 800 800 800
6800 3800 2800 2000 2800
a b c
Volume of the washing tank was 3500 L. 120 mg/L free chlorine. 1 mg/L free chlorine.
buffered peptone water (BPW, 2 g/L) (Oxoid, Basingstoke, UK). Samples were plated on plate count agar (PCA, Scharlab, Barcelona, Spain) and incubated at 30 °C for 36–48 h. Non-neutralized PWW samples were transported from the fresh produce processing plant to the CEBAS-CSIC lab in refrigerated conditions and used for the evaluation of chemical oxygen demand (COD) and chlorate content. COD was determined by the standard photometric method (APHA, 1998) using a photometer (Spectroquant NOVA 60, Merck). The concentration of chlorate was assessed using a UPLC-MS as described in Gil et al. (2016), and the results expressed in mg/L. Processing lines were monitored over 2–6 h.
15–20 min in PWW samples (100 mL) taken from the washing tanks. FC concentrations were evaluated by the DPD method (APHA, 1998) using the Spectroquant NOVA 60 photometer (Merck, Darmstadt, Germany) and the adequate test kits. Temperature and pH were measured with a portable multimeter sensION + MM150 (Hach, Loveland, Colorado, USA). The theoretical HClO concentration was calculated based on FC, T, and pH values (Randtke, 2010). The analyses of other physicochemical characteristics and total aerobic bacteria (TAB) were done in samples taken approximately every hour. Three samples were taken for TAB and three more for the study of physicochemical characteristics at each sampling time. For these analyses, six bottles (2 L) were filled up at each sampling point. For the enumeration of TAB, three of the bottles contained sodium thiosulphate pentahydrate (Scharlau, Barcelona, Spain) to quench the residual disinfectant. Neutralized water samples were taken to the laboratory located in the processing plant for the microbial analysis. Enumeration of TAB was performed by membrane filtration (0.45 μm) and surface plating. Serial dilutions were prepared when needed with
2.5. Fresh produce sampling and analysis Three samples of unwashed produce, three of washed produce, and three of rinsed produce were taken at each of the 3–6 time points of each trial. Fresh produce samples (200 g each) were taken in sterile bags and directly moved to the laboratory located in the processing 3
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plant. Each sample was centrifuged with a clean kitchen centrifuge for 1 min. Between sampling points, centrifuges were disinfected with ethanol (70%) and rinsed with tap water. For microbiological analyses, a portion (25 g) of each centrifuged sample was transferred to sterile stomacher bags and mixed with 100 mL of BPW (2 g/L) containing sodium thiosulphate (0.1 g/L) to neutralize disinfectant residuals. For the detection of TAB, pour and surface plating of the homogenized samples was performed using the same media and incubation conditions described for PWW analyses. The presence of chlorate in produce was analyzed in 30 g of centrifuged fresh produce sample, chopped during 10 s using a meat mincer (Moulinex A320, Moulinex, Ecully, France). Ten grams were mixed with 10 mL of a solution of formic acid (1%) in methanol. The mixture was sonicated for 2 min, briefly vortexed, and centrifuged (1900 g for 5 min). The supernatant was filtered through 0.22 μm filters (Sartorius Minisart PES; Sartorius, Gottingen, Germany) and diluted (1:10) using a solution of formic acid (1%) in methanol. Chlorate content was analyzed by UPLC-MS (Gil et al., 2016) and results expressed in mg/kg fresh weight (FW).
14
Chlorate (mg/L)
B
Gas+liquid shredded Gas shredded Gas+liquid chopped Gas chopped
16
12 10 8 6 4 2 0 0
500
1000
1500
2000
COD (mg/L) Fig. 2. Scatterplot of the presence of chlorate vs the chemical oxygen demand (COD) in process wash water for washing shredded and chopped lettuce with chlorine gas combined or not with liquid chlorine.
2.6. Statistical analysis
The most relevant results to discuss the recurrent problems that the fresh produce industry is facing regarding washing and chlorination are presented.
The presence of chlorate has been reported as the main disadvantage of using chlorine for the PWW disinfection. López-Gálvez et al. (2018) reported the presence of chlorate in commercial processing lines in which solid chlorine (calcium hypochlorite) in combination with liquid chlorine (sodium hypochlorite) were used as antimicrobial agents. Alfredo, Stanford, Roberson, and Eaton (2015) reported higher chlorate levels in drinking water treated with liquid chlorine (sodium hypochlorite) compared with chlorine gas. The formation of chlorate occurs during the fabrication and storage of highly concentrated solutions of liquid chlorine (Breytus, Kruzic, & Prabakar, 2017). Processing companies that use highly concentrated liquid solutions as wash water disinfectant should evaluate other mitigation strategies such as using a less concentrated solution, low storage temperature, short storage time, and the on-site generation of chlorine to reduce the build-up of chlorate levels before the use (ADAS, 2016). The fact that chlorine gas is not widely used by the fresh produce industry could be explained by the higher complexity of its application and the worker's safety issues associated with this form of chlorine (Breytus et al., 2017).
3.1. Chlorine source and the presence of chlorate
3.2. Chlorination management system
Fresh produce industry whose edible products are at risk of exceeding the proposed chlorate MRL (e.g. 0.15 mg/kg for leafy greens including lettuce, EFSA, 2019) should consider seriously using an alternative to chlorine. Chlorine alternatives include peroxyacetic acid and hydrogen peroxide, that lower the risk of contamination from disinfection by-products but do not have the same antimicrobial effect (Van Haute et al., 2015). Optimum chlorination management and residue mitigation in fresh produce are the best strategies to continue using chlorine. Our results showed that when chlorine gas was used, the concentration of chlorate in PWW was always below the limit of quantification (< 0.001 mg/L) while when liquid chlorine was added in combination with chlorine gas, high accumulation of chlorate as its degradation product in PWW occurred (Fig. 2). Higher COD levels were also linked to 60% of water reduction (Table 2). As the concentration of organic matter increased, the chlorine demand increased. Consequently, liquid chlorine was needed to be added to the PWW to maintain the desired residual FC. Under these circumstances, chlorate was accumulated in the PWW and was uptaken by the product, remaining even after product rinse (Fig. 3). As far as we know, this is the first study reporting that there is no chlorate accumulation in PWW when chlorine gas is used as chlorine source during industrial produce washing, suggesting that it could be used for residue mitigation on the product.
Hypochlorous acid (HClO) is the most active form of chlorine, and its concentration in chlorinated water depends to a great extent on the pH and T of the wash water (Randtke, 2010). Ideally, a pH below 6.5 should be maintained to increase the concentration of HClO in the PWW. Residual FC and the theoretical HClO present considering pH and T of the PPW are presented. When the chlorination management was performed using the manual procedure without FC and pH control, the differences in the concentrations of FC and HClO caused by the pH were clearly observed (Fig. 4A and 4C). According to the pH range between 7.0 and 8.5 and taking into account the T of the PWW (≈6 °C), the percentage of FC in the form of HClO was in the range 15–85% and 5–64% in the first and the second tanks, respectively. In contrast, when the chlorination management was performed using the automatic procedure, 95–100% of FC was in the form of HClO, which explains the overlap of FC and HClO as pH was maintained in the range 5.5–6.5 (Fig. 4B and 4D). Similar results were observed for chlorine gas. When the chlorination management was performed using the manual procedure, FC fluctuated between high concentrations (maximum FC = 67 mg/L) and almost absence (minimum FC = 0.9 mg/L) at a pH between 6.5 and 7 (Fig. 5A and 5C). When T of water (≈5 °C) was taken into account, the percentage of HClO was in the range 85–95% for shredded lettuce and 64–85% for chopped lettuce. In contrast, when the chlorination
Data on microbial populations were log-transformed. Graphs were made using Sigma Plot 14.0 Systat Software, Inc. (Addilink Software Scientific S.L., Barcelona, Spain). IBM SPSS statistics 24 was used for statistical analysis. Shapiro Wilk test and Levene's test were employed to assess the normality and equality of variance, respectively. When normality could be assumed, T-tests were used to compare two treatments. Assuming also normality, one-way ANOVA was performed to compare more than two treatments, with Tukey's HSD or Dunnett's as post hoc tests depending on the homogeneity of the variances. When data was not following a normal distribution, nonparametric tests (Mann–Whitney U and Kruskal–Wallis) were applied to determine the differences between treatments. 3. Results and discussion
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A
B a
3
4
a
3
2
2
1
1 <0.001
0
Unwashed
<0.001
<0.001
b <0.001
Washed
Rinsed
Unwashed
Chlorate (mg/kg)
Chlorate (mg/kg)
4
0 Washed
Rinsed
Fig. 3. Presence of chlorate in unwashed, washed, and rinsed shredded lettuce with chlorine gas (A), and chlorine gas combined with liquid chlorine (B). Different letters indicate significant differences (p < 0.05). Values are the mean ± standard deviation of n = 9 samples. See Table 1 for samplings 4 (graph A) and 6 (graph B).
58 ± 39 mg/L for baby leaves). Apart from the product type, differences in COD could be caused by the different product throughput as it was almost four times higher in the case of cut lettuce (≈800 kg/h) compared with baby leaves (≈230 kg/h) (Table 1). Differences in COD due to the product type have been reported in previous studies (LópezGálvez et al., 2018; Luo et al., 2018). Luo et al. (2018) reported significantly lower COD in chopped romaine lettuce and shredded iceberg lettuce wash water when compared with diced cabbage. López-Gálvez et al., 2018 observed significantly higher COD in a shredded vegetables processing line compared with chopped lettuce and baby leaves processing lines. In PP2, differences in COD were also due to the cut size as, despite the higher water consumption (Table 2), COD was significantly higher (p < 0.05) in shredded lettuce than in chopped lettuce PWW. The average COD when shredded lettuce was washed was more than twice as high as when chopped lettuce was washed both for the manual chlorine management procedure (1087 ± 435 vs 543 ± 54 mg/L, respectively), and the automatic procedure (1744 ± 292 vs 840 ± 41 mg/L, respectively). In general, higher COD leads to a higher demand for disinfectants and this adds difficulties to chlorination management. At the same time, if higher volumes of disinfectant are added to the PWW to maintain the fixed residual FC, there is a higher risk of the presence of DBP residues (Gómez-López, Marín, Medina-
management was performed using the automatic procedure, FC was correctly maintained and the pH ranged between 5.5 and 6.5 for shredded lettuce and between 6.0 and 6.5 for chopped lettuce, leading to theoretical percentages of HClO in the range 95–99% (Fig. 5B and 5D). The use of an inefficient chlorination management system created higher FC fluctuations in the PWW when processing shredded lettuce with a very high FC demand. The better adjustment of FC to satisfy chlorine demand and the regulation of pH under the automatic procedure had consequences on the efficacy of the inactivation of microorganisms in the PWW. In general, the population of TAB was significantly lower (p < 0.05) when the PWW disinfection was managed using the automatic procedure compared with the manual one (data not shown). 3.3. Product type and cut size Our results showed that the physicochemical and microbiological characteristics of PWW were affected by product type and cut size. Changes in the COD levels over time in PP1 when different fresh products were washed are shown in Fig. 6. The COD values detected, as mean of all the samplings, were significantly higher (p < 0.05) when the cut product was washed compared with whole leaves (mean ± standard deviation of 358 ± 238 mg/L for cut lettuce compared with
Batavia leaf
Chopped iceberg
50
40
40 Free chlorine HClO
Free chlorine HClO
30
30
20
20
10
10
0 10
C Baby leaves
Batavia leaf
Chopped iceberg
9
pH
Baby leaves
Chopped iceberg
D
Baby leaves
Chopped iceberg
0 10 9
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1 0
0 8:00
9:00
10:00
11:00
Time
12:00
13:00
8:00
9:00
10:00
11:00
Time 5
12:00
13:00
pH
Free chlorine/HClO (mg/L)
Baby leaves
Free chlorine/HClO (mg/L)
B
A 50
Fig. 4. Impact of chlorination management with liquid chlorine on the changes of free chlorine, hypochlorous acid (HClO), and pH in the process wash water. Samplings 1 (graphs A and C, manual procedure) and 2 (graphs B and D, automatic procedure). Vertical dotted lines indicate the change in the type of product washed. Values are the mean ± standard deviation of n = 2 samples for each sampling time. See Table 1 for sampling characteristics.
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Fig. 5. Impact of chlorination management with chlorine gas combined or not with liquid chlorine on the changes of free chlorine, hypochlorous acid (HClO), and pH of process wash water. Samplings 4 (graphs A and C, manual procedure) and 6 (graphs B and D, automatic procedure). Values are the mean ± standard deviation of n = 2 samples in each sampling time. See Table 1 for sampling characteristics.
between these two fresh products is so different is not apparent. One reason could be a hypothetical difference in the resistance of the microbiota to the antimicrobial treatment. This different resistance could be caused by differences in microbiome composition from the two types of produce washed (e.g., endospore-producing bacteria might be more prevalent on certain types of produce or in produce grown in a certain area or at certain seasons). Furthermore, it has been reported that the microbiota of baby leaves is more exposed to environmental stress than the microbiota of whole heads with a more closed structure (e.g.
Martínez, Gil, & Allende, 2013; López-Gálvez et al., 2018; Warriner & Namvar, 2013). The type of product being washed also has an impact on the microbiological quality of the PWW. This is illustrated in Fig. 7, which shows the changes in the populations of the TAB when the baby leaves and cut lettuce were washed. In all the cases, the TAB values detected in PWW were significantly higher (p < 0.05) when baby leaves were washed compared with cut lettuce (4.2 ± 0.3 and 2.4 ± 0.7 log cfu/ 100 mL, respectively). The reason why the microbial load of PWW
Fig. 6. Impact of product type and cut size on the chemical oxygen demand (COD) in the process wash water. Samplings 1, 2, and 3 (graphs A, B and C for tank 1 and graphs D, E and F for tank 2, respectively). Vertical grey dotted lines indicate the change in the type of product washed. See Table 1 for sampling characteristics. 6
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Fig. 7. Impact of product type and cut size on the population of total aerobic bacteria (TAB) in the process wash water. Samplings 1, 2, and 3 (graphs A, B and C for tank 1 and graphs D, E and F for tank 2, respectively). Vertical grey dotted lines indicate the change in the type of product washed. See Table 1 for sampling characteristics.
and/or in the water used for vacuum cooling. It is important to mention that the chlorate content in these samples was lower than the proposed chlorate MRL of 0.15 mg/L (EFSA, 2019). Our results also showed that differences in chlorate content between the washed and rinsed products were not always significant. In the case of chopped lettuce, the content of chlorate was significantly lower when the product was rinsed (p < 0.05) (Fig. 8B). In contrast, in the case of baby leaves and shredded lettuce, these differences were not significant (p > 0.05) (Fig. 8A and 8C). López-Gálvez et al. (2018) also reported high variability in the impact of the rinsing step as a residue mitigation of chlorate in the product at commercial processing facilities. These results highlight the variation of the outcome of the process at industrial scale and suggest that processing companies should further study and optimize this final tap water rinse as a residue mitigation strategy.
iceberg lettuce), and this higher exposition can induce tolerance to other stress factors, including chlorine treatment (Gruzdev, Pinto, & Sela, 2011). When different cut sizes were compared for the same type of produce (iceberg lettuce) in PP2, TAB populations in chlorinated PWW under manual chlorination management were significantly higher (p < 0.05) when washing shredded lettuce than chopped lettuce. The average TAB population in shredded lettuce PWW was 4.9 ± 0.7 log cfu/100 mL, whereas only 2.8 ± 0.6 log cfu/100 mL were detected in chopped lettuce PWW. This difference between different cut sizes observed for the same product could not be explained by the different product flow (kg/h), as it was higher when processing chopped lettuce than shredded lettuce (Table 1, samplings 4 and 5). Furthermore, water replenishment was higher when shredded lettuce was washed compared with chopped lettuce under manual chlorination management (Table 2, samplings 4 and 5). When lettuce was washed using the automatic procedure for the chlorination management, differences between the TAB population of PWW from shredded lettuce (3.7 ± 0.5 log cfu/100 mL) and chopped lettuce (2.5 ± 0.3 log cfu/100 mL) were reduced. A possible explanation for these results is that the organic matter released by the shredded product was higher than that of the chopped product, which might hamper the efficacy of disinfectants. Differences in physicochemical and microbiological characteristics of the PWW will generate different demand for disinfectants to maintain the quality of the PWW. A case-by-case study of each processing line is needed in order to establish an appropriate chlorination management system. The uptake of chlorate by the washed product is also affected by product type and cut size. Chlorate uptake has been reported to be higher when increasing the cut grade, being greater in shredded than in chopped or large cut lettuce (Garrido, Marín, Tudela, Allende, & Gil, 2019). In the present study, when the concentration of chlorate in the washed product was compared with the unwashed one, only in the case of chopped lettuce the concentration of chlorates was significantly higher (p < 0.05) (Fig. 8B), while no significant differences were observed for baby leaves and shredded lettuce (Fig. 8A and 8C). The chlorate detected in the unwashed product could be due to different causes, like the presence of chlorate in the water used for irrigation
4. Conclusions To obtain empirical evidence of basic concepts of chlorination management at industrial scale is not an easy task. This work provides and discusses industrial-scale data that illustrate some important factors related to produce washing. The shown concepts include the impact of the chlorine source on the occurrence of chlorate, the importance of proper pH regulation, the changes in the water characteristics due to the product type and cut size, and the effect of the rinsing step on the presence of chlorate in the product. The poor control of the chlorination management when manual procedures were used emphasize the need for automatic monitoring and control of the chlorination management. The fluctuating conditions regarding product type and cut size cause considerable variability in the physicochemical and microbiological characteristics of the PWW within one processing line. For each line, critical operational parameters should be identified as the type of product, the equipment design and the type of sanitizer selected will influence the chlorination management. Acknowledgments Authors are thankful for financial support from the MINECO (Project AGL2016-75878-R), the Center for Produce Safety Grant 7
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0.15
Chlorate (mg/kg)
B
Chopped iceberg lettuce
Shredded iceberg+romaine lettuce
ns
C 0.15
ns ns a
0.10 ns 0.05
0.00
ns
ns
c
hed y wash Unwas Primar
0.10
b
Rinsed
hed y wash Unwas Primar
Rinsed
hed y wash Unwas Primar
Chlorate (mg/kg)
Baby leaves
0.05
Rinsed
0.00
Fig. 8. Impact of product rinse on the chlorate residue mitigation in unwashed, primary washed and rinsed baby leaves (A), chopped lettuce (B) and shredded lettuce (C). Different letters indicate significant differences (p < 0.05). Values are the mean of each product evaluated in samplings 1, 2 and 3. See Table 1 for sampling characteristics.
Agreement (Project 2017–01) and CSIC (Intramural 201670E056). We also thank Silvia Andújar, Natalia Hernández, Alicia Marín and Yolanda Garrido for their technical assistance.
trihalomethane generation during dynamic washing of fresh-cut spinach. Food Control, 42, 132–138. Gómez-López, V. M., Marín, A., Medina-Martínez, M. S., Gil, M. I., & Allende, A. (2013). Generation of trihalomethanes with chlorine-based sanitizers and impact on microbial, nutritional and sensory quality of baby spinach. Postharvest Biology and Technology, 85, 210–217. Gruzdev, N., Pinto, R., & Sela, S. (2011). Effect of desiccation on tolerance of Salmonella enterica to multiple stresses. Applied and Environmental Microbiology, 77, 1667–1673. Hamilton, A. J., Mebalds, M. I., Aldaoud, R., & Heat, M. (2005). Physical, chemical and microbial characteristics of wastewater from carrot washing in Australia: Implications for reuse and environmental discharge. International Journal of Vegetable Science, 11, 57–72. Holvoet, K., Jacxsens, L., Sampers, I., & Uyttendaele, M. (2012). Insight into the prevalence and distribution of microbial contamination to evaluate water management in the fresh produce processing industry. Journal of Food Protection, 75, 671–681. López-Gálvez, F., Tudela, J. A., Allende, A., & Gil, M. I. (2018). Microbial and chemical characterization of commercial washing lines of fresh produce highlights the need for process water control. Innovative Food Science & Emerging Technologies, 51, 211–219. Luo, Y., Nou, X., Yang, Y., Alegre, I., Turner, E., Feng, H., et al. (2011). Determination of free chlorine concentrations needed to prevent Escherichia coli O157:H7 crosscontamination during fresh-cut produce wash. Journal of Food Protection, 74, 352–358. Luo, Y., Zhou, B., Van Haute, S., Nou, X., Zhang, B., Teng, Z., et al. (2018). Association between bacterial survival and free chlorine concentration during commercial freshcut produce wash operation. Food Microbiology, 70, 120–128. Maffei, D. F., Sant'Ana, A. S., Franco, B. D. G. M., & Schaffner, D. W. (2017). Quantitative assessment of the impact of cross-contamination during the washing step of ready-toeat leafy greens on the risk of illness caused by Salmonella. Food Research International, 92, 106–112. Manzocco, L., Ignat, A., Anese, M., Bot, F., Calligaris, S., Valoppi, F., et al. (2015). Efficient management of the water resource in the fresh-cut industry: Current status and perspectives. Trends in Food Science & Technology, 46, 286–294. Munther, D., & Wu, J. (2013). Enhanced surveillance on food-borne disease outbreaks: Dynamics of cross-contamination in biocidal wash procedure. Journal of Theoretical Biology, 321, 28–35. Randtke, S. J. (2010). Chemistry of aqueous chlorine. In Black, & V. Corporation (Eds.). White's handbook of chlorination and alternative disinfectants (pp. 68–173). (5th ed.). Hoboken, USA: John Wiley and Sons. Shen, C., Norris, P., Williams, O., Hagan, S., & Li, K. (2016). Generation of chlorine byproducts in simulated wash water. Food Chemistry, 190, 97–102. Suslow, T. (2001). Water disinfection. A practical approach to calculating dose values for preharvest and postharvest applications. Publication 7256 (pp. –). Oakland, CA: Division of Agriculture and Natural Resources, University of California. Teng, Z., Luo, Y., Alborzi, S., Zhou, B., Chen, L., Zhang, J., et al. (2018). Investigation on chlorine-based sanitization under stabilized conditions in the presence of organic load. International Journal of Food Microbiology, 266, 150–157. Tudela, J. A., López-Gálvez, F., Allende, A., Hernández, N., Andújar, S., Marín, A., et al. (2019). Operational limits of sodium hypochlorite for different fresh produce wash water based on microbial inactivation and disinfection by-products (DBPs). Food Control, 104, 300–307. Van Haute, S., López-Gálvez, F., Gómez-López, V. M., Eriksson, M., Devlieghere, F., Allende, A., et al. (2015). Methodology for modeling the disinfection efficiency of fresh-cut leafy vegetables wash water applied on peracetic acid combined with lactic acid. International Journal of Food Microbiology, 208, 102–113. Warriner, K., & Namvar, A. (2013). Postharvest washing as a critical control point in fresh produce processing: Alternative sanitizers and wash technologies. In J. Hoorfar (Ed.). Global safety of fresh produce: A handbook of best practice, innovative commercial solutions and case studies (pp. 71–102). Cambridge, UK: Woodhead Publishing. Zhou, B., Luo, Y., Nou, X., & Millner, P. (2014). Development of an algorithm for feedforward chlorine dosing of lettuce wash operations and correlation of chlorine profile with Escherichia coli O157:H7 inactivation. Journal of Food Protection, 77, 558–566.
References ADAS, Agricultural Development and Advisory Service (2016). Chlorine and its oxides, chlorate and perchlorate review. CP 154a. Agriculture and Horticulture Development Board. Alfredo, K., Stanford, B., Roberson, J.A., & Eaton, A. (2015). Chlorate challenges for water systems. Journal of the American Water Works Association., 107, E187–E196. APHA, American Public Health Association (1998). Standard methods for the examination of water and wastewater (20th ed.). Washington, DC: American Public Health Association. Azimi, V., Munther, D., Fakoorian, S. A., Nguyen, T. T., & Simon, D. (2017). Hybrid extended Kalman filtering and noise statistics optimization for produce wash state estimation. Journal of Food Engineering, 212, 136–145. Breytus, A., Kruzic, A.P., & Prabakar, S. (2017). Chlorine decay and chlorate formation in two water treatment facilities. Journal of the American Water Works Association., 109, E110–E120. Casani, S., Rouhany, M., & Knøchel, S. (2005). A discussion paper on challenges and limitations to water reuse and hygiene in the food industry. Water Research, 39, 1134–1146. Coroneo, V., Carraro, V., Marras, B., Marrucci, A., Succa, S., Meloni, B., et al. (2017). Presence of Trihalomethanes in ready-to-eat vegetables disinfected with chlorine. Food Additives & Contaminants: Part A, 34, 2111–2117. CPS (Center for Produce Safety). (2016). Project 2017-107F. Establishment of operating standards for produce wash systems through the identification of specific metrics and test methods. Available at:: https://www.centerforproducesafety.org/ researchproject/402/awards/Establishment_of_operating_standards_for_produce_ wash_systems_through_the_identification_of_specific_metrics_and_test_methods.html. Danyluk, M. D., & Schaffner, D. W. (2011). Quantitative assessment of the microbial risk of leafy greens from farm to consumption: Preliminary framework, data, and risk estimates. Journal of Food Protection, 74, 700–708. EFSA (2015). CONTAM Panel (EFSA Panel on Contaminants in the Food Chain). Scientific opinion on risks for public health related to the presence of chlorate in food. EFSA Journal, 13(4135), 103. EFSA. (2019). European commission regulation, amending annex III to regulation (EC) No 396/2005 of the european parliament and of the council as regards maximum residue levels for chlorate in or on certain products. Regulation - Ares(2019)334046 - 21/01/ 2019. Fan, X., & Sokorai, K. J. (2015). Formation of trichloromethane in chlorinated water and fresh-cut produce and as a result of reaction with citric acid. Postharvest Biology and Technology, 109, 65–72. Fu, T.-J., Li, Y., Awad, D., Zhou, T.-Y., & Liu, L. (2018). Factors affecting the performance and monitoring of a chlorine wash in preventing Escherichia coli O157:H7 crosscontamination during postharvest washing of cut lettuce. Food Control, 94, 212–221. Garrido, Y., Marín, A., Tudela, J. A., Allende, A., & Gil, M. I. (2019). Chlorate uptake during washing is influenced by product type and cut piece size, as well as washing time and wash water content. Postharvest Biology and Technology, 151, 45–52. Gil, M. I., Marín, A., Andujar, S., & Allende, A. (2016). Should chlorate residues be of concern in fresh-cut salads? Food Control, 60, 416–421. Gil, M. I., Selma, M. V., López-Gálvez, F., & Allende, A. (2009). Fresh-cut product sanitation and wash water disinfection: Problems and solutions. International Journal of Food Microbiology, 134, 37–45. Gombas, D., Luo, Y., Brennan, J., Shergill, G., Petran, R., Walsh, R., et al. (2017). Guidelines to validate control of cross-contamination during washing of fresh-cut leafy vegetables. Journal of Food Protection, 80, 312–330. Gómez-López, V. M., Lannoo, A.-S., Gil, M. I., & Allende, A. (2014). Minimum free chlorine residual level required for the inactivation of Escherichia coli O157:H7 and
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Chlorination management in commercial fresh produce processing lines Juan A. Tudela, Francisco López-Gálvez, Ana Allende, María I. Gil
∗
T
Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, Campus Universitario de Espinardo, 30100, Murcia, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Leafy vegetable Antimicrobial treatment Microbial safety Processing wash water Chlorate Disinfection by-product
Despite all the information available and the fact that the fresh produce industry has been active for more than 30 years, chlorination management of process wash water (PWW) still needs to be further optimized. It is necessary to adjust the chlorination management to the demanding goals of avoiding microbial cross-contamination and the presence of disinfection by-products (DBPs). In the present study, two commercial processing plants were examined, and the impact of chlorination management was studied by comparing two chlorine sources (chlorine gas and liquid chlorine) under manual and automatic control procedures. The effect of the reduction of water replenishment rates, product type and cut size, as well as product rinse, were also evaluated. Results showed that there was no presence of chlorate in PWW (< 0.001 mg/L) and in the product (< 0.001 mg/ kg) when chlorine gas alone was used, while chlorate accumulation occurred in water (up to 16 mg/L) and product (> 2 mg/kg) when liquid chlorine was combined to cope with the chlorine demand. Chlorination management using the automatic control system led to better free chlorine and pH regulation. pH levels using the automatic control were in the range 5.5–6.5, while pH was in the range 6.5–8.5 when using manual control systems. Fluctuations in the physicochemical and microbiological characteristics of PWW were linked to changes in product type and cut size. Chemical oxygen demand (COD) of PWW was twice as high when shredded lettuce was washed (1000–1800 mg/L) compared with chopped lettuce (500–900 mg/L). The microbial load in PWW was higher when washing baby leaves (≈4.0 log cfu/100 mL) compared with cut lettuce (≈2.5 log cfu/100 mL), and higher when washing shredded lettuce compared with chopped lettuce. Product type and cut size significantly affected chlorate residues removal by produce rinse. Tap water rinsing led to significant reductions (p < 0.05) in chlorate concentration in the case of chopped lettuce, but not in the case of shredded lettuce and baby leaves. This study highlights the importance of adequate chlorination management for chlorate residue mitigation, including the selection of chlorine source, control system, and proper water rinse. The concept that ‘one size does not fit all’ should be present when establishing the operational procedures for different commodities and product formats.
1. Introduction In the fresh produce industry, disinfection of process wash water (PWW) is necessary to prevent the transfer of pathogenic microorganisms through the water between product lots (Danyluk & Schaffner, 2011; Gil, Selma, López-Gálvez, & Allende, 2009; Gombas et al., 2017; Maffei, Sant'Ana, Franco, & Schaffner, 2017). Fecal bacterial indicators have been detected in PWW at industrial scale when no antimicrobial agents are in place (Hamilton, Mebalds, Aldaoud, & Heat, 2005; Holvoet, Jacxsens, Sampers, & Uyttendaele, 2012). Chlorine-based antimicrobial treatments are widely used by the fresh produce industry to improve the microbiological status of PWW (Fu, Li, Awad, Zhou, & Liu, 2018; Teng et al., 2018). Control systems should maintain a desired free
∗
chlorine level at a suitable pH for the effective antimicrobial action of chlorine during industrial produce washing (Munther & Wu, 2013; Suslow, 2001). Several lab-scale studies have been performed to assess the antimicrobial efficacy of chlorine in produce washing (GómezLópez, Lannoo, Gil, & Allende, 2014; Luo et al., 2011). In contrast, few studies have addressed the same topic at industrial scale. Luo et al. (2018) assessed the minimum effective dose of free chlorine in industrial leafy greens wash water, and suggested a minimum level of 10 mg/L. López-Gálvez, Tudela, Allende, and Gil (2018), also at industrial scale, observed the presence of microorganisms in produce wash water with free chlorine concentrations > 20 mg/L, probably due to a non-optimal pH regulation. Recent results obtained at lab scale (Tudela et al., 2019) suggest that different products require different
Corresponding author. E-mail address: [email protected] (M.I. Gil).
https://doi.org/10.1016/j.foodcont.2019.106760 Received 16 April 2019; Received in revised form 8 June 2019; Accepted 7 July 2019 Available online 09 July 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
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shredded (6 mm-wide pieces) iceberg lettuce.
free chlorine concentrations in order to maintain the microbial quality of the wash water. Apart from the antimicrobial efficacy, the chemical risk associated with the formation and accumulation of disinfection by-products (DBPs) is considered as a primary concern that the fresh produce industry is facing nowadays (Gil, Marín, Andujar, & Allende, 2016; Shen, Norris, Williams, Hagan, & Li, 2016). Coroneo et al. (2017) and Gil et al. (2016) detected trihalomethanes and chlorates, respectively, in ready to eat vegetables produced in companies that use chlorinated wash water. Chlorate is a DBP resulting from the use of chlorine disinfectants in food and drinking water processing, whose presence in fresh produce poses a potential risk for consumers (EFSA, 2015; EFSA, 2019). The European Food Safety Authority (EFSA), using data collected between 2014 and 2018, indicated that chlorate residues are present at levels that frequently exceed the default maximum residue levels (MRLs) of 0.01 mg/kg, but the general trend towards decreasing levels suggests that manufacturing practices are improving (EFSA, 2019). An optimum chlorination management procedure aims to reduce the microbiological risk without increasing the chemical risk due to the chlorate residues (Tudela et al., 2019). Despite the existing body of knowledge, chlorination management is often poorly performed in many fresh produce processing plants (López-Gálvez et al., 2018). Scientific research seeks advances in the monitoring and control of fresh produce washing operations (Azimi, Munther, Fakoorian, Nguyen, & Simon, 2017; CPS, 2016; Zhou, Luo, Nou, & Millner, 2014). Water reuse is a regular practice by the fresh produce industry to reduce the high-water demand (Casani, Rouhany, & Knøchel, 2005). In particular, the washing process is the processing step that requires the highest water needs (Manzocco et al., 2015). For water saving interventions, reducing water inflow in the washing tank could be a potentially appropriate strategy for the washing operations. The working procedures established for chlorination management have to be flexible enough to be adapted to fluctuating conditions of high and low chlorine demand depending on the product. In general, different types of products are handled in the same production line without considering the specific characteristics of each one (López-Gálvez et al., 2018). It is expected that different products affect the microbiological and physicochemical characteristics of PWW, including DBPs (Fan & Sokorai, 2015) differently. Therefore, proper chlorination management for industrial produce washing is still challenging (Luo et al., 2018; Warriner & Namvar, 2013). The present study aimed to gather relevant data on the chlorination management of PWW at commercial processing conditions to help the fresh produce industry understand the implications of the selection of the operational standards. The obtained data were used to elaborate on some important concepts on chlorination management and chlorate residue mitigation, including the impact of: i) chlorine source, ii) control system, and iii) product type and cut size.
2.2. Processing line design The processing line in PP1 included two tanks (primary wash and secondary wash) (Fig. 1) of 1500 L of cold (≈6 °C) chlorinated tap water. Water was recirculated from the second tank to the first tank. The product was moved along the tanks by the water flow. The product was transferred from the first to the second tank and went out of the second tank via perforated conveyor belts. Between the first and the second tank, the product was rinsed by showers of chlorinated water coming from the second tank. After the secondary wash, the product was rinsed by showers of cold tap water. The washing time from the produce entrance to the showers was 60 s. In PP2, there was only one washing tank with a volume of 3500 L. To reduce the water needs, in samplings 6, 7 and 8 (automatic procedure) water used for the rinsing step was recycled into the washing tank, while in samplings 4 and 5 (manual procedure) the rinsing water was discarded (Table 2). At the exit of the tank, the product was rinsed with cold tap water showers. The washing time from the produce entrance to the showers was 30 s. In both processing plants, there were water catch tanks below the washing tanks to collect the water from the perforated conveyor belts as well as the overflow water from the washing tanks. Except in the case of the manual procedure in PP2, this water was incorporated into the washing tanks at the point where the product was introduced. The flow of the water ensured the homogeneous distribution of disinfectant in the washing tank. 2.3. Chlorination management procedures Sodium hypochlorite (NaClO) (Industrias Gamer, Murcia, Spain) as liquid chlorine and chlorine gas (Cl2) (Air Liquide, Spain) were the sources of free chlorine (FC). Two different chlorination control procedures were compared: i) a manual procedure, which leads to an inaccurate control of free chlorine and pH levels and represents the management performed nowadays in many fresh produce processing plants; and ii) an automatic control system, which was able to maintain specific chlorine and pH levels during the industrial produce washing. This automatic procedure was characterized by the accurate control of FC and pH through the ASAP™ unit (Automated SmartWash Analytic Platform, Smart Wash Solutions, Salinas, USA). In PP1, liquid chlorine was used as a source of FC under both the manual and the automatic chlorination procedures. When the manual procedure was used, liquid chlorine was added in both washing tanks within the range of 40–80 mg/L FC without pH control. In the automatic procedure, the FC goal was set at 10–20 mg/L, and the pH was set at 6.0 by adding phosphoric acid as an acidulant. In PP2, the main difference was that chlorine gas was used as a chlorine source. Chlorinated water was generated by injecting Cl2 into cold water. Chlorinated water (FC = 120 mg/L) was stored in a big container located in an adjacent room before use. As in PP1, two chlorination control procedures were used, manual and automatic procedures. In the manual procedure, the target residual FC was 80 mg/ L, chlorine demand was dealt with by replenishment with chlorinated water (Cl2, FC = 120 mg/L), and no pH regulator was used. In the automatic control procedure, the tank was filled and replenished with chlorinated water from chlorine gas (maximum chlorine 120 mg/L), but when there was a high FC demand, liquid chlorine was dosed to keep the desired FC concentration (10–20 mg/L). Also, under the automatic control procedure, the pH was adjusted to a target value of 6.0 with phosphoric acid.
2. Materials and methods 2.1. Processing plants and plant material Two different processing plants, PP1 and PP2, from the same fresh produce processor were selected for the study. Table 1 shows the commercial conditions of the eight samplings performed in PP1 (1–3) and PP2 (4–8). The eight samplings were distributed in three visits to PP1, and three more visits to PP2. Leafy vegetables such as baby leaves and cut lettuce, including Romaine and Iceberg lettuce, as the main product types for prepared salads, were examined. The products processed in PP1, samplings 1–3, included baby leaves such as lamb's lettuce (Valerianella locusta L.), rocket (Eruca sativa), tatsoi (Brassica rapa subsp. narinosa) and baby spinach (Spinacia oleracea L.), as well as chopped romaine and iceberg lettuce (Lactuca sativa L. var. longifolia and var. capitata) of 30–40 mm-wide pieces. The products processed in PP2, samplings 4–8, included chopped (30-40 mm-wide pieces) and
2.4. Water sampling and analysis Free chlorine (FC), pH, and temperature (T) were assessed each 2
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Table 1 Summary of the conditions of samplings (1–8) at the two processing plants (PP1 and PP2) when washing baby leaves and cut lettuce using liquid chlorine (NaClO) and chlorine gas (Cl2) under manual and automatic chlorination procedures. Sampling
Processing plant (PP)
Plant material
Product size
Chlorine Source
Chlorination Management
Product flow (kg/h)
1 2 3 4 5 6 7 8
1 1 1 2 2 2 2 2
Baby leaves + lettuce Baby leaves + lettuce Baby leaves + lettuce Lettuce Lettuce Lettuce Lettuce Lettuce
Whole + Chopped Whole + Chopped Whole + Shredded Shredded Chopped Shredded Chopped Shredded
Liquid Liquid Liquid Gas Gas Gas + Liquid Gas + Liquid Gas + Liquid
Manual Automatic Automatic Manual Manual Automatic Automatic Automatic
198a+190b + 730c 285a+792c 220a+351d 1,136e 1,765c 1,365e 581c 1,568e
In the context of this study, Baby leaves and whole leaf Batavia were considered whole product. a Baby leaves. b Whole leaf Batavia. c Chopped iceberg lettuce. d Shredded romaine and iceberg lettuce. e Shredded iceberg lettuce.
Fig. 1. Scheme of processing plant 1 (PP1) with two washing tanks (primary wash and secondary wash). Table 2 Water consumption of samplings (4–8) at the processing plant 2 (PP2) when washing shredded and chopped lettuce using chlorine gas (Cl2) combined or not with liquid chlorine (NaClO) under manual and automatic chlorination procedures. Sampling
Product size
Chlorine Source
Chlorination Management
Tank refillingsa (/h)
Chlorinated waterb inflow (L/h)
Rinse waterc flow (L/h)
Total consumption (L/h)
4 5 6 7 8
Shredded Chopped Shredded Chopped Shredded
Gas Gas Gas + Liquid Gas + Liquid Gas + Liquid
Manual Manual Automatic Automatic Automatic
1 0 0 0 0
3000 3000 2000 1200 2000
800 800 800 800 800
6800 3800 2800 2000 2800
a b c
Volume of the washing tank was 3500 L. 120 mg/L free chlorine. 1 mg/L free chlorine.
buffered peptone water (BPW, 2 g/L) (Oxoid, Basingstoke, UK). Samples were plated on plate count agar (PCA, Scharlab, Barcelona, Spain) and incubated at 30 °C for 36–48 h. Non-neutralized PWW samples were transported from the fresh produce processing plant to the CEBAS-CSIC lab in refrigerated conditions and used for the evaluation of chemical oxygen demand (COD) and chlorate content. COD was determined by the standard photometric method (APHA, 1998) using a photometer (Spectroquant NOVA 60, Merck). The concentration of chlorate was assessed using a UPLC-MS as described in Gil et al. (2016), and the results expressed in mg/L. Processing lines were monitored over 2–6 h.
15–20 min in PWW samples (100 mL) taken from the washing tanks. FC concentrations were evaluated by the DPD method (APHA, 1998) using the Spectroquant NOVA 60 photometer (Merck, Darmstadt, Germany) and the adequate test kits. Temperature and pH were measured with a portable multimeter sensION + MM150 (Hach, Loveland, Colorado, USA). The theoretical HClO concentration was calculated based on FC, T, and pH values (Randtke, 2010). The analyses of other physicochemical characteristics and total aerobic bacteria (TAB) were done in samples taken approximately every hour. Three samples were taken for TAB and three more for the study of physicochemical characteristics at each sampling time. For these analyses, six bottles (2 L) were filled up at each sampling point. For the enumeration of TAB, three of the bottles contained sodium thiosulphate pentahydrate (Scharlau, Barcelona, Spain) to quench the residual disinfectant. Neutralized water samples were taken to the laboratory located in the processing plant for the microbial analysis. Enumeration of TAB was performed by membrane filtration (0.45 μm) and surface plating. Serial dilutions were prepared when needed with
2.5. Fresh produce sampling and analysis Three samples of unwashed produce, three of washed produce, and three of rinsed produce were taken at each of the 3–6 time points of each trial. Fresh produce samples (200 g each) were taken in sterile bags and directly moved to the laboratory located in the processing 3
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plant. Each sample was centrifuged with a clean kitchen centrifuge for 1 min. Between sampling points, centrifuges were disinfected with ethanol (70%) and rinsed with tap water. For microbiological analyses, a portion (25 g) of each centrifuged sample was transferred to sterile stomacher bags and mixed with 100 mL of BPW (2 g/L) containing sodium thiosulphate (0.1 g/L) to neutralize disinfectant residuals. For the detection of TAB, pour and surface plating of the homogenized samples was performed using the same media and incubation conditions described for PWW analyses. The presence of chlorate in produce was analyzed in 30 g of centrifuged fresh produce sample, chopped during 10 s using a meat mincer (Moulinex A320, Moulinex, Ecully, France). Ten grams were mixed with 10 mL of a solution of formic acid (1%) in methanol. The mixture was sonicated for 2 min, briefly vortexed, and centrifuged (1900 g for 5 min). The supernatant was filtered through 0.22 μm filters (Sartorius Minisart PES; Sartorius, Gottingen, Germany) and diluted (1:10) using a solution of formic acid (1%) in methanol. Chlorate content was analyzed by UPLC-MS (Gil et al., 2016) and results expressed in mg/kg fresh weight (FW).
14
Chlorate (mg/L)
B
Gas+liquid shredded Gas shredded Gas+liquid chopped Gas chopped
16
12 10 8 6 4 2 0 0
500
1000
1500
2000
COD (mg/L) Fig. 2. Scatterplot of the presence of chlorate vs the chemical oxygen demand (COD) in process wash water for washing shredded and chopped lettuce with chlorine gas combined or not with liquid chlorine.
2.6. Statistical analysis
The most relevant results to discuss the recurrent problems that the fresh produce industry is facing regarding washing and chlorination are presented.
The presence of chlorate has been reported as the main disadvantage of using chlorine for the PWW disinfection. López-Gálvez et al. (2018) reported the presence of chlorate in commercial processing lines in which solid chlorine (calcium hypochlorite) in combination with liquid chlorine (sodium hypochlorite) were used as antimicrobial agents. Alfredo, Stanford, Roberson, and Eaton (2015) reported higher chlorate levels in drinking water treated with liquid chlorine (sodium hypochlorite) compared with chlorine gas. The formation of chlorate occurs during the fabrication and storage of highly concentrated solutions of liquid chlorine (Breytus, Kruzic, & Prabakar, 2017). Processing companies that use highly concentrated liquid solutions as wash water disinfectant should evaluate other mitigation strategies such as using a less concentrated solution, low storage temperature, short storage time, and the on-site generation of chlorine to reduce the build-up of chlorate levels before the use (ADAS, 2016). The fact that chlorine gas is not widely used by the fresh produce industry could be explained by the higher complexity of its application and the worker's safety issues associated with this form of chlorine (Breytus et al., 2017).
3.1. Chlorine source and the presence of chlorate
3.2. Chlorination management system
Fresh produce industry whose edible products are at risk of exceeding the proposed chlorate MRL (e.g. 0.15 mg/kg for leafy greens including lettuce, EFSA, 2019) should consider seriously using an alternative to chlorine. Chlorine alternatives include peroxyacetic acid and hydrogen peroxide, that lower the risk of contamination from disinfection by-products but do not have the same antimicrobial effect (Van Haute et al., 2015). Optimum chlorination management and residue mitigation in fresh produce are the best strategies to continue using chlorine. Our results showed that when chlorine gas was used, the concentration of chlorate in PWW was always below the limit of quantification (< 0.001 mg/L) while when liquid chlorine was added in combination with chlorine gas, high accumulation of chlorate as its degradation product in PWW occurred (Fig. 2). Higher COD levels were also linked to 60% of water reduction (Table 2). As the concentration of organic matter increased, the chlorine demand increased. Consequently, liquid chlorine was needed to be added to the PWW to maintain the desired residual FC. Under these circumstances, chlorate was accumulated in the PWW and was uptaken by the product, remaining even after product rinse (Fig. 3). As far as we know, this is the first study reporting that there is no chlorate accumulation in PWW when chlorine gas is used as chlorine source during industrial produce washing, suggesting that it could be used for residue mitigation on the product.
Hypochlorous acid (HClO) is the most active form of chlorine, and its concentration in chlorinated water depends to a great extent on the pH and T of the wash water (Randtke, 2010). Ideally, a pH below 6.5 should be maintained to increase the concentration of HClO in the PWW. Residual FC and the theoretical HClO present considering pH and T of the PPW are presented. When the chlorination management was performed using the manual procedure without FC and pH control, the differences in the concentrations of FC and HClO caused by the pH were clearly observed (Fig. 4A and 4C). According to the pH range between 7.0 and 8.5 and taking into account the T of the PWW (≈6 °C), the percentage of FC in the form of HClO was in the range 15–85% and 5–64% in the first and the second tanks, respectively. In contrast, when the chlorination management was performed using the automatic procedure, 95–100% of FC was in the form of HClO, which explains the overlap of FC and HClO as pH was maintained in the range 5.5–6.5 (Fig. 4B and 4D). Similar results were observed for chlorine gas. When the chlorination management was performed using the manual procedure, FC fluctuated between high concentrations (maximum FC = 67 mg/L) and almost absence (minimum FC = 0.9 mg/L) at a pH between 6.5 and 7 (Fig. 5A and 5C). When T of water (≈5 °C) was taken into account, the percentage of HClO was in the range 85–95% for shredded lettuce and 64–85% for chopped lettuce. In contrast, when the chlorination
Data on microbial populations were log-transformed. Graphs were made using Sigma Plot 14.0 Systat Software, Inc. (Addilink Software Scientific S.L., Barcelona, Spain). IBM SPSS statistics 24 was used for statistical analysis. Shapiro Wilk test and Levene's test were employed to assess the normality and equality of variance, respectively. When normality could be assumed, T-tests were used to compare two treatments. Assuming also normality, one-way ANOVA was performed to compare more than two treatments, with Tukey's HSD or Dunnett's as post hoc tests depending on the homogeneity of the variances. When data was not following a normal distribution, nonparametric tests (Mann–Whitney U and Kruskal–Wallis) were applied to determine the differences between treatments. 3. Results and discussion
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Fig. 3. Presence of chlorate in unwashed, washed, and rinsed shredded lettuce with chlorine gas (A), and chlorine gas combined with liquid chlorine (B). Different letters indicate significant differences (p < 0.05). Values are the mean ± standard deviation of n = 9 samples. See Table 1 for samplings 4 (graph A) and 6 (graph B).
58 ± 39 mg/L for baby leaves). Apart from the product type, differences in COD could be caused by the different product throughput as it was almost four times higher in the case of cut lettuce (≈800 kg/h) compared with baby leaves (≈230 kg/h) (Table 1). Differences in COD due to the product type have been reported in previous studies (LópezGálvez et al., 2018; Luo et al., 2018). Luo et al. (2018) reported significantly lower COD in chopped romaine lettuce and shredded iceberg lettuce wash water when compared with diced cabbage. López-Gálvez et al., 2018 observed significantly higher COD in a shredded vegetables processing line compared with chopped lettuce and baby leaves processing lines. In PP2, differences in COD were also due to the cut size as, despite the higher water consumption (Table 2), COD was significantly higher (p < 0.05) in shredded lettuce than in chopped lettuce PWW. The average COD when shredded lettuce was washed was more than twice as high as when chopped lettuce was washed both for the manual chlorine management procedure (1087 ± 435 vs 543 ± 54 mg/L, respectively), and the automatic procedure (1744 ± 292 vs 840 ± 41 mg/L, respectively). In general, higher COD leads to a higher demand for disinfectants and this adds difficulties to chlorination management. At the same time, if higher volumes of disinfectant are added to the PWW to maintain the fixed residual FC, there is a higher risk of the presence of DBP residues (Gómez-López, Marín, Medina-
management was performed using the automatic procedure, FC was correctly maintained and the pH ranged between 5.5 and 6.5 for shredded lettuce and between 6.0 and 6.5 for chopped lettuce, leading to theoretical percentages of HClO in the range 95–99% (Fig. 5B and 5D). The use of an inefficient chlorination management system created higher FC fluctuations in the PWW when processing shredded lettuce with a very high FC demand. The better adjustment of FC to satisfy chlorine demand and the regulation of pH under the automatic procedure had consequences on the efficacy of the inactivation of microorganisms in the PWW. In general, the population of TAB was significantly lower (p < 0.05) when the PWW disinfection was managed using the automatic procedure compared with the manual one (data not shown). 3.3. Product type and cut size Our results showed that the physicochemical and microbiological characteristics of PWW were affected by product type and cut size. Changes in the COD levels over time in PP1 when different fresh products were washed are shown in Fig. 6. The COD values detected, as mean of all the samplings, were significantly higher (p < 0.05) when the cut product was washed compared with whole leaves (mean ± standard deviation of 358 ± 238 mg/L for cut lettuce compared with
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Fig. 4. Impact of chlorination management with liquid chlorine on the changes of free chlorine, hypochlorous acid (HClO), and pH in the process wash water. Samplings 1 (graphs A and C, manual procedure) and 2 (graphs B and D, automatic procedure). Vertical dotted lines indicate the change in the type of product washed. Values are the mean ± standard deviation of n = 2 samples for each sampling time. See Table 1 for sampling characteristics.
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Fig. 5. Impact of chlorination management with chlorine gas combined or not with liquid chlorine on the changes of free chlorine, hypochlorous acid (HClO), and pH of process wash water. Samplings 4 (graphs A and C, manual procedure) and 6 (graphs B and D, automatic procedure). Values are the mean ± standard deviation of n = 2 samples in each sampling time. See Table 1 for sampling characteristics.
between these two fresh products is so different is not apparent. One reason could be a hypothetical difference in the resistance of the microbiota to the antimicrobial treatment. This different resistance could be caused by differences in microbiome composition from the two types of produce washed (e.g., endospore-producing bacteria might be more prevalent on certain types of produce or in produce grown in a certain area or at certain seasons). Furthermore, it has been reported that the microbiota of baby leaves is more exposed to environmental stress than the microbiota of whole heads with a more closed structure (e.g.
Martínez, Gil, & Allende, 2013; López-Gálvez et al., 2018; Warriner & Namvar, 2013). The type of product being washed also has an impact on the microbiological quality of the PWW. This is illustrated in Fig. 7, which shows the changes in the populations of the TAB when the baby leaves and cut lettuce were washed. In all the cases, the TAB values detected in PWW were significantly higher (p < 0.05) when baby leaves were washed compared with cut lettuce (4.2 ± 0.3 and 2.4 ± 0.7 log cfu/ 100 mL, respectively). The reason why the microbial load of PWW
Fig. 6. Impact of product type and cut size on the chemical oxygen demand (COD) in the process wash water. Samplings 1, 2, and 3 (graphs A, B and C for tank 1 and graphs D, E and F for tank 2, respectively). Vertical grey dotted lines indicate the change in the type of product washed. See Table 1 for sampling characteristics. 6
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Fig. 7. Impact of product type and cut size on the population of total aerobic bacteria (TAB) in the process wash water. Samplings 1, 2, and 3 (graphs A, B and C for tank 1 and graphs D, E and F for tank 2, respectively). Vertical grey dotted lines indicate the change in the type of product washed. See Table 1 for sampling characteristics.
and/or in the water used for vacuum cooling. It is important to mention that the chlorate content in these samples was lower than the proposed chlorate MRL of 0.15 mg/L (EFSA, 2019). Our results also showed that differences in chlorate content between the washed and rinsed products were not always significant. In the case of chopped lettuce, the content of chlorate was significantly lower when the product was rinsed (p < 0.05) (Fig. 8B). In contrast, in the case of baby leaves and shredded lettuce, these differences were not significant (p > 0.05) (Fig. 8A and 8C). López-Gálvez et al. (2018) also reported high variability in the impact of the rinsing step as a residue mitigation of chlorate in the product at commercial processing facilities. These results highlight the variation of the outcome of the process at industrial scale and suggest that processing companies should further study and optimize this final tap water rinse as a residue mitigation strategy.
iceberg lettuce), and this higher exposition can induce tolerance to other stress factors, including chlorine treatment (Gruzdev, Pinto, & Sela, 2011). When different cut sizes were compared for the same type of produce (iceberg lettuce) in PP2, TAB populations in chlorinated PWW under manual chlorination management were significantly higher (p < 0.05) when washing shredded lettuce than chopped lettuce. The average TAB population in shredded lettuce PWW was 4.9 ± 0.7 log cfu/100 mL, whereas only 2.8 ± 0.6 log cfu/100 mL were detected in chopped lettuce PWW. This difference between different cut sizes observed for the same product could not be explained by the different product flow (kg/h), as it was higher when processing chopped lettuce than shredded lettuce (Table 1, samplings 4 and 5). Furthermore, water replenishment was higher when shredded lettuce was washed compared with chopped lettuce under manual chlorination management (Table 2, samplings 4 and 5). When lettuce was washed using the automatic procedure for the chlorination management, differences between the TAB population of PWW from shredded lettuce (3.7 ± 0.5 log cfu/100 mL) and chopped lettuce (2.5 ± 0.3 log cfu/100 mL) were reduced. A possible explanation for these results is that the organic matter released by the shredded product was higher than that of the chopped product, which might hamper the efficacy of disinfectants. Differences in physicochemical and microbiological characteristics of the PWW will generate different demand for disinfectants to maintain the quality of the PWW. A case-by-case study of each processing line is needed in order to establish an appropriate chlorination management system. The uptake of chlorate by the washed product is also affected by product type and cut size. Chlorate uptake has been reported to be higher when increasing the cut grade, being greater in shredded than in chopped or large cut lettuce (Garrido, Marín, Tudela, Allende, & Gil, 2019). In the present study, when the concentration of chlorate in the washed product was compared with the unwashed one, only in the case of chopped lettuce the concentration of chlorates was significantly higher (p < 0.05) (Fig. 8B), while no significant differences were observed for baby leaves and shredded lettuce (Fig. 8A and 8C). The chlorate detected in the unwashed product could be due to different causes, like the presence of chlorate in the water used for irrigation
4. Conclusions To obtain empirical evidence of basic concepts of chlorination management at industrial scale is not an easy task. This work provides and discusses industrial-scale data that illustrate some important factors related to produce washing. The shown concepts include the impact of the chlorine source on the occurrence of chlorate, the importance of proper pH regulation, the changes in the water characteristics due to the product type and cut size, and the effect of the rinsing step on the presence of chlorate in the product. The poor control of the chlorination management when manual procedures were used emphasize the need for automatic monitoring and control of the chlorination management. The fluctuating conditions regarding product type and cut size cause considerable variability in the physicochemical and microbiological characteristics of the PWW within one processing line. For each line, critical operational parameters should be identified as the type of product, the equipment design and the type of sanitizer selected will influence the chlorination management. Acknowledgments Authors are thankful for financial support from the MINECO (Project AGL2016-75878-R), the Center for Produce Safety Grant 7
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Fig. 8. Impact of product rinse on the chlorate residue mitigation in unwashed, primary washed and rinsed baby leaves (A), chopped lettuce (B) and shredded lettuce (C). Different letters indicate significant differences (p < 0.05). Values are the mean of each product evaluated in samplings 1, 2 and 3. See Table 1 for sampling characteristics.
Agreement (Project 2017–01) and CSIC (Intramural 201670E056). We also thank Silvia Andújar, Natalia Hernández, Alicia Marín and Yolanda Garrido for their technical assistance.
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