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Food Control 127 (2021) 108112

Contents lists available at ScienceDirect

Food Control

journal homepage: www.elsevier.com/locate/foodcont

Characterization and mitigation of chemical oxygen demand and chlorine

demand from fresh produce wash water
a,b a b a
zi teng , Yaguang Luo a,* , bin zhou , Qing Wang , Cathleen J Hapeman
a
US Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Beltsville, MD, United States
b
Department of Nutrition and Food Science, University of Maryland, College Park, MD, United States

ARTICLE INFO ABSTRACT

Keywords: Washing is essential in fresh-cut produce industry to remove debris and ensure desirable shelf life. The intensive water
Fresh-cut produces usage during washing and water scarcity in the production regions necessitate proper water reuse and reconditioning.
produce washing However, the organic compounds accumulated in the water have elevated chlorine demand (CLD) and chemical
Chemical oxygen demand
oxygen demand (COD), thus complicating food sanitation and subsequent water treatment. Hereby, we examined the
Chlorine demanded
major sources of COD and CLD during fresh-cut produce washing, and explored the mitigation of CLD by treating
organic exudates
water treatment select water constituents for the first time. Results showed that sugars as the predominant composition contribute to
over 80% of COD in the wash water, whereas minor constituents including proteins/ peptides, acids, and phenolic
compounds account for over 80% of total CLD. The actual CLDs by those com pounds depend highly on produce type.
Furthermore, while removing large molecules from the wash water led to the greatest CLD reduction for romaine
lettuce, absorption of anionic molecules proved the most effective for iceberg lettuce, onion, and carrot wash water.
This study suggests the possibility of mitigating CLD by more targeted and cost-effective water treatment procedures
without the need for removing sugars. It also recommends prudent and product-specific design for produce sanitization
and water treatments.

1.Introduction cross-contamination (Luo et al., 2018). Although the CLD could be

Fresh-cut produce is popular among consumers due to its freshness
and convenience. Washing is an essential process in fresh-cut produce
industry to remove debris and to ensure desirable product shelf life (Luo
et al., 2018). Due to the great water usage of produce washing, as well
as the water scarcity in the regions where the produce is harvested,
wash water would ideally be reconditioned and reused in subsequent
wash ings (Lehto et al., 2014; Van Haute et al. , 2013). However, the
practice is complicated by the organic exudates released from cut
produce (Teng, van Haute, et al., 2018). The exudates can increase
chemical oxygen demand (COD) up to 10,000 mg/L of O2 (Luo et al.,
2018), which is far above the waste water release limit set by the US
Environmental Protection Agency (USEPA, 45 mg /L biochemical oxygen
demand which is equivalent to 50–100 mg/L COD depending on the
chemical composition) (USEPA, 2017). In addition, the exudates are
vulnerable to electrophilic attack by the free chlorine (FC, with
hypochlorous acid as the major functional component), the major sanitizer
used in fresh produce industry. This creates high chlorine demand (CLD),
which leads to stable FC levels associated with elevated risk of pathogenic
overcome by more aggressive chlorination, such a practice leads to more
significant generation of toxic disinfection byproducts (Gallard & von
Gunten, 2002). Thus, the wash water will require extensive and proper
treatment before it can be discharged or reused.
The selection of water treatment procedures depends on the target
and time frame. Sugars are the target compounds for COD treatment,
according to our previous study on cabbage wash water (Teng, et al.,
2018). Those compounds abound in the wash water and lack the
properties amenable to various water treatment procedures, such as
ultrafiltration and ion-exchange. In addition, while sugars and COD need
to be eliminated after washing to prevent microbial growth, their impact
during produce washing is limited under proper FC level maintenance
(Luo et al., 2018). Consequently, sugars do not need to be removed
immediately after their release into wash water. Therefore, multistage
treatments involving reverse osmosis (RO) are generally selected for
COD reduction due to their desirable efficacy of sugar removal (Born
horst et al., 2018). On the other hand, over 80% of the CLD of cabbage
wash water arises from its minor constituents: proteins, peptides, acids,
and phenolic compounds (Teng, et al., 2018). Unlike COD, CLD has a

* Corresponding author.
E-mail address: Yaguang.Luo@usda.gov (Y. Luo).

https://doi.org/10.1016/j.foodcont.2021.108112
Received 2 November 2020; Received in revised form March 7, 2021; Accepted 22 March 2021
Available online 27 March 2021
0956-7135/Published by Elsevier Ltd.
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Z. Teng et al.
major impact during washing as FC can be depleted rapidly upon addition
(Weng et al., 2016). Therefore, timely removal of wash water constituents
is vital for CLD reduction, with completeness of removal being a second
priority. Such a goal may be achieved by ultrafiltration, physical absorption,
or ion-exchange, all of which can absorb the abovementioned CLD
contributors with favorable throughput and better cost-effectiveness than
RO (Galloway & Mahoney, 2004; Mukho padhyay, 1999). Lastly, the
diverse compositions among different types of produce should be
investigated, as they dictate the source of COD and CLD in the wash
water, thus determining the choice of water treatment techniques.
The major objectives of this study were (1) to identify and compare the
major sources of COD and CLD in wash water generated by different types
of produce, and (2) to determine the effectiveness of CLD reduction by
removing compounds with select properties (high molecular weight, low
polarity, or negative charge) from the wash water. Four major types of
vegetables belonging to different families were chosen: romaine lettuce,
iceberg lettuce, carrot, and onion (Luo et al., 2018). The major com pounds
in the wash water, including sugars, proteins/peptides, acids, and phenolic
compounds, were analyzed for their abundances and contributions to COD
and CLD. The effect of treating select compounds on the CLD of wash
water was then evaluated. To our knowledge, this is the first study on
potential procedures and the underlying chemistry for CLD mitigation during
fresh produce washing.
2. Materials and methods
2.1. materials
Romaine lettuce (Lactuca sativa L. var. longifolia), iceberg lettuce
(Lactuca sativa L. var. capitata), white onions (Allium cepa L.), and carrots
(Daucus carota subsp. sativus) were obtained from a local wholesale
market ( Jessup, MD, USA), and stored at 4 ÿC until being processed.
HPLC grade standards and water purchased from Sigma-Aldrich (St.
Louis, MO, USA) were used for chromatographic analyses. Distilled water
was used for all non-chromatographic assays, including CLD, COD, total
phenolic content (TPC), and total Kjeldahl nitrogen (TKN) tests. All other
chemicals were of analytical grade and purchased from Sigma-Aldrich (St
Louis, MO, USA).
2.2. Preparation of wash water samples
Water samples were prepared by washing consecutive batches of cut
products with the same batch of water. Produce was cut using an in
industrial vegetable slicer (Nichimo Seven Chefs ECD-302, Tokyo, Japan)
into the following shapes: carrot sticks (3 mm × 3 mm x 26 mm), romaine
and iceberg lettuce shreds (3 mm width), and onion dices (3 mm × 3 mm).
Batches of cut products, 800 g each, were washed sequentially in 10 L of
distilled water at 4 ÿC for 1 min with manual agitation. After each batch of
washing, the product was drained and removed, and approximately 200
mL distilled water was supplemented to compensate the water loss. The
total number of batches being washed was ten for iceberg and romaine
lettuce, and five for carrot and onion.
Each experiment was repeated three times. Water samples were collected
at the end of washing and refrigerated (4 ÿC for less than 4 h), freeze-
dried, or frozen (ÿ 20 ÿC) immediately depending on the type of test
performed. The samples were then stored at ÿ80 ÿC before analyses.
23. Measurement of COD and CLD
A scheme for water sample handling and analyzes is presented in
supplementary Fig S1. Refrigerated water samples were measured for
their COD and CLD within 2 h after wash water preparation. COD was
determined using the Hach© method 10236 after proper dilution (typically
two to five times) with distilled water (Hach Company, 2014).
For CLD measurements, wash water samples were diluted with
two
Food Control 127 (2021) 108112
phosphate buffer (PB; pH 6.5, 50 mmol/L, same hereinafter) to a COD
level of 1500 mg/L to negate the effect of the initial FC/COD ratio on the
equilibrium of chlorination reaction. The resulting dispersions were
incubated at 4 ÿC for 30 min and chlorinated by adding concentrated
sodium hypochlorite solution (Sigma-Aldrich, St. Louis, MO, USA) to an FC
level of 1000 mg/L FC. The initial and residual FC levels were measured
by N,N-diethyl-p-phenylenediamine (DPD) method (Hach Company, 2014)
at preset intervals up to 120 min. The CLD was calculated as the difference
between initial and residual FC levels.
2.4. Characterization of sugars, organic acids, phenolic compounds, and
proteins
Freeze-dried wash water samples were reconstituted in HPLC-grade
water and subjected to sugar and organic acid analyzes using an Agi lent
Infinity® 1260 HPLC system (Agilent Technologies, Santa Clara, CA,
USA), equipped with a 1260 Infinity II Refractive Index Detector and 1260
Infinity II Diode Array Detector. All compounds were identified and
quantified with calibration curves established using external standards.
Frozen wash water samples were thawed and measured for TPC and
TKN. TPC was measured using the Fast Blue BB (FBBB) assay (Xiao et
al., 2015). Crude protein was measured by TKN assay using Hach©
method 10242 (Hach Company, 2017). Detailed procedures of the above
assays can be found in our previous publication (Teng, et al., 2018).
2.5. CLD removal by wash water treatment
A schematic diagram for wash water treatment is presented in Fig S2.
Freeze-dried wash water samples were reconstituted in distilled water to a
COD level of 5000 mg O2/L. A 15-mL aliquot was drawn and centri fuged
at 15,000×g for 10 min to remove insoluble substance. The su pernatant,
defined as the soluble fraction, was subjected to three treatments as
described below.
2.5.1. Removal of compounds with high molecular weight (MW)
The soluble fraction (15 mL) was centrifuged (5000×g, 30 min) in a
Macrosep® centrifugal dialysis tube (Pall Corp., Port Washington, New
York, USA) with molecular weight cutoff (MWCO) of 30 kDa, and the Filter
was centrifuged under the same condition in tubes with MWCO of 3 kDa.
The two-stage centrifugation procedure was employed to avoid clogging of
the dialysis membrane. The retentates collected from the two steps were
combined and restored to 15 mL with PB. This fraction was defined as the
high-MW fraction, which contained mostly proteins/ peptides according to
the Hach© total Kjeldahl nitrogen (TKN) assay.
The filtrate obtained after the second centrifugation was restored to 15 mL
with PB and was defined as the low-MW portion. Both fractions were frozen
immediately at ÿ80 ÿC before further assays.
2.5.2. Removal of compounds with low polarity
A Waters Sep-Pak C-18 cartridge (Waters Ltd., Mississauga, Ontario,
Canada) was equilibrated with methanol and water per manufacturer's
instruction (Waters, 2001), after which 2 mL of the soluble fraction was
passed through the cartridge. Elution was performed thereafter, first with 8
mL water, followed by 4 mL of 40% methanol in water, and lastly with 8 mL
of 80% methanol in water. The water eluate was defined as
the high-polarity fraction, and the combined methanolic eluate was defined
as the low-polarity fractions. Both fractions were measured for their
absorbance at 280 nm (OD280) and total solid content (degree Brix). Both
fractions were freeze-dried and then stored at ÿ80 ÿC until further assays.
2.5.3. Removal of compounds with negative charge
One milliliter of the soluble fraction was passed through a Discovery ®
DSC-SAX strong anionic exchange cartridge (Sigma-Aldrich, St. Louis,
MO, USA), that was pretreated with methanol and water per manufac
turer's instruction (Sigma-Aldrich). The cartridge was eluted with 2 mL
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Z. Teng et al. Food Control 127 (2021) 108112

of water, and the eluate was defined as the neutral/cationic (N/C) fraction. No
further elution was performed since the anionic compounds are bound
irreversibly to the cartridge via strong ionic bonds, as described by the
manufacturer (Sigma-Aldrich).
2.6. Determination of COD and CLD by compounds and fractions
The fractions prepared as described in the previous section were diluted or
reconstituted as follows. Three milliliters of high- and low MW fractions were
withdrawn and diluted with 7 mL of PB. High- and low-polarity fractions in the
form of dry powder were reconstituted in 10 mL PB. Lastly, 2 mL of the N/C
fraction was diluted with 3 mL PB. The CLD and COD of each fraction of the
wash water were measured by DPD and Hach methods as previously
described. Appropriate conversion factors that accounted for the dilution in
Sections 2.5 and 2.6 were adopted to calculate the contribution to CLD from
all fractions in the raw wash water samples.
The COD and CLD of each compound were determined as follows.
The COD of sugars or acids were calculated using their specific COD values
derived from their oxidative reactions. For instance, the specific COD of
glucose is determined as 192 g O2 per 180 g glucose (Eqn. (1))
proteins and peptides, organic acids, and phenolic compounds (Table 1).
The amount of water-insoluble compounds (eg, carotenoids and fatty acids)
was expected to be low, and their reactivity with FC is much lower than that of
water-soluble compounds due to their inefficient mass transfer (Deborde & Von
Gunten, 2008) .
Table 1 shows the contents of sugars, proteins/peptides, organic acids,
and phenolics compounds recovered from the wash water samples. The total
content of those compounds was twice as large in onion and carrot wash water
as in iceberg and romaine lettuce wash water.
This is similar to results on whole produce where the solid concentrations
were twice as high in carrot (0.12 g/g) and onion (0.11 g/g) compared to
romaine (0.05 g/g) and iceberg lettuce (0.04 g/g )
(USDA-ARS., 2019a; USDA-ARS., 2019b; 2019c; 2019d). In addition, carrot
and onion contain approximately twice as much the amount of sugars and
somewhat more organic acids (USDA-ARS., 2019a; USDA-ARS., 2019b;
2019c; 2019d), both of which are small and soluble molecules and diffuse more
efficiently during washing than proteins and peptides (Aletor et al., 2002).
Onions generated more total solid content in wash water than carrots despite
its lower solid content. This was probably due to the smaller size of diced onion
compared to stick-cut carrot, which produced larger injured surface and allowed
more exudate to be released (Li et al., 2019).

(C6H12O6 + 6 O2 ÿ 6 CO2 + 6 H2O). eqn. 1 Among the analyzed compounds, sugars were the predominant chemicals
in all wash water samples. Romaine and iceberg lettuce as leafy greens
The CLD of sugars and organic acids were calculated by applying their
contained the highest percentage of fructose followed by glucose, while
measured concentrations to their respectively regression models as reported
sucrose was not detected. In comparison, sucrose was the most abundant
before (Teng, et al., 2018). The CLD and COD of phenolic compounds were
compound in carrot wash water accounting for 46% of the total solid content.
estimated by applying the TPC level (as gallic acid equivalent) to the regression
Onion wash water also contained large and approximately equal amounts of
model for CLD and specific COD value for gallic acid, respectively.
fructose and glucose, whereas only moderate levels of sucrose were observed.

The CLD and COD of proteins and peptides were determined according
to Teng, et al., 2018. In brief, the soluble fraction of wash water was separated
by molecular weight as described in Section 2.5.1. The high-MW fraction Table
(HMW) was collected for later analysis, while the low-MW fraction was further 1 Chemical composition of wash water generated from romaine lettuce, iceberg
subjected to treatment by the C-18 car tridge as described in Section 2.5.2. lettuce, carrot, and onion in mg/L (percentage)a .
The methanol eluate obtained from the above treatment was freeze-dried and type Romaine Iceberg carrot onion
defined as the low-MW, low-polarity fraction (LMLP). Both HMW and LMLP 2600 ± 75d 2370 ± 52c 5690 ± 113b 7170 ± 231a
sugars
fractions exhibit high contents of proteins and peptides, as suggested by their (81.4 ± 2.3)b (85.0 ± 1.9) (94.8 ± 1.9) (89.4 ± 2.9)
OD280 readings. Therefore, those fractions were combined, diluted properly fructose 1850 ± 46b 1480 ± 16d 1650 ± 46c 2990 ± 90a
(58.5 ± 1.4) (52.8 ± 0.6) (28.4 ± 0.8) (38.1 ± 1.1)
with PB, and measured for their CLD and COD directly (defined as CLD1 and
glucose 770 ± 46c 885 ± 16c 1130 ± 34b 3170 ± 65a
COD1 respectively). In addition, the water eluate obtained from the C-18
(22.9 ± 1.4) (32.2 ± 0.6) (20.3 ± 0.6) (38.9 ± 0.8)
cartridge treatment, defined as the low-MW, high-polarity fraction (LMHP), was sucrose NDc NA 2600 ± 12a 980 ± 25b
measured for its TKN. The CLD and COD of the LMHP fraction (defined as (46.0 ± 0.2) (12.4 ± 0.3)
CLD2 and COD2 respectively) was estimated by reconstituting soy protein
Proteins/ 392 ± 36ab 354 ± 72b 94.9 ± 9.0c 493 ± 45a
hydrolysate (SPH) in PB at a concentration that provided the same TKN level
peptides (12.2 ± 1.1) (12.7 ± 2.6) (1.7 ± 0.2) (6.1 ± 0.6)
as the LMHP fraction. The total CLD and COD from proteins were calculated
as CLD = CLD1 + CLD2 and COD = COD1 + COD2, respectively. organic acids 189 ± 13b 46.2 ± 5.2c 172 ± 14b 240 ± 28a
(5.9 ± 0.4) (1.6 ± 0.2) (3.0 ± 0.3) (3.0 ± 0.3)
Citric acid 32.1 ± 3.1b 1.7 ± 1.1d 10.0 ± 1.1c 58.4 ± 6.2a
(1.0 ± 0.1) (0.4 ± 0.0) (0.2 ± 0.0) (0.7 ± 0.1)
malic acid 126 ± 16b 24.0 ± 1.9c 160 ± 12a 162 ± 12a
2.7. Statistics (3.9 ± 0.4) (0.9 ± 0.1) (2.8 ± 0.2) (2.0 ± 0.2)
ascorbic acid 31.1 ± 2.4a 11.4 ± 0.7c NA 15.7 ± 1.7b
(1.0 ± 0.1) (0.4 ± 0.0) (0.2 ± 0.0)
All treatments and assays were performed in triplicate. Data in the tartaric acid NA NA 2.70 ± 0.1b 3.70 ± 0.20a
supplementary tables (Tables S1 and S2) are presented as mean ± (0.1 ± 0.0) (0.1 ± 0.0)
standard error. Raw data from each replicate of CLD assays are presented in
Phenolic 16.0 ± 1.4c 17.5 ± 0.8c 31.1 ± 4.4b 115 ± 4.7a (0.5 ±
supplementary Figures S3, S4, S5, and S6. Significant difference (p < 0.05)
compounds (0.5 ± 0.0) (0.6 ± 0.0) 0.0) (1.4 ± 0.0) 5680
8020
± 120b
± 230a
was identified by analysis of variance followed by Tukey test, using Sigma Plot Totald 3210 ± 96c 2780 ± 55d
version 12.0 (Systat Inc., Chicago, IL, USA).
a
Non-parenthesized numbers in the table indicate the concentrations of
3. Results and discussion individual compounds in their respective water samples, in milligrams per liter
water. Parenthesized numbers represent the percentages of those compounds
in the water samples.
3.1. Chemical composition of wash water b
All data are presented as mean ± standard error (n = 3). Different super
scripts designate statistically significant difference (P < 0.05) according to
Wash water contains various compounds released from the cut surface of ANOVA with Tukey analysis.
c Not detected.
produce. Of key interest are the water-soluble components, mainly because
d
wash water is an aqueous system. These include sugars, Sum of sugar, protein/peptide, organic acid, and phenolic contents.

3
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Proteins and peptides were the second most abundant identified compounds in
iceberg and romaine lettuce wash water, representing over 12% of the total solid
content. Leafy greens are known for their relatively high abundance of proteins,
especially enzymes and storage proteins (Choi et al., 2008). Similarly, proteins and
peptides were the second most abundant compounds in the solids of the onion
wash water, although at a lower percentage of 0.061 g/g. Carrots, which are known
for their low protein content (Bao & Chang, 1994), yielded a much lower percentage
of proteins and peptides (0.017 g/g) in the solids of the wash
toilet.
Organic acids were the third most abundant compounds identified in the solids
of romaine lettuce, iceberg lettuce, and onion wash water, while they ranked second
in abundance in carrot wash water after sugars. Comparing among the four types
of vegetables, romaine lettuce wash water showed the highest percentage of
organic acids (0.059 g/g), followed by carrot, onion (both at 0.03 g/g), and iceberg
lettuce (0.016 g/g). Among those acids, citric and malic acids were the primary acids
detected in all wash water samples, in line with previous studies on whole produce
(Altunkaya & Gokmen, ¨ 2009; Rodriguez Galdon et al., 2008).
The total content of phenolic compounds was estimated by Fast Blue BB assay
and are presented as gallic acid equivalents (GAE). The reason for not performing
comprehensive LC/MS measurement is the complex phenolic profile of plant-derived
samples (Mattila & Hellstrom, ¨ 2007; Teng, et al., 2018), which renders it impractical
to
identify individual phenolic species and measure the CLD of each identified
compound. As shown in Table 1, phenolic compounds quantified in GAE were the
fourth most abundant compounds in the solid content of wash water.
Onion wash water contained the greatest amount of phenolic compounds in the
total solids (0.014 g/g), while the solids of the other three wash water samples
contained ~0.005 g/g phenolic compounds.
3.2. Chemical oxygen demand (COD) of wash water
As introduced in Section 1, the COD level of wash water needs to be reduced
substantially before the water can be reused for produce washing or discharged.
The COD from a certain compound is determined by its abundance in wash water
presented in Table 1, as well as its specific COD. The latter was derived from its
complete oxidation exemplified in Eqn. (1) and verified by Hach COD assay.
According to our preliminary study (Supplementary Fig S3), all identified compounds
exhibit similar specific COD values. Therefore, a water sample with highest solid
content should exhibit the highest COD, and sugars being the most abundant
compound in wash water were expected to be the major contributor to COD.
Fig. 1. Chemical oxygen demand from different compounds in romaine lettuce,
iceberg lettuce, carrot, and onion wash water. Results from statistical analyzes
are presented in Supplementary Table S1.
4
Food Control 127 (2021) 108112
Fig. 1 compares the COD of different wash water samples together with its
breakdown by compound types. Total COD ranked by vegetable type as onion >
carrot > romaine > iceberg. Sugar accounted for over 80% of the total COD in all
wash water samples. The second major contributor to COD was proteins and
peptides in romaine, iceberg, and onion wash water. Carrot wash water with less
proteins and peptides exhibited lower percentage of COD by those compounds.
Phenolic compounds measured in GAE accounted for less than 1% of the total
COD in all wash water samples due to their low abundance. The above compounds
explained over 88% of the total COD in the water samples.
Other unidentified compounds that could potentially affect COD may include dietary
fibers, fatty acids, inorganic salts, and other micronutrients such as beta-carotene
(USDA-ARS., 2019a; USDA-ARS., 2019b; 2019c; 2019d).
3.3. Chlorine demand (CLD) of wash water
Compared to COD which mainly affects post-washing water treatments, CLD
can influence water quality and product safety instantly and substantially during
washing via rapid FC depletion. As shown in Fig. 2, the CLD of wash water
increased remarkably in the first 5 min of chlorination, during which over 50% of the
total CLD was achieved, followed by a sustained increase over the next 2 h. The
rapid consumption of free chlorine at early stages of chlorination was probably due
to the highly reactive compounds such as free amino acids, phenolic compounds
with certain substitution groups, and sulfurous compounds such as cysteine
containing proteins (Weng et al., 2016). This phenomenon underscores the
importance of timely intervention to mitigate CLD and maintain sanitizer levels.
Furthermore, water-soluble fraction recovered via centri fugation accounted for over
85% of the CLD in all wash water samples, which implies limited reduction of CLD
from wash water by filtration.
Among different produce types, onion exhibited the highest CLD during the tested
period, followed by romaine and iceberg lettuce. Notably, carrot wash water
exhibited the lowest CLD despite its high solid content and COD.
The contributions to CLD from different compounds are presented in Fig. 3. The
CLD from each class of compounds (eg, organic acids) was calculated by summing
the CLD from each constituent in the class (eg, malic acid). The latter value was
derived by applying its measured concentration in the wash water to its respective
regression model that has been reported before (Teng, et al., 2018). Sugars
accounted for up to 8.3% of CLD during the first 15 min of chlorination despite their
major contribution to COD (Fig. 1). The predominant functional groups of sugars,
namely hydroxyl and carbonyl moieties, react at limited rate with hypochlorous acid
(Deborde & Von Gunten, 2008). On the other
Fig. 2. Average chlorine demand (CLD) of romaine lettuce, iceberg lettuce,
carrot, and onion wash water, and their water-soluble constituents (centri
fuged). Data for individual experiments are shown in Figure S3.
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Z. Teng et al.
Fig. 3. Contribution to chlorine demand at 15 (A) and 120 (B) minutes of
chlorination by different classes of compounds in romaine lettuce, iceberg let
tuce, carrot, and onion wash water. Data from all replicates are presented in
Fig S2.
hand, sugars showed steady and remarkable increase in CLD over time
owing to their abundance and slow depletion rate. At 120 min of
chlorination, sugars contributed to as much as 34.8% of CLD for carrot
wash water and 13–16% for other water samples.
Proteins and peptides account for over 50% of the total CLD in romaine
and iceberg lettuce wash water, both at 15 and 120 min (Fig. 3).
This is ascribed to the relative high abundance of these compounds (Table
1), together with the considerable reactivity of their functional groups,
including amine, thiol, phenolic, and carboxyl groups (Peskin & Winterbourn,
2001). These groups exhibit moderate to high reaction rates with
hypochlorous acid, which allows proteins and peptides to consume FC
effectively at different stages of chlorination (Pattison & Davies, 2001). In
comparison to lettuce, onion and carrot wash water with lower percentages
of proteins and peptides exhibited lower CLD contribution from those
compounds, both at 15 min (31% and 19% for onion and carrot,
respectively) and 120 min (36% and 23 % for onion and carrot, respectively).
Organic acids as the third most type of molecules in wash water
contributed to 19–41% of the total CLD (Fig. 3). The percentages of their
contribution increased over time, suggesting a sustained consumption of
FC by these molecules. Organic acids abound in the wash water with
varying specific CLD (Teng, et al., 2018), which depends on the number of
carboxylic groups as well as the substitution groups present on the
molecular backbone (Deborde & Von Gunten, 2008). Carrot wash water
showed the highest percentage of CLD from organic acids as it contained
the highest percentage of those compounds in the wash water (Fig. 3).
Although the amount of phenolic compounds in wash water was below
1%, they contributed to 7–17% of CLD in the first 15 min (Fig. 3).
The high reactivity of phenolic compounds with FC is attributable to the p-
ÿ aromatic ring bond interactions, which facilitates the electrophilic attack
by hypochlorous acid (Deborde & Von Gunten, 2008). As the phenolic
compounds were depleted rapidly, their CLD peaked at early stages and
remained relatively stable over time, leading to a decline in their percentage
of contribution to CLD. It should be noted that phenolic compounds vary
substantially in molecular weight, number of aromatic rings, type of
substitution groups, and status of conjugation (Heredia & Cisneros-
Zevallos, 2009; Llorach et al., 2008; Simin et al., 2013). , all of which lead
to a pronounced variation in reactivity. As such, the actual CLD from
phenolic compounds may deviate considerably from the estimate using
gallic acid as a model compound, since the latter is a free phenolic acid
with very high reactivity with hypochlorous acid (Toi vonen & Lu, 2013).
Collectively, sugars, proteins/peptides, organic acids, and phenolic
compounds accounted for over 70% of CLD at 15 min for onion, carrot,
and iceberg lettuce wash water, and over 88% of total CLD in romaine
lettuce wash water (Fig. 3). At 120 min, those compounds represented
over 90% of the total CLD measured in all four types of wash water
samples. These data suggest that other unidentified compounds may
5
Food Control 127 (2021) 108112
contribute to the CLD, especially at the early state of chlorination. These
compounds may include isothiocyanates (Hanley et al., 1988), ally sulfides
(Kim et al., 2005), and acids not identified in this study, such as glutamic
and succinic acid (Howard et al., 1995).
3.4. Reduction of CLD by removing select groups of compounds
As discussed in Section 1, timely, partially removal of major CLD
contributors is of great importance for food safety and water quality during
fresh-cut produce washing. Hereby, three techniques were applied to
remove select wash water constituents by molecular weight, polarity, and
electric charge, respectively. The CLDs of the removed and remaining
portions of the wash water are compared in the following paragraphs.
First, reconstituted wash water was fractionated by MW through dialysis
centrifugation, a technique that retains high-MW molecules in a similar way
to ultrafiltration. The retentate comprises mainly proteins, high-MW
peptides, and polysaccharides, whereas the eluate contains low-MW
compounds such as free phenolic compounds, organic acids, low-MW
peptides or amino acids. As indicated in Fig. 4A, low-MW fractions
contributed to over 50% of CLD for carrot wash water, over 60% for onion
and romaine lettuce wash water, and over 70% for iceberg lettuce wash
water. In addition, the low-MW fractions in iceberg and romaine lettuce
wash water showed more steadily increase of CLD over time compared to
those in other water samples. High-MW fraction generally accounted for
smaller percentages of CLD, possibly due to the steric hindrance on larger
molecules (mainly high-MW proteins) that prevents the hypochlorous acid
from diffusing to and reacting with the functional groups. An exception to
that observation was the high-MW fraction in carrot wash water, whose
CLD increased steadily over time and accounted for nearly 50% of the
total CLD after 120 min of chlorine. Understanding this phenomenon
requires further study on the chemical composition of that fraction.
Next, reconstituted wash water was treated by reverse phase SPE,
which removes low-polarity (LP) compounds such as phenolic compounds
and proteins rich in hydrophobic residues. As shown in Fig. 4B, the LP
fraction contributed to over 40% of total CLD for iceberg lettuce and carrot,
about 30% for onion, and less than 20% for romaine lettuce wash water.
In addition, the CLD of this fraction remained stable after the first 15 min,
compared to that of the HP fraction which increased continuously. One
possible explanation for that observation is the rapid depletion of phenolic
compounds at early stages of chlorination (Teng, et al., 2018), which
prevented CLD of the LP fraction from increasing over time. Notably, the
highest percentage of CLD by the LP was observed in iceberg lettuce and
carrot wash water (Fig. 4B), which also exhibited the highest percentage
of CLD attributable to phenolic com pounds (Fig. 3).
Lastly, reconstituted wash water was subjected to anionic ion exchange
treatment. The negatively charged fraction that was irreversibly retained
by the cartridge comprises mostly organic acids (pKa = 2–3) and proteins/
peptides (pI ~ 4.5) (Aletor et al., 2002). The CLD of that fraction was
estimated by subtracting the total CLD from the CLD by the positively or
non-charged fraction. As shown in Fig. 4C, both positively/non-charged
and negatively charged fractions exhibited continuously increasing CLD
over the chlorination process. Those results further suggest that the
negatively charged compounds account for over 50% of CLD in iceberg
lettuce wash water compared to 40–45% for carrot and onion. These
results can be explained by the greater contribution to CLD by proteins
and acids in iceberg lettuce wash water.
Fig. 4D summarizes the reduction in CLD at 15 min by removing the
above three groups of compounds, which is highly dependent on product
type. Removal of negatively charged compounds led to the greatest
reduction in CLD for iceberg lettuce (60%), carrot (48%), and onion (54%)
wash water, while treatment on high-MW constituents reduced the CLD of
romaine lettuce wash water most dramatically (37%). Sur prisingly,
treatment on anionic compounds led to much inferior results
Machine Translated by Google
Z. Teng et al.
for romaine lettuce than for iceberg lettuce. The exact reason underlying
such a discrepancy needs further investigation, but two possible causes
are discussed below.
First, romaine lettuce contains a greater amount of positively charged
amino acids, namely, lysine and arginine compared to iceberg lettuce,
while the contents of negatively charged amino acids (aspartic and
glutamic acids) between the two lettuce types are similar (USDA-ARS,
2019a; 2019b). The abundant positively-charged amino acids and proteins/
peptides are most likely to be eluted in the cati onic/neutral fraction.
Furthermore, these amino acids contain extra amino groups which react
with hypochlorous acid rapidly, thus adding to the CLD of the positively/
non-charged fraction of wash water.
Second, the greater organic acid content in romaine wash water might
also have played a role, since the acids compete with negatively charged
amino acids and proteins/peptides for the available binding sites on the
ion-exchange cartridge, rendering the latter more difficult to be irreversibly
retained. As a result, some of the negatively charged proteins/peptides
might have remained in and contributed to the CLD of the the positively/
non-charged fraction.
3.5. Factors that affect COD and CLD during fresh produce washing
As discussed in Sections 3.3 and 3.4, COD and CLD are distinct water
quality parameters associated with different groups of compounds. COD
which measures the oxygen demand at complete oxidation is determined
mined solely on the total number of oxidizable atoms, mainly carbon,
hydrogen, nitrogen, and sulfur. Therefore, sugars that provide the most
of those atoms in the water contribute most significantly to COD, and the
wash water that contains most sugar exhibits the highest COD. CLD is
determined by not only the number but also the arrangement of those
atoms. For instance, the reactivity of amino acids and certain phenolic
compounds are several orders of magnitude higher than that of sugars
(Deborde & Von Gunten, 2008). Similarly, proteins with more reactive
residuals, such as lysine and arginine with additional amino groups and
cysteine with a thiol group, are more reactive at equal weight than other
amino acid species (Deborde & Von Gunten, 2008). Lastly, certain
compounds (eg, glucosinolate in brassicas) are destroyed instantly when
they are released from damaged cells and mixed with other chemicals
(eg, myrosinase), thus losing their CLD during washing
6
Food Control 127 (2021) 108112
Fig. 4. Chlorine demand breakdown by select groups
of compounds in romaine lettuce, iceberg lettuce,
carrot, and onion wash water. A: time-dependent CLD
of low- (LMW) and high-molecular weight (HMW)
compounds. B: time-dependent CLD of low- (LP) and
high-polarity (HP) compounds. C: time-dependent
CLD of positively charged or neutral (P/N) com pounds
as well as negatively charged (Neg) com pounds. D:
reduction of CLD observed at 15 min by removing
abovementioned groups of compounds.
Data from all replicates are presented in Figs S5, S6,
and S7.
(Kliebenstein et al., 2005).
Additionally, several external factors can affect the CLD of wash
water. For instance, higher CLD has been observed at lower pH (Teng,
et al., 2018) or higher temperature (Ndiongue et al., 2005) at the same
reactant/FC ratio. The application of organic acids (eg, citric acid) as an
acidulant during washing will further increase the CLD of the wash water.
All these factors need to be considered when evaluating or predicting the
CLD during produce washing.
4. Conclusion
This study characterized the major sources of COD and CLD in fresh
cut produce wash water, as well as potential methods for CLD reduction.
Sugars contributed to 80–95% of the total COD in wash water. On the
other hand, proteins, acids, and phenolic compounds accounted for over
80% of total CLD despite their low abundance. The exact percentage of
CLD from each compound depends on produce type and chlorination
time. The above findings allowed the design of effective water treatments
to mitigate CLD without the need of removing sugars from the system.
The efficacy of such treatments varied significantly with produce type:
treating negatively charged compounds resulted in the greatest reduction
in CLD for iceberg lettuce, carrot, and onion wash water, while removing
high-MW components demonstrated the best performance for romaine
lettuce wash water. This systematic study emphasized the great variation
in the magnitude and source of COD/ CLD in processing different types
of vegetables, and it provides produce processors with essential guidance
to develop safe and effective strategies for product sanitization and water
treatment or reuse.
Disclaimer
All opinions expressed in this paper are the authors and do not
necessarily reflect the policies and views of USDA, ARS, DOE, or ORAU/
ORISE. Mention of trade names or commercial products in this publication
is solely for the purpose of providing specific information and does not
imply recommendation or endorsement by the USDA. USDA is an equal
opportunity provider and employer.
Machine Translated by Google
Z. Teng et al.
Competing financial interest
The authors declare no competing financial interest.
Declaration of interests
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
CREDIT authorship contribution statement
Zi Teng: Conceptualization, Methodology, Investigation, Validation,
Formal analysis, Writing – review & editing. Yaguang Luo: Funding
acquisition, Conceptualization, Project administration, Supervision,
Writing – review & editing. Bin Zhou: Methodology, Investigation,
Validation, Writing – review & editing. Qin Wang: Project administration,
Supervision, Writing – original draft. Cathleen J. Hapeman: Su
pervision, Writing – review & editing.
Acknowledgments
The authors would like to thank Ms. Lin Chen for assistance in
chemical analyses. This research was supported in part by the US
Department of Agriculture (USDA), Agricultural Research Service (ARS)
intramural projects in the National Programs 108 (Food Safety), and
306 (Product Quality and New Uses) and by the USDA, National Institute Turner, ER, Wang, Q., & Millner, PD (2018). Association between bacterial survival and free
chlorine concentration during commercial fresh-cut produce wash operation. Food
of Food and Agriculture, Specialty Crop Research Initiative , Award no. Microbiology, 70, 120–128. https://doi.org/10.1016/j.fm.2017.09.013 Mattila, P., &
2016-51181-24283. This research was also supported by an Hellstrom, ¨J. (2007). Phenolic acids in potatoes, vegetables, and some of their products.
appointment to the USDA-ARS Research Participation Program Journal of Food Composition
j.jfca.2006.05.007
apparatus for high efficiency reverse osmosis
administered by the Oak Ridge Institute for Science and Education
(ORISE) through an interagency agreement between the US Department
of Energy (DOE) and the USDA. ORISE is managed by Oak Ridge operation. US Patent US5925255A. Available at https://patents.google.com/patent/
Associated Universities (ORAU) under DOE contract number DE-SC0014664. US5925255A/en .
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi. org/10.1016/
j.foodcont.2021.108112.
References
Aletor, O., Oshodi, A., & Ipinmoroti, K. (2002). Chemical composition of common leafy
vegetables and functional properties of their leaf protein concentrates. Food Chemistry,
78(1), 63–68. https://doi.org/10.1016/S0308-8146(01)00376-4 Altunkaya, A., & Gokmen,
¨ V. (2009). Effect of various anti-browning agents on phenolic
Compound profile of fresh lettuce (L. sativa). Food Chemistry, 117(1), 122–126.
https://doi.org/10.1016/j.foodchem.2009.03.085
Bao, B., & Chang, K. (1994). Carrot pulp chemical composition, color, and water-holding
capacity as affected by blanching. Journal of Food Science, 59(6), 1159–1161. https://
doi.org/10.1111/j.1365-2621.1994.tb14666.x
Bornhorst, ER, Luo, Y., Park, E., Vinyard, BT, Nou, X., Zhou, B., Turner, E., &
Millner, P.D. (2018). Immersion-free, single-pass, commercial fresh-cut produce
washing system: An alternative to flume processing. Postharvest Biology and
Technology, 146, 124–133. https://doi.org/10.1016/j.postharvbio.2018.08.008
Choi, J.-S., Cho, S.-W., Kim, T.-S., Cho, K., Han, S.-S., Kim, H.-K., Woo, S.- H., &
Chung, K.-Y. (2008). Proteome analysis of greenhouse-cultured lettuce with the
natural soil mineral conditioner illite. Soil Biology and Biochemistry, 40(6), 1370–1378.
https://doi.org/10.1016/j.soilbio.2007.04.032
Deborde, M., & Von Gunten, U. (2008). Reactions of chlorine with inorganic and organic
compounds during water treatment—kinetics and mechanisms: A critical review.
Water Research, 42(1), 13–51. https://doi.org/10.1016/j.watres.2007.07.025
Gallard, H., & von Gunten, U. (2002). Chlorination of natural organic matter: Kinetics of
chlorination and of THM formation. Water Research, 36(1), 65–74. https://doi.org/10.1016/
S0043-1354(01)00187-7 _ Galloway, M., & Mahoney, J. (2004). Ultrafiltration for seawater
reverse osmosis
pretreatment. Membrane Technology, 2004(1), 5–8. https://doi.org/10.1016/
S0958-2118(04)00063-1 Hach Company. (2014). Oxygen demand, chemical - mercury-
free reaction digestion
method. Method, 10236. DOC316.53.01254, Edition 2). Available at https://www.
hach.com/asset-get.download-en.jsa?id=7639982524.
7
Food Control 127 (2021) 108112
Hach Company. (2017). Nitrogen, simplified TKN (s-TKN™), method, 10242.
DOC316.53.01258, Edition 4). Available at https://www.foodprocessing.com/assets/
knowledge_centers/hach/assets/DOC316.53.01258.pdf .
Hanley, A., Heaney, R., Lewis, J., Sones, K., Spinks, E., Wilkinson, A., & Fenwick, G.
(1988). Benzyl isothiocyanate in onion (Allium cepa L.) and mushroom (Agaricus
bisporus)—a re-examination. Food Chemistry, 30(2), 157–162. https://doi.org/
10.1016/0308-8146(88)90153-7 _ Heredia, JB, & Cisneros-Zevallos, L. (2009). The
effect of exogenous ethylene and methyl jasmonate on pal activity, phenolic profiles and
antioxidant capacity of carrots (Daucus carota) under different wounding intensities.
Postharvest Biology and Technology, 51(2), 242–249. https://doi.org/10.1016/
j.postharvbio.2008.07.001 Howard, L., Braswell, D., Heymann, H., Lee, Y., Pike, L., &
Aselage, J. (1995). Sensory attributes and instrumental analysis relationships for strained
processed carrot flavor. Journal of Food Science, 60(1), 145–148. https://doi.org/10.1111/
j.1365-2621.1995.tb05625.x _
Kim, J.-H., Seo, H.-Y., No, K.-M., Han, B.-J., Lee, S.-J., Seo, Y.-S., & Kim , K.-S. (2005).
Changes of volatile odor components in onion by freeze-drying. Journal of the Korean
Society of Food Science and Nutrition, 34(2), 230–235. https://doi.org/10.3746/
jkfn.2005.34.2.230 _
Kliebenstein, DJ, Kroymann, J., & Mitchell-Olds, T. (2005). The
glucosinolate–myrosinase system in an ecological and evolutionary context. Current
Opinion in Plant Biology, 8(3), 264–271. https://doi.org/10.1016/j.pbi.2005.03.002 Lehto,
M., Sipila, ¨ I., Alakukku, L., & Kymal ¨ ¨ Ainen, H.-R. (2014). Water consumption and
wastewaters in fresh-cut vegetable production. Agricultural and Food Science, 23(4),
246–256. Available at https://helda.helsinki.fi/bitstream/handle/10138/165412 /
41306_38272_1_PB.pdf?sequence=1.
Li, J., Teng, Z., Weng, S., Zhou, B., Turner, ER, Vinyard, BT, & Luo, Y. (2019).
Dynamic changes in the physicochemical properties of fresh-cut produce wash water as
impacted by commodity type and processing conditions. Plos One, 14(9), Article
e0222174. https://doi.org/10.1371/journal.pone.0222174 Llorach, R., Martínez-Sanchez,
´ ´ ´
A., Tomas-Barberan, FA, Gil, MI, & Ferreres, F. (2008).
Characterization of polyphenols and antioxidant properties of five lettuce varieties and
escarole. Food Chemistry, 108(3), 1028–1038. https://doi.org/10.1016/j.
foodchem.2007.11.032 Luo, Y., Zhou, B., Van Haute, S., Nou, X., Zhang, B., Teng, Z.,
and Analysis, 20(3),
152–160. https://doi.
Mukhopadhyay, D. (1999).org/10.1016/
Method and
Ndiongue, S., Huck, P., & Slawson, R. (2005). Effects of temperature and biodegradable
organic matter on control of biofilms by free chlorine in a model drinking water distribution
system. Water Research, 39(6), 953–964. https://doi.org/10.1016/j.
watres.2004.12.019
Pattison, DI, & Davies, MJ (2001). Absolute rate constants for the reaction of
hypochlorous acid with protein side chains and peptide bonds. Chemical Research in
Toxicology, 14(10), 1453–1464. https://doi.org/10.1021/tx0155451 Peskin, AV, &
Winterbourn, CC (2001). Kinetics of the reactions of hypochlorous acid and amino acid
chloramines with thiols, methionine, and ascorbate. Free Radical Biology and Medicine,
30(5), 572–579. https://doi.org/10.1016/S0891-5849(00) 00506-2
Rodriguez Galdon, B., Tascon Rodriguez, C., Rodríguez Rodríguez, E., & Diaz Romero, C.
(2008). Organic acid contents in onion cultivars (Allium cepa L.). Journal of
Agricultural and Food Chemistry, 56(15), 6512–6519. https://doi.org/10.1021/
jf800282h _
Sigma-Aldrich. Solid phase extraction: Ion exchange methodology. Available at https://
www.sigmaaldrich.com/analytical-chromatography/sample-preparation/spe/ione xchange
-methodology.html.
Simin, N., Orcic, D., Cetojevic-Simin, D., Mimica-Dukic, N., Anackov, G., Beara, I., Mitic
Culafic, D., & Bozin, B. (2013). Phenolic profile, antioxidant, anti-inflammatory and
cytotoxic activities of small yellow onion (Allium flavum L. subsp. flavum, Alliaceae). LWT-
Food Science and Technology, 54(1), 139–146. https://doi.org/10.1016/j.lwt.2013.05.023 .
Available at.
Teng, Z., Luo, Y., Alborzi, S., Zhou, B., Chen, L., Zhang, J., Zhang, B., Millner, P., &
Wang, Q. (2018). Investigation on chlorine-based sanitization under stabilized
conditions in the presence of organic load. International Journal of Food Microbiology,
266, 150–157. https://doi.org/10.1016/j.ijfoodmicro.2017.11.027 Teng, Z., van Haute, S.,
Zhou, B., Hapeman, CJ, Millner, PD, Wang, Q., & Luo, Y.
(2018). Impacts and interactions of organic compounds with chlorine sanitizer in
recirculated and reused produce processing water. Plos one, 13(12), e0208945. DOI.
https://doi.org/10.1371/journal.pone.0208945.
Toivonen, PM, & Lu, C. (2013). Differential quenching of free chlorine by organic
compounds potentially exuded from injured plant tissues. Postharvest Biology and
Technology, 86, 192–194. https://doi.org/10.1016/j.postharvbio.2013.06.035 USDA-
ARS. (2019a). Food data central search results - lettuce, cos or romaine, raw.
Available at https://fdc.nal.usda.gov/fdc-app.html#/food-details/169247/nutrients
.
USDA-ARS. (2019b). Food data central search results - lettuce, iceberg (includes
crisphead types), raw. Available at https://fdc.nal.usda.gov/fdc-app.html#/food-
details/169248/nutrients .
USDA-ARS. Food data central search results - carrots, raw. Available at https://fdc.nal.
usda.gov/fdc-app.html#/food-details/170393/nutrients , 2019c.
Machine Translated by Google
Z. Teng et al.
USDA-ARS. Food data central search results - onions, raw. Available at https://fdc.nal.
usda.gov/fdc-app.html#/?query=carrots,%20raw , 2019d.
USEPA. (2017). NPDES permit writers' maunal, Chapter 5. Available at https://www3.
epa.gov/npdes/pubs/pwm_chapt_05.pdf .
Van Haute, S., Sampers, I., Holvoet, K., & Uyttendaele, M. (2013). Physicochemical
quality and chemical safety of chlorine as a reconditioning agent and wash water
disinfectant for fresh-cut lettuce washing. Applied and Environmental Microbiology, 79
(9), 2850–2861. https://doi.org/10.1128/AEM.03283-12
Food Control 127 (2021) 108112
Waters. (2001). A Sample preparation primer and guide to solid phase extraction
methods development. Available at https://www.waters.com/webassets/cms/libra ry/
docs/wa20300.pdf .
Weng, S., Luo, Y., Li, J., Zhou, B., Jacangelo, JG, & Schwab, KJ (2016). Assessment and
speciation of chlorine demand in fresh-cut produce wash water. Food Control, 60, 543–
551. https://doi.org/10.1016/j.foodcont.2015.08.031
Xiao, Z., Lester, GE, Park, E., Saftner, RA, Luo, Y., & Wang, Q. (2015). Evaluation and
correlation of sensory attributes and chemical compositions of emerging fresh produce:
Microgreens. Postharvest Biology and Technology, 110, 140–148. https://doi. org/
10.1016/j.postharvbio.2015.07.021