Impact of transportation, storage, and retail shelf conditions on lettuce quality and phytonutrients losses in the supply chain

Abstract This study was initiated to investigate the impact of transportation, storage, and retail shelf conditions on lettuce quality and phytonutrients losses in the urban fresh produce market supply chain. Reducing postharvest losses is a priority to reduce the loss of the dietary‐based phytonutrients and to improve the health of the consumers. Limited information is available in South Africa related to the postharvest and nutrition loss in the urban fresh produce market supply chain. In this study, we quantified the postharvest losses, changes in phytochemicals, and loss of minerals in lettuce at different points of Tshwane Fresh Produce Market supply chain. Lettuce supply to the Tshwane Fresh Produce Market from two different provinces, Gauteng and North West, were included in this study for comparison. Lettuce from the two provinces was collected from five different farms. The loss of fresh weight, changes in visual quality, phytonutrition properties, and economic loss of lettuce at the supply chain points: (a) transport; (b) storage; (c) and at the retail shelf was investigated. Five boxes of lettuce per supply chain point from Gauteng and North West provinces were randomly selected. The results indicated that the high temperature (25°C) and low RH (40%) at the retail shelf affected the weight, overall quality, and phytonutrition properties of lettuce. Cumulative economic loss was higher at the retail shelf due to the inferior quality of lettuce. The study identified where major quality and phytonutrition losses occur during marketing. The study demonstrated to identify the where major food and nutritional loss losses occur during marketing. This information will significantly benefit food sustainability by introducing technologies to manage food and nutrition losses.

Limited information is available in South Africa related to the postharvest and nutrition loss in the urban fresh produce market supply chain. In this study, we quantified the postharvest losses, changes in phytochemicals, and loss of minerals in lettuce at different points of Tshwane Fresh Produce Market supply chain. Lettuce supply to the Tshwane Fresh Produce Market from two different provinces, Gauteng and North West, were included in this study for comparison. Lettuce from the two provinces was collected from five different farms. The loss of fresh weight, changes in visual quality, phytonutrition properties, and economic loss of lettuce at the supply chain points: (a) transport; (b) storage; (c) and at the retail shelf was investigated. Five boxes of lettuce per supply chain point from Gauteng and North West provinces were randomly selected. The results indicated that the high temperature (25°C) and low RH (40%) at the retail shelf affected the weight, overall quality, and phytonutrition properties of lettuce. Cumulative economic loss was higher at the retail shelf due to the inferior quality of lettuce. The study identified where major quality and phytonutrition losses occur during marketing. The study demonstrated to identify the where major food and nutritional loss losses occur during marketing. This information will significantly benefit food sustainability by introducing technologies to manage food and nutrition losses.

| INTRODUC TI ON
The production of vegetables is constrained by postharvest losses, which in turn limit the volumes of good quality produce reaching consumers. Food losses and waste contribute to postharvest losses while the reduction of postharvest losses is reported as a critical component of ensuring future global food security (Aulakh, Regmi, Fulton, & Alexander, 2013). Postharvest losses are also associated with malnutrition (FAO, 2011). Therefore, to sustain food security, food availability needs to be increased via reduction of postharvest losses during the supply chain (Aulakh, Regmi, Fulton, & Alexander, 2013). At the same time, reducing food waste improves marketing and investment for the growers (Kader, 2005). Postharvest food losses include quantitative (decrease in weight) and qualitative (reduction or changes in nutritional values, taste, color, texture, or appearance) or economic losses (Buzby & Hyman, 2012).
Consumption of leafy vegetables such as lettuce is very popular nowadays not only due to their freshness but also for nutritive value and health benefits (Ansorena et al., 2012). Crisphead lettuce which is also known as Iceberg lettuce (Lactuca sativa L.) is considered to be one of the furthermost popular, economically important and commonly consumed vegetables. The health benefits of consuming lettuce on a daily basis are incontestable because lettuce constitutes a rich source of active bioactive compounds which include flavonoids, phenols, antioxidants, vitamins (C, A, and folate), and minerals (potassium, phosphorus, and calcium) (Cruz et al., 2014).
Several major causes of the losses at the retail shelf include poor storage conditions, high temperatures, and rough handling of lettuce heads by market intermediaries and consumers (Kanlayanarat & Acedo, 2011). Insufficient pest control management and nutrient supply during pre-harvest periods are also cited as exerting a negative impact on the postharvest quality of lettuce (Sharma & Singh, 2011). Frequently, such product that does not meet the required market standards and it is rejected at the market level (DAFF, 2016).
Although the postharvest loss of vegetables has been assessed in certain reports in various countries, to our knowledge the impact of supply chain factors on the loss of bioactive compounds and minerals in lettuce was not studied in detail. In light of these considerations, the aims of the present study were (a) to quantify the loss and changes in predominant bioactive compounds such as phenolic acids (chlorogenic, chicoric, caftaric, and caffeic acid), flavonoids (quercetin), carotenoids, total chlorophyll, and ascorbic acid at different points (transport, storage, and the retail shelf) of the supply chain at Tshwane Fresh Produce Market; (b) to identify the supply chain points where the predominant losses occur; and (c) to do an economic analysis of postharvest losses of lettuce.

| Experimental plan and sampling
Five commercial farmers from Gauteng and North West provinces, who are regular suppliers of crisphead lettuce to the Tshwane Fresh Produce Market, were included in this study. These two provinces were selected because they fall among the top lettuce producers in South Africa, and they are the major lettuce suppliers to the Tshwane market throughout the year. Five replicate boxes each containing eight heads total weight of 5 kg of lettuce from each province (North West and Gauteng) were drawn randomly from each point of the supply chain: (a) after the transport (the transportation was done using a refrigerated truck at 10°C and 80% RH); (b) during storage at the market (fluctuated from 15 to 20°C); and (c) at the retail shelf for quantitative and qualitative loss assessments. A completely randomized design was adopted in this study.
Thereafter, two heads of lettuce from each box were snap frozen in liquid nitrogen and held at −80°C to assess the changes in phytonutrition properties. Also, quantitative and quantitative loss and the changes in phytonutrition properties at the retail shelf from Day 1 to Day 4 were conducted to determine the saleable date of the lettuce to ensure that consumers purchase lettuce that is of good quality and high nutritional value.

| Temperature and RH assessments
Temperature (°C) and RH (%) were recorded using a Tinytag T/RH data loggers (Gemini data loggers Ltd., UK) during storage and at the retail shelf.

| Crisphead quality analysis
The percentage weight loss was determined by subtracting sample weights from their initial recorded weight and presented as a percentage of the initial weight (Aguero, Ponce, Moreira, & Roura, 2011). Lettuce head color was impartially measured with a Minolta CR-400 chromameter (Minolta, Osaka, Japan). Color value measurements were taken at three main points on each head, from the outer part, the middle part, and the inner part of the lettuce head. The color changes of crisphead lettuce were quantified in the L*, a*, b*, C, and h° color values (Ren, Li, Qian, Fan, & Du, 2017). The quality defects yellowing, rusty brown, stem discoloration and wilting, and the overall acceptance (based on color, free from defects or decay) were evaluated and scored following a five structure hedonic rating scale (Zhan, Hub, Li, & Panga, 2012) where 5: severe, 4: moderately severe, 3: moderate, 2: slight, 1: fairly free, and 0: none. Overall acceptance rating of three was considered the acceptable limit for sale or consumption (Zhan et al., 2012).  The ABTS + assay was performed using a method described by Arnao, Cano, and Acosta (2001). Samples of 0.2 g of lettuce (snap frozen sample) were extracted twice with 1.5 ml of 80% methanol.

| Determination of bioactive compounds
The stock solutions included 7.4 mM ABTS + solution and 2.6 mM potassium persulfate solution. Thereafter, the working solution was prepared by mixing the two stock solutions in equal net and allowing them to react for 12 hr at room temperature in a dark place.
The solution was then diluted by mixing 1 ml ABTS + solution with 22.5 ml methanol to obtain an absorbance of 1.1 ± 0.002 units at 734 nm using a multiplate reader and ensuring that fresh ABTS + solution was prepared for each assay. Lettuce extracts (15 μl) were allowed to react with 285 μl of the ABTS + solution for 2 hr in a dark place and the absorbance was taken at 734 nm using a multiplate reader. Calibration curves were constructed for each assay using Trolox as the standard. The antioxidant activity (ABTS assay) was expressed as TEAC.

| Mineral analysis
Lettuce leaves were oven dried at 70°C for 48 hr and thereafter ground and sieved to quantify the leaf N, P, K, Ca, Mg, Zn, and Fe content using the ICP OES (inductively coupled plasma optical emission spectrometry) according to Mahlangu, Maboko, Sivakumar, Soundy, and Jifon (2016).

| Economic losses
Economic loss was calculated by computing the monetary value of the physical loss acquired by expressing the percentage of physical loss as a fraction of the actual selling price of each lettuce box per kilogram.

| Statistical analysis
The experiment was conducted twice during 2016 and 2017 winter season and since there are no significant variations between the seasons (year), the data were pooled together for the statistical analysis.
All data were analyzed with the aid of the Genstat program version 15.1, and to determine the mean differences, Fisher's protected least significant differences (LSD) at p < 0.05 level of significance was performed.

| Temperature, RH fluctuation, and weight loss
Cumulative temperature and RH measurements taken during storage and the retail shelf for four consecutive days are shown in However, the overall temperature and RH varied between 21.4-24.2°C and 25.6%-39.9%, respectively at the market shelf (Table 1).
Temperature and RH are major environmental factors that are crucial in maintaining quality and extending the shelf life of fresh fruit and vegetables (Kitinoja, 2002). The total weight loss of crisphead lettuce at three stages of the supply chain within two provinces exhibited great variation with produce accumulating the greatest weight loss at the retail shelf (Table 2). Significant weight loss (45.31%) occurred at the retail shelf (Day 3) than in storage where the weight loss was 25.55%, while the produce after transportation did not show any weight loss (0.00%). However, the magnitude of weight loss of lettuce failed to differ significantly between the two provinces ( Table 2). Weight loss increased significantly with increasing time (days) at the retail shelf and on Day 4 the weight loss was 64.6% (Table 3). It is evident from these results that weight loss is the most important cause of postharvest deterioration because it is associated with the saleable weight. The observed fluctuation between the temperature and the RH (Table 1) is responsible for the observed weigh loss in this study.

| Quality losses
Quality losses due to yellowing, rusty brown, stem discoloration, and wilting damage were significantly higher in lettuce at the retail shelf (Table 2). Also, there were no significant differences observed between the two locations where the lettuce was obtained with respect to the above-mentioned quality defects (Table 2). Rusty brown and stem discoloration slightly increased during storage (3 days), but the quality was still regarded as acceptable for sale and consumption according to the hedonic scale less than 3 (Table 2). At the retail shelf (Day 3), browning and rib discoloration increased severely exhibiting a hedonic scale more or less 3 and the product and shows limited marketability became unmarketable (Table 2). However on Days 3 and 4, the visual quality decreased due to increased browning, pinking, and wilting exceeding the hedonic scale score of 4. It is interesting to note that on Day 3 and Day 4, the lettuce heads at retail shelf the average losses for ascorbic acid were 48% and 68.9%, respectively ( Table 3). The major reason for wilting was water loss due to transpiration through the stomata when the concentration gradient of the water vapor between the air spaces in the leaf and the  (Table 4).
Significant change (lower) in h° in lettuce heads withdrawn at the retail shelf indicated that lettuce heads were becoming more yellow in color (Table 4). The color coordinate a* showed a higher negative value in lettuce heads withdrawn at the transport point and it decreased significantly at the retail shelf indicating the onset of browning (Table 4). The significant decrease in color coordinate b* value in lettuce from the retail self was mainly as a consequence of higher browning (Table 4)

| Variation in bioactive compounds
A significant decrease in ascorbic acid, total chlorophyll content, and carotenoids was noted in the lettuce heads withdrawn at the retail shelf point (Table 5). The ascorbic acid content was 54.91 mg 100 g/FW after transport and 25% of the ascorbic acid content in TA B L E 1 Temperature and RH in storage and at the retail shelf at the Tshwane Fresh Produce Market  to improve the lightness of strawberries, peaches, and apples (Rababah, Ereifej, & Howard, 2005). Previous reports stated that 5%-12% loss of ascorbic acid occurs at 30 and 40°C, respectively, during the 24 hr delay between harvesting and processing (Kader & Morris, 1978). The daily requirement of ascorbic content for a man is 90 mg/d and for women, 75 mg/d (Frei & Trabe, 2001). The study clearly demonstrated that around 81% of the ascorbic acid content was lost on the retail shelf on the Day 4 (Table 3). In the present study, the similar reduction trends were observed with light intensity (L* values) and the ascorbic acid content of lettuce at the retail shelf (Tables 2 and 3). Presence of higher ascorbic acid content was reported to reduce browning and it acts as an anti-browning agent (Landi, Degl'Innocenti, Guglielminetti, & Guidi, 2013). The observed decline in ascorbic acid content at the retail shelf also explains the increased browning (rusty browning and rib discoloration in lettuce in this study Table 3). The observed loss of visual quality is on the other hand associated with the loss of ascorbic acid that acts as an anti-browning agent and also due to the increase activity of the PPO activity (Tomás- Barberán et al., 1997).
The total carotenoid content was also reduced by 22.9% and 47.8% at the storage point and the retail shelf, respectively (Table 5).  (Table 5).
An increasing trend was noted in quercetin content, the antioxidant capacity FRAP, and the ABTS + in lettuce heads at the retail shelf (Table 5). However, the observed variation in ascorbic acid, total chlorophyll content, carotenoids, quercetin content, and the antioxidant capacity (FRAP and the ABTS + ) in lettuce heads withdrawn at different points of the supply chain from the two provinces (Gauteng × North West) were not significant (Table 5). Crisphead lettuce contained higher levels of chicoric, caftaric, and chlorogenic acids than the other phenolic acids ( Table 6). The levels of phenolic acids significantly increased after transport and remained at higher concentrations at the retail shelf (Table 5). Abiotic stress conditions, including temperature and RH variation, often favor the accumulation of flavonoids (Dixon & Paiva, 1995). The observed increase in the concentration of quercetin, phenolic acids (chicoric and chlorogenic acids), and antioxidant activity at the retail shelf could be due to temperature and RH fluctuations (Tables 5 and 6). Chicoric and chlorogenic acids act as substrates for enzymatic browning or pinking in lettuce (Luna et al., 2012;Monaghan, Vickers, Grove, & Beacham, 2017). However, in this study, observed increase in phenolic acid at the retail shelf was more less similar to the observation reported by Zhao, Carey, Young, and Wang (2007). The phenylalanine ammonialyase activity could have increased in response to microbial decay, TA B L E 5 Variation of ascorbic acid, quercetin, total chlorophyll, carotenoids, and antioxidant properties of Crisphead lettuce at the different supply chain points of Tshwane Fresh Produce Market   (Cantos, Espín, & Tomás-Barberán, 2001) and could have resulted in an increased biosynthesis of phenolics during storage and at the retail shelf. On the other hand, the increased concentration of phenolic acids (Table 6) also confirms that there is another mechanism involved in lettuce browning other than the PPO activity as suggested by Mai and Glomb (2013).
The increase in phenolic acids was responsible for observed higher antioxidant power (FRAP) and the antioxidant capacity (ABTS + ) ( Table 5). Correlation between the phenolic compounds and FRAP or ABTS + has been established (Wootton-Beard, Moran, & Ryan, 2011). Therefore, the unmarketable lettuce can be freeze dried and used as functional food ingredient for food supplementation programs.

| Loss of mineral elements
Loss of mineral elements such as K, P, Ca, Mg, Fe, Zn, Mn, Cu, and B was significantly higher at the retail shelf (Table 7A,B) and there were no significant differences observed between the crisphead lettuce obtained from the two locations (Gauteng and North West).
The levels of K, P, and Ca in lettuce heads did not vary significantly between the transport and storage points (  (Table 7B). The loss of mineral elements at the retail shelf was also reported in leafy vegetables (Gogoa, Opiyob, Ulrichs, & Huyskens-Keila, 2017). The loss of mineral elements in this study was mainly due to the higher temperature at the retail shelf that had favored the metabolic changes and senescence (Gogoa et al., 2017).
Also the minerals such as iron and calcium in vegetables can chemically altered and interact with other compounds (Buescher, Howard, & Dexter, 1999). Sometimes, the storage conditions can influence the oxidation state of minerals (Fe and Cu) that can reduce the solubility and the bioavailability of these minerals (Buescher et al., 1999).
The loss of ascorbic acid during storage could have affected the iron availability (Frossard, Bucher, Machler, Mozafar, & Hurrell, 2000) in the lettuce in this study.

| Economic losses
The cumulative losses at the storage and the retail shelf (Day 2) were 36.7% and 53.1%, respectively (Table 8A). The total economic losses at storage and retail shelf were ZR 144 and ZR 146 (Table 7A). Crisphead lettuce during supply chain was higher at the retail shelf (Table 8B).
The geographic site of production, distance from the Tshwane Fresh Produce Market, conditions during agricultural production, postharvest handling, and storage conditions at the farm could have played a vital role in determining the quality (Gogoa et al., 2017).
It is evident in this study that economic losses associated with crisphead lettuce increased along the supply chain. Although these varied between the provinces, the cumulative losses (R/kg) were higher at the retail shelf (Table 7), where the price value was determined by the weight loss and overall quality (Gogoa et al., 2017).
This study illustrated the impact of temperature accumulated at the retail shelf depends on the duration (number of days) (Hertog, Lammertyn, Scheerlinck, & Nicolaï, 2007) and it is evident in this study that the impact of accumulated temperature difference significantly affected the visual quality and the nutritional value with an increase in the number of days. In addition, this study further showed the importance of maintaining the cold chain on the retail shelf to reduce the 45% weight loss of crisphead lettuce in this study. Also, this type of study can be adopted for different commodities and the information generated will benefit in improving the cold chain technologies to reduce food loss and nutrition.

ACK N OWLED G EM ENT
The authors wish to thank the Tshwane Fresh Produce Market officials and the market agents from Gauteng and the North West Provinces for allowing us to conduct this study.

CO N FLI C T O F I NTE R E S T
Non conflict of interest between the authors.

E TH I C A L S TATEM ENT
This study does not include any animal or human testing or questionnaire analysis on market survey.