Postharvest responses of hydroponically grown lettuce varieties to nitrogen application rate

Bevly M. Mampholo, Martin Maboko, Puffy Soundy, Dharini Sivakumar

1 Phytochemical Food Network Research Group, Department of Crop Sciences, Tshwane University of Technology, Pretoria 0001,South Africa

2 Agricultural Research Council-Roodeplaat, Vegetable and Ornamental Plant, Private Bag X293, Pretoria 0001, South Africa

Abstract Limited information is available on the influence of preharvest N application rates on postharvest quality of different lettuce genotypes. Two green leafy lettuce (Multigreen 1 and Multigreen 3) and red leafy lettuce (Multired 4) were grown in gravel film technique and fertigated with five different N application rates: 60, 90, 120, 150 and 180 mg L-1. The 120 mg L-1 N application is commercially recommended for lettuce. After harvest, lettuce samples were packed in a bioriented poly propylene packaging (5% O2 and 5% CO2) and held at 5°C and 85% RH for 3, 6, 9 and 12 days. The genotypes,preharvest N application rates and storage time affected the leaf colour coordinates, phenolic acids (dicaffeoyltataric acid,caffeoyl tartaric acid, 3-caffeoylquinic acid and 3,4-dihydroxycinnamic acid) and browning enzyme activities (phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POD)). Lower rates of N application at preharvest stage showed higher weight loss with the storage time increasing in Multigreen 3. In Multigreen 1, colour coordinate b＊ value decreased remarkably with N application rates from 60 to 120 mg L-1 due to the onset of browning during storage. While in Multigreen 3 and N application higher than 60 mg L-1 influenced the decrease in b＊ value. Browning occurred due to the increased activity of PAL enzyme and the availability of the substrates caftaric, chlorogenic, caffeic acids, PPO activity and production of browning pigments due to the activity of POD. Higher, N application rates (＞120 mg L-1) influenced the browning mechanism and showed brownish red leaves in Multired 4 during storage. Higher ascorbic acid concentration played a role in reducing the onset of browning in the fresh cuts leaves of Mulitired 4 and Multigreen 3 fertilized with lower preharvest lower N application rates (＜120 mg L-1). Preharvest N application at 90 mg L-1 retained the colour, ascorbic acid content and the phenolic acid components and extended the shelf life of Multired 4 lettuce up to 6 days.

Keywords: Lactuca sativa, ascorbic acid, phenylalanine ammonia-lyase, polyphenol oxidase, tissue browning

1. lntroduction

Lettuce (Lactuca sativa) is a rich source of antioxidants such as polyphenols, ascorbic acid and carotenoids (Sofo et al. 2016). However, antioxidant content in plants can be manipulated by growing conditions and agronomic practices (Pérez-López et al. 2018). At the same time,preharvest factors such as fertiliser application play a major role in determining product quality and shelf life of fruits and vegetables (Kader 2008). Shortage or excess of nitrogen can positively or negatively affect quality parameters and nutritional components of lettuce (Kader 2008; Sofo et al. 2016). Although information is available on the influence of nitrogen in soil (Hoque et al. 2010) or hydroponic systems (Bonasia et al. 2013) on quality, the information on postharvest quality and bioactive compounds is limited. Nitrogen application of 225 kg ha-1showed the least postharvest decay in romaine lettuce and defects in iceberg lettuce (Hoque et al. 2010). Higher N application rates (337 kg ha-1) have been reported to negatively affect postharvest quality in romaine lettuce (Hoque et al. 2010).Furthermore, Luna et al. (2013) recommended moderate levels of N to obtain better postharvest quality in lettuce.Growing salad vegetables with a short growing cycle like lettuce in a hydroponic system is a popular practice, which has many advantages such as providing good quality and sanitary products without soil contaminants, while benefiting the environment by reducing water and nutrient usage(Fontana and Nicola 2008).

Fresh cut lettuce salads are very popular due to convenience and higher consumer acceptance. Weight loss,colour, texture and appearance greatly affect postharvest quality, consumer acceptance and the saleable price of fresh cut lettuce (Hoque et al. 2010). The term appearance describes the size, shape, wholeness, presence of defects and consistency for fresh cut vegetables (Bonasia et al.2013). Texture of vegetables is influenced by cellular turgor pressure, which determines the consistency or weight loss of the product (Bonasia et al. 2013). In current marketing practice, fresh cut lettuce are packed in modified atmosphere packaging (MAP) and maintained at low temperature storage to retain quality and nutritional components similar to the whole original product at harvest(Tudela et al. 2016). Shelf life of fresh cut vegetables is limited due to enzymatic browning that alters the colour of the product due to the production of brown pigments(Hisaminato et al. 2001). Browning in fresh cut lettuce is one of the primary causes of quality loss (Mai and Glom 2013). Furthermore, browning of lettuce affects sensory and biochemical properties which affect consumer acceptance of the product (Mai and Glom 2013). Phenylalanine ammonialyase (PAL) activity, polyphenol content (chlorogenic acid,isochlorogenic acid and dicaffeoyl tartaric acid), polyphenol oxidase and peroxidase activities are involved in the production of o-quinones browning pigments (Hisaminato et al. 2001). Although several chemical treatments have been recommended to control the phenolic metabolism associated with browning, there are concerns about chemical toxicity related to food safety with regards to the recommended anti-browning agents or treatments(Altunkaya and G?kmen 2008).

Agronomic practices such as nitrogen (N) application rates were demonstrated as a tool to manipulate the enhancement of phytochemicals (Mampholo et al. 2018).Furthermore, N application rates can influence the quality and shelf life of fresh cut products (Bonasia et al. 2013).Since N application influences cell size, number of leaves and fresh mass, N available during the growth phase of the plant can also influence postharvest quality and shelf life(Weston and Barth 1997).

It is also important to understand the impact of nitrogen application on storage loss and phytochemical properties in red and green loose leafy lettuce cultivars used for fresh cuts. Optimum N application rates to obtain desirable yields with higher bioactive compounds and quality attributes differ between varieties (Becker et al.2015). However, in practice, limited standard protocols are available for lettuce breeding companies in relation to fresh cut processing on cultivar selection or recommended preharvest N application. Therefore, the aim of this study was to determine the influence of different N application rates on green and red lettuce varieties grown for fresh cuts, packed in standard MAP and held at 5°C up to 12 days on the retention of overall quality (weight loss,browning, colour values, L＊, a＊, b＊), browning enzymes PAL,polyphenol oxidase (PPO), peroxidase (POD), browning substrates (3,4-dihydroxycinnamic acid, caffeoyl tartaric acid, 3-caffeoylquinic acid and dicaffeoyltartaric acid) and ascorbic acid content.

2. Materials and methods

2.1. Plant materials

Lettuce varieties, two green Mutigreen 1 and Multigreen 3(Green Curly) and one red Multired 4 (Red Oak) seeds purchased from Starke Ayres Pty. Ltd., Kempton Park, South Africa were grown as stated by Mampholo et al. (2018). The conditions for growing the lettuce, the fertilizer application in 24 hydroponic tables (gravel-film technique under 40% white shade-net structure during winter season (June to July) in 2015 and 2016 were elaborated by Mampholo et al. (2018).The N fertilizer was applied at six different concentrations of 60, 90, 120, 150 and 180 mg L-1as ammonium nitrate(NH4NO3). The treatments were replicated four times and a split-plot design was used to evaluate the performance.The gravel-film technique culture included 24 tanks filled with 1 000 L of nutrient. The nutrients were dissolved in g 1 000 L-1of water as stated by Mampholo et al. (2018): calcium chloride (CaCl2) (67 mg L-1Ca and 132 mg L-1Cl), mono potassium phosphate (MKP) (45 mg L-1P and 57 mg L-1K),magnesium sulfate heptahydrate (MgSO4·7H2O) (44 mg L-1Mg and 57 mg L-1S), potassium sulfate (K2SO4) (118 mg L-1K and 50 mg L-1S), potassium chloride (KCl) (65 mg L-1K and 59 mg L-1Cl) and Hidrospoor (1.74 mg L-1Fe,0.36 mg L-1Mn, 0.22 mg L-1Zn, 0.024 mg L-1Cu, 0.46 mg L-1B and 0.04 mg L1molybdenum (Mo). A pH 6.0 to 6.5 was maintained in the nutrient solution.

2.2. Sample preparation and storage conditions

Lettuce leaves (young leaves) from the respective N application treatments that were free from decay, defects and mechanical damage were separated from the petiole.Thereafter, 200 g lettuce leaves were packed in MAP bioriented polypropylene bags with micro perforations(Knilam Packaging (Pty) Ltd., Cape Town, South Africa)used for marketing fresh cut lettuce. The thickness of the bags was 35 μm (size 40 cm×18 cm). The MAP bags were thermally sealed at the top with a sealer as mentioned by Mampholo et al. (2013) in order to create a suitable internal atmosphere. The gas composition within the MAP remained at 5% O2and 5% CO2. Thereafter, the MAP packed lettuce samples obtained from different preharvest N application treatments were held at 5°C for up to 12 days. Before packaging (day 0) weight of the leaves, visual characteristics(colour values), ascorbic acid, components of phenolic acids and anti-browning enzymes (PAL, PPO and POD) were all determined. At 3 day intervals, samples were withdrawn from the low temperature storage and evaluations were made on weight loss, and above mentioned parameters were determined in five replicates.

2.3. Weight loss

Weight loss was determined according to the method described by Mampholo et al. (2013). The weight of the packages were weighed before and after 3, 6, 9 and 12 days of storage and the data were expressed in percentage weight loss.

2.4. Evaluation of browning

Browning at the green leaf stalk and the blade were visually evaluated using a 1-4 hedonic scale (1=no browning;2=slight browning; 3=definite browning; 4=extreme browning) as described by Hisaminato et al. (2001).Browning at the red leaf stalk and the blade were visually evaluated using a 1-3 hedonic scale (1=no browning;2=slight reddish brown; 3=darker reddish brown).

2.5. Leaf colour values L＊, a＊ and b＊

Leaf surface colour was measured as described by Mampholo et al. (2013), using a Minolta CR-400 chromameter (Minolta,Osaka, Japan). Calibration of the chromameter was performed using a standard white tile. In the CIE colour system, positive a＊ values describe the intensity of red colour, while positive b＊ values describe the intensity of yellow colour and the L＊ (Luminosity) describes lightness.Measurements were taken from five points on two lettuce leaves as described by Mahlangu et al. (2016).

2.6. Ascorbic acid content

Determination of ascorbic acid was performed as previously described (Mahlangu et al. 2016) using 5 g of fresh leaves.

2.7. Phenolic acid composition

For the quantification of phenolic acids (3-caffeoylquinic acid(chlorogenic acid), 3,4-dihydroxycinnamic acid (caffeic acid),dicaffeoyltartaric acid (chichoric acid), caffeoyl tartaric acid(caftaric acid)) 5 g of freeze dried samples were extracted in 16 mL of methanol-water-formic acid mixture (25:24:3,v/v) using an ultrasonic extraction devise as described previously (Ntsoane et al. 2016; Malejane et al. 2018).The phenolic acids were quantified at 330 nm using HPLC(Ntsoane et al. 2016).

2.8. Phenylalanine ammonia-lyase (PAL) activity

The PAL activity was determined according to Sellamuthu et al. (2013). An aliquot of 75 μL enzyme extract was mixed with 150 μL of borate buffer (50 mmol L-1, pH 8.8)containing 20 mmol L-1l-phenylalanine for 60 min at 37°C.After incubation, the reaction was stopped by adding 75 μL of 1 mol L-1HCl and the production of cinnamate was measured at 290 nm (Zenyth 200rt Microplate Reader Biochrom Ltd.,UK). The specific activity of the enzyme was expressed in nmol cinnamic acid mg-1protein h-1.

2.9. Polyphenol oxidase (PPO) activity

The PPO activity was determined using a reaction mixture containing 75 μL of catechol solution prepared in 0.05 mol L-1sodium phosphate buffer at pH 6.5 according the method described by Sellamuthu et al. (2013). The reaction mixture was incubated for 5 min at 30°C and the enzyme activity was measured at 420 nm using a spectrophotometer reader(Zenyth 200rt Microplate Reader Biochrom Ltd, UK) at 420 nm and at 25°C, and expressed as unit μg-1protein min-1.

2.10. Peroxidase (POD) activity

The POD activity was determined according to the method of Sellamuthu et al. (2013). An aliquot of 36 μL of enzyme in 144 μL of 0.1 mol L-1sodium phosphate buffer (pH 7) and 4% guaiacol was incubated for 5 min at 30°C. Subsequently,72 μL of H2O2(100 mmol L-1) was added and the increase in absorbance at 460 nm for 120 s was measured (Zenyth 200rt Microplate Reader Biochrom Ltd., UK). The specific activity of the enzyme was expressed in unit of enzyme activity as ΔOD460mg-1protein min-1. Protein determination was carried out by the method described by Bradford (1976)using bovine serum albumin as standard.

2.11. Statistical analysis

The two-year data were subjected to analysis of variance(ANOVA) using a statistical program GenStat?ver. 11.1 (VSN International Ltd., Hemel Hempstead, UK). Treatment mean values were separated using Fisher’s protected t-test least significant difference (LSD) at the 5% level of significance.The polynomial model procedure was tested using GenStat,ver. 11.1 to analyse the effect of N application rates on the postharvest parameters such as weight loss, colour values and browning enzyme activities after postharvest storage.

3. Results and discussion

Weight loss, phenolic content, browning enzymes, leaf colour changes and ascorbic acid content did not vary significantly with the rates of N application during both years and were not affected by N rate in all three lettuce varieties(V) during both years. The interactions between Y×N (the year and N application rates) and Y×N×V (V, varieties) were not significant during this trial for the above-mentioned parameters.

3.1. lmpact of N supply and weight loss during postharvest storage

Percentage weight loss increased with storage time irrespective of the rates of pre-harvest N application in Multigreen 1 (Fig. 1-A) and Multired 4 (Fig. 1-C). However,in Multigreen 3, lower rates of N application at preharvest stage showed higher weight loss with the storage time increasing (Fig. 1-B). When compared the three varieties of fresh cut lettuce, the red variety, Multired 4 revealed higher percentage of weight loss. Percentage weight loss in Multigreen 1 was lower than in Multired 4 with the storage time increasing. In green variety Multigreen 3, the weight loss (%) increased around 1 to 2% with preharvest application less than 90 mg L-1N. Weight loss is a vital factor associated with the saleable weight during marketing and weight loss higher than 5 to 10% has been reported to reduce the saleable value of fresh produce due to wilting(Brown and Bourne 1988). Weight loss is associated with water loss due to transpiration (Ben-Yehoshua 1990) which can occur through damage of the barriers that protect against transpiration during fresh cut processing (Ben-Yehoshua 1990). Weight loss within the MAP is affected by transpiration and moisture condensation within the packaging (Volpe et al. 2018). However, in this study, the response to preharvest N application rates during storage differed between the different varieties. Lower rates of N application influenced weight loss with the storage time increasing in fresh cut green lettuce Multigreen 3 (Fig. 1-B).From our findings, the percentage weight loss was higher in Multired 4 due to lower thickness of the cuticle. According to previous reports, the influence of N application rate was very low on weight loss in crisphead cultivars Marius and Saladin(Poulsen et al. 1994). Unfertilised Butterhead lettuce(cv. Faustina-ISEA) planted in soil showed higher weight loss than lettuce fertilised with 100 kg ha-1N during 12 days of storage (Bonasia et al. 2013).

Fig. 1 Influence of preharvest nitrogen application on percentage weight loss of fresh cut lettuce cultivars during postharvest storage. A, Mutigreen 1. B, Multigreen 3. C, Multired 4.

3.2. lmpact of N supply on biochemical mechanism of browning, phenolic profile and leaf colour changes during postharvest storage

Leaf colour of fresh cuts of the different types of lettuce varieties varied in response to preharvest N application during storage. Colour value L＊ (light intensity), was not significantly affected by the different N application rates in fresh cuts of variety Multigreen 1 (Fig. 2-A). When the light intensity decreased, leaves became darker with the storage time increasing (Fig. 2-A). Although Multigreen 3 fertilized with lower preharvest N application retained the lightness or glossiness of the leaf up to 3 days during postharvest storage (Fig. 2-B), the light intensity decreased irrespective of lower or higher preharvest N application rates with the storage time increasing (12 days) (Fig. 2-B). However, in red variety Multired 4, light intensity was not remarkably influenced by storage time or preharvest N application rates (Fig. 2-C).

The chromaticity (a＊) relates to the green colour of the lettuce and this was affected during storage in Multigreen 1(Fig. 3-A). However, lower and higher N application rates remarkably reduced the a＊ value and showed darker green leaves (Fig. 3-A). With the storage time increasing, the leaves became more darker by reducing the a＊ value. On the other hand, b＊ value decreased remarkably with N application rates from 60 to 120 mg L-1with the storage time increasing (6 to 9 day) indicating the onset of browning(scales 1-2) in Multigreen 1 (Fig. 1).

Fig. 2 Influence of preharvest nitrogen application on colour coordinate L＊ of fresh cut lettuce during postharvest storage. A,Multigreen 1. B, Multigreen 3. C, Multired 4.

Fig. 3 Influence of preharvest nitrogen application on leaf colour coordinates and browning enzyme activities in Multigreen 1 fresh cut lettuce during postharvest storage. A, a＊. B, b＊. C, phenylalanine ammonia-lyase (PAL) activity. D, polyphenol oxidase (PPO)activity. E, peroxidase (POD) activity.

On the contrary, higher b＊ value and absence of browning was noted at lower and higher N application rates at shorter storage time (day 3) (Fig. 3-B). Also, the PAL activity was higher in fresh cuts of Multigreen 1 subjected to preharvest N application of 60 to 90 mg L-1and gradually declined with higher (＞120 mg L-1) N application rates during storage(Fig. 3-C). The higher PAL activity can be linked with the higher concentrations of 3,4-dihydroxycinnamic, caffeoyl tartaric and 3-caffeoylquinic acids observed with lower rates of preharvest N (＜120 mg L-1) application (Fig. 3-C).

It is interesting to note that the PPO activity in fresh cuts of Multigreen 1 subjected to higher preharvest N application rates (＞90 mg L-1) showed a noticeable increase with storage time (Fig. 3-D) than those applied with lower N application rates (60 to 90 kg ha-1). The lower b＊ value relates to the higher browning (scale 4) (Fig. 3-B) at N application rates(＞90 mg L-1) that coincided with the higher PPO activity(Fig. 3-D). Although POD activity increased slightly with storage time, it was higher at preharvest N application(＜90 mg L-1) and declined gradually with increasing rates of N application in fresh cut Multigreen 1 (Fig. 3-E). It is noteworthy to mention that the preharvest application of N rates showed a close relationship with the accumulation of dicaffeoyltartaric acid (caftaric acid), 3-caffeoylquinic(chlorogenic acids) in Multigreen 1 (Mampholo et al. 2018).

However, caftaric chlorogenic and caffeic acids (Tables 1,2 and 3) could have acted as substrates of PPO activity in fresh cuts of Multigreen 1 applied with higher N application rates ＞90 mg L-1at preharvest stage on day 3 in storage.A sharp decline in concentration of caffeoyl tartaric and 3-caffeoylquinic acids were noted on day 6 in storage(Tables 1 and 2). Higher N application rates showed a significantly lower concentration of caffeoyl tartaric,3-caffeoylquinic, and caffeic acids in Multigreen 1 (Tables 1, 2 and 3) indicating that they were used as substrates for the PPO activity. The POD activity was higher at preharvest lower N application rates and declined with increasing N application rates without much influence from the storage time (Fig. 3-E). However, the dicaffeoyltartaric acid was noted to increase in Multigreen 1 during storage with respect to the N application treatments.

In fresh cuts of Mulitigreen 3, preharvest application of lower N rates with shorter storage time decreased the chromaticity a＊ value indicating retention of green colour in the leaves (Fig. 4-A). Preharvest application of lower N rates with increasing storage showed less variation in the a＊ value (Fig. 4-A). Although higher N application rates slightly reduced the a＊ value on day 3, with the storage time increasing, the a＊ value marginally decreased and indicated dark green leaves (Fig. 4-A). Moreover,preharvest N application rates＞120 mg L-1showed slightly higher a＊ values indicating degradation of the green colour.In this variety, the lower rates (60 mg L-1) of preharvest N application showed remarkably higher b＊ values indicating absence of browning on day 3 of storage (Figs. 1 and 4-B). The chromaticity (b＊) value decreased with higher N application rates of 90 to 150 mg L-1and slightly decreased with the storage time increasing (9 to 12 days), leading to the browning (scale 4) of fresh cut leaves (Figs. 1 and 4-B).

A prominent decline in the chromaticity (b＊) value was noted with preharvest N application rates＞60 mg L-1possibly due to the onset of browning in the leaves (Fig.4-B). The influence of 120 mg L-1preharvest application rate on browning was higher on day 3 and marginally increased with the storage time increasing (Fig. 4-B).Previous research on preharvest application of N rates on phenolic acids demonstrated a close relationship between N and caffeoyl tartaric acid at harvest (Mampholo et al.2018). However, the concentrations of caffeoyl tartaric and 3,4-dihydroxycinnamic acids in the fresh cut Multigreen 3 were higher at preharvest 60 to 90 mg L-1and decreased subsequently at 120 mg L-1and slowly increased at 150 and 180 mg L-1(Tables 1-3). This variation in the changes in phenolic acid concentrations corresponds well with the trend observed in the PAL activity in Multigreen 3 (Tables 1-3; Fig. 4-C). PAL activity was higher in fresh cut lettuce that received lower supply of N (＜120 mg L-1). Decreasing phenolic acid concentrations and increasing PAL activity were observed with the storage time increasing (Tables 1-3; Fig. 4-C). The observed trend in PAL activity also corresponded well with the PPO activity (Fig. 4-D) and further confirmed the observed onset of browning (declining chromaticity b＊ value) during storage in fresh cuts of Multigreen 3 applied with preharvest N treatment of 60 and 120 mg L-1. POD activity was increased under N treatment from 60 to 150 mg L-1on day 3 and declined with the storage time increasing, where as in those leaves subjected to preharvest N treatment of 60 mg L-1the POD activity was higher from day 6 onwards (Fig. 4-E).

Table 1 Influence of nitrogen on caffeoyl tartaric acid in lettuce varieties during postharvest storage (mg 100 g-1 DW)1)

Table 2 Influence of nitrogen on 3-caffeoylquinic acid (mg 100 g-1 DW) in lettuce varieties during postharvest storage1)

Positive chromaticity a＊ value relates to the red colour in red lettuce variety Multired 4 and preharvest N application at lower rates showed slightly higher a＊ values during storage (3 to 12 days) (Fig. 5-A). However, with higher rates of N application, an opposite trend showing a slight decrease in a＊ values was noted (Fig. 5-A). Preharvest 90 to 120 mg L-1N application rates led to a mild decline in a＊ and b＊ values in fresh cuts of Multired 4 on day 6 (Fig. 5-A and B). But with the storage time increasing, the chromaticity b＊ value further decreased (Fig. 5-B) and revealed brownish red leaves (scale 2). Also, the higher N application rates(＞120 mg L-1), showed a similar trend, revealing lower b＊value on day 3 (Fig. 5-B) but the b＊ value continued to increase slightly with the storage time increasing indicating more bluish red colour (Fig. 5-B). Dicaffeoyltartaric acid is the predominant acid in Multired 4 and the accumulation of this phenolic compound was noted to increase with higher N application rates＞120 mg L-1at harvest (Mampholo et al. 2018) and during postharvest storage (Table 4).Dicaffeoyltartaric acid showed the highest concentration with 180 mg L-1N on day 12 during storage (Table 4). This increasing trend in the accumulation of dicaffeoyltartaric,chlorogenic, 3,4-dihydroxycinnamic and caffeoyl tartaric acids coincided well with an increase in PAL activity (Tables 1-4; Fig. 5-C). However, the PPO and the POD activities were higher in fresh cut lettuce of Multired 4 applied with lower N application rates (＜120 mg L-1) on day 3 of storage.This notably corresponds with the declining concentration of chlorogenic, 3,4-dihydroxycinnamic and caffeoyl tartaric acids which probably could have been used as substrates(Tables 1-4). However, overall, the concentrations of phenolic acids declined during storage with increasing time (Tables 1-4). The POD activity could conceivably be responsible for the degradation of phenolic compounds.The currently recommended application of 120 mg L-1at preharvest stage helped to reduce the dark brown colour(higher b＊) up to day 3 (Fig. 5-C).

Retention of ascorbic acid content during postharvest storage followed the trend: Multired 4＞Multigreen 3＞Multigreen 1 (Table 5). Ascorbic acid concentration significantly decreased during storage in this study (Table 5).Ascorbic acid content in all three fresh cut lettuce typesdecreased with higher rates of preharvest N application at the preharvest stage (Mampholo et al. 2018). When comparing the three leafy lettuce varieties, the red lettuce variety Multired 4 retained more ascorbic acid content up the day 6 (Table 5). The fresh-cuts of green varieties can be stored up to day 3. Preharvest N application of 90 mg L-1has the potential to provide about 19 mg of ascorbic acid in 100 g FW of Mulltired 4 leaves on day 6 of storage (Table 5).

Table 3 Influence of nitrogen on 3,4-dihydroxycinnamic acid in lettuce varieties during postharvest storage (mg 100 g-1 DW)1)

Fig. 4 Influence of preharvest nitrogen application on leaf colour coordinates and browning enzyme activities in Multigreen 3 fresh cut lettuce during postharvest storage. A, a＊. B, b＊. C, phenylalanine ammonia-lyase (PAL) activity. D, polyphenol oxidase(PPO) activity. E, peroxidase (POD) activity.

Fig. 5 Influence of preharvest nitrogen application leaf colour coordinates and browning enzyme activities in Multired 4 fresh cut lettuce during postharvest storage. A, a＊. B, b＊. C, phenylalanine ammonia-lyase (PAL) activity. D, polyphenol oxidase (PPO)activity. E, peroxidase (POD) activity.

Table 4 Influence of nitrogen on dicaffeoyltartaric acid in lettuce varieties during postharvest storage (mg 100 g-1 DW)1)

Prehavest N application rates did not affect the colour value L＊ in fresh cuts of Multired 4 (red leafy lettuce) and Multigreen 1 and this corresponds with the fresh cuts of Butterhead lettuce (cv. Faustina-ISEA) (Bonasia et al.2013). However, the colour value L＊ was influenced by lower N application at preharvest stage in fresh cuts of Multigreen 3.The changes in colour value L＊ (low) and b＊ (low) could possibly be attributed to browning, which both affect the visual quality of the fresh cuts. The severity of browning varies between cultivars (Lopez-Galvez et al. 1996). The application of higher than recommended N rates have been reported to reduce shelf life mainly due to an increase in susceptibility to mechanical injury, physiological disorders and decay (Kader 2002). Higher rates of N application have been associated with discolouration during storage in cabbage and potato. Berard (1990) demonstrated the influence of higher N application rates on the incidence and severity of black midrib in cold storage in the susceptible cultivar Safekeeper.

Table 5 Influence of nitrogen on ascorbic acid in lettuce varieties during postharvest storage (mg 100 g-1 DW)1)

Furthermore, electrolyte leakage in the leaf tissue was reduced at lower N application rate and electrolyte leakage and change in membrane structure increased at higher N application rates (Bonasia et al. 2013). But, according to the previous authors, these results were not consistent during the two years of investigation (Bonasia et al. 2013).However, in the current study, red and green lettuce varieties showed different trends in their browning response related to the chromaticity b＊ value with respect to the N application rates (Figs. 3-B, 4-B and 5-B). The application of higher than recommended N rates in gravel film technique (GFT)affected the fresh cut visual quality and coincides with the previous findings of Poulsen et al. (1994) and Bonasia et al.(2013). In Multigreen 1, the higher N application rates for a shorter time showed lower browning (higher b＊ value).Therefore, it is evident from this study that the response to preharvest N application rates on browning depends on the variety.

Wounding increases the PAL activity (Couture et al.1993). Physiological attributes related to quality attributes and storage life of minimally processed lettuce coincided with increasing concentration of predominantly phenolic acids in different fresh cuts of lettuce cultivars. Increase in specific phenolic acid concentration differed according to the N application in different cultivars in this study (Luna et al. 2012). Furthermore, the decrease in phenolic acids and increasing PPO is likely to explain the browning or deep blush brown colour in red Multired 4 during storage with higher N application rates (Luna et al. 2012). The reduction in b＊ value related to the onset of browning in the fresh cuts of Mulitired 4 and Multigreen 3 were minimised by lower preharvest N application rates (＜120 mg L-1) due to fairly higher concentrations of ascorbic acid (Table 5).However, Luna et al. (2013) reported that the ascorbic acid concentrations are higher in red lettuce cultivars compared to the green cultivars. The higher ascorbic acid concentrations in Multired 4 could probably have controlled the browning and maintained the higher b＊ value (Table 5).Multired 4 showed improved shelf life and overall quality due to the higher concentration of phenolic compounds and lower PPO activity than the two green lettuce cultivars(Cantwell and Kasmire 2002; Nicolle et al. 2004; Luna et al.2013). Reducing trend (80% reduction) in dicaffeoyltartaric acid concentration was reported during postharvest storage at 5°C and 85% RH (Wills and Stuart 2000). However, a divergence in observation with regards to the increase in dicaffeoyltartaric acid content in Multigreen 1 at similar storage conditions could deny its participation as a substrate for browning mechanism.

4. Conclusion

The GFT hydroponic system can be recommended as a soil less culture system to manage N application and to obtain desirable quality after postharvest storage. N application rates＜120 mg L-1can be recommended to regulate the observed slight changes in browning related to the chromatic value b＊. Remarkable changes in phenolic acid compositions and ascorbic acid content during postharvest storage were noted in the present study. Overall, the red variety Multired 4 performed better during postharvest storage than the green leaf lettuce varieties up to 6 days in colour, ascorbic acid content and phenolic acid compositions.

Acknowledgements

The authors wish to acknowledge the National Research Foundation Grant (98352) for Phytochemical Food Network to Improve Nutritional Quality for Consumers, South Africa.