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re-harvest treatment of spinach with Ascophyllum nodosum extractmproves post-harvest storage and quality
i Fana, Saveetha Kandasamya, D. Mark Hodgesb,lan T. Critchleyc, Balakrishnan Prithiviraj a,∗
Department of Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro, NS B2B 5E3, CanadaAtlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada, 32 Main Street, Kentville, Nova Scotia B4N IJ5, CanadaAcadian Seaplants Limited, 30 Brown Avenue, Dartmouth, NS B3B 1X8, Canada
r t i c l e i n f o
rticle history:eceived 28 August 2013eceived in revised form 7 February 2014ccepted 28 February 2014vailable online 22 March 2014
eywords:pinach leavesscophyllum nodosum
a b s t r a c t
Fresh spinach (Spinacia oleracea L.) leaves contain high concentrations of beneficial phytochemicals. How-ever, spinach does not store well and is highly perishable especially during handling and post-harveststorage. In this study, we investigated the effect of pre-harvest root-treatment of spinach with Ascophyl-lum nodosum extract (ANE) at different concentrations (0, 0.1, 1.0, or 5.0 g L−1) on post-harvest qualityof fresh-cut spinach over a 35-day storage period. At the time of harvest there was no significant differ-ence in dry weight, chlorophyll, ascorbate and lipid peroxidation between ANE-treated and non-treatedplants. However, the loss in fresh weight and visual quality (color and turgor) of spinach leaves duringstorage was reduced by pre-harvest application of ANE. Lipid peroxidation was significantly reduced in
isual qualityeight loss
ipid peroxidationhelf life
ANE-treated leaves. The total chlorophyll content and ascorbate content in the control and treated leaveswas however identical over the storage period and decreased at a similar rate. A negative correlation wasobserved between visual quality and lipid peroxidation. The results show that pre-harvest ANE applica-tion through root drench, especially at 1.0 g L−1, enhanced post-harvest storage quality of spinach leaves.
The incidence of various human chronic diseases, such as can-er and cardiovascular disease, is increasing. The public has realizedhat a nutritious diet promotes the maintenance of good health andeduces the incidence of certain diseases. Epidemiological studiesave documented that a high intake of fruit and vegetables is asso-iated with a reduced occurrence of chronic diseases (Hung et al.,004). As a result, there is an increase in the demand for freshruit and vegetables that are a rich source of bioactive compoundsarticularly anti-oxidative phytochemicals (Kang et al., 2005).
The quality (e.g. color and firmness) and nutrient content ofruit and vegetables decrease during post-harvest storage. Pre- andost-harvest physical damage, undesirable environmental factors,icrobial pathogens and insect pests contribute to the decrease
n quality and shelf life (Kader, 2002). Post-harvest quality lossn fruit and vegetables can often be related to a number of pre-arvest factors like temperature, light intensity, water supply, and
agro-chemicals. Furthermore physiological stresses during the pre-harvest period reduce post-harvest quality (Hodges et al., 2004).The growth stage/maturity at harvest can also influence responseto abiotic stress during storage. Harvesting baby spinach a few daysearly improved visual quality and nutritional value during stor-age (Bergquist et al., 2006). Additionally, timing during the day ofharvest affects bioactive composition and quality of fruits and veg-etables, possibly due to the changes in light intensity and watercontent (Veit et al., 1996).
Post-harvest losses may also result from unfavourable storageconditions which can manifest as loss of flavor, texture and pig-ments; softening of tissues, induction of decay, dehydration/weightloss and a decline in nutrient levels (Kader, 2002; Nishikawa et al.,2003). Unfavorable storage conditions can exacerbate oxidativestress. For example, less-than-optimal storage temperature caninduce oxidative stress, leading to such dysfunctions as loss ofmembrane integrity and accelerated senescence. Senescence is aslow progress of cell death often associated by deterioration of
cellular membranes and is largely related to excess reactive oxy-gen species (ROS) (Hodges et al., 2004). Oxidative stress-associatedpost-harvest senescence includes repression of enzyme activities,loss of pigments and disintegration of membranes and is often
anifested as shortened shelf life and decline of nutritional qualityHodges et al., 2004).
The brown alga, Ascophyllum nodosum is a widely-researchedeaweed species traditionally used as a fertilizer and soil condi-ioning agent. The application of an extract of A. nodosum (ANE)mproved a number of physiological characteristics in plants, suchs increased root growth, elevated capsidiol concentration (Norriend Hiltz, 1999) and improved resistance to environmental stressesKhan et al., 2009). A few investigations looked at the potentialse of ANE to minimize post-harvest losses in fruit and vegeta-les. The deterioration of watermelon and nectarines was delayednd their shelf life was significantly prolonged in ANE-treatedruits compared to the control (Norrie and Hiltz, 1999). Pre-harvestpplication of ANE significantly reduced pear fruit weight loss andncidence of decay during post-harvest handling (Abdel-Hafeez,005).
Spinach is an annual, cool season, green, leafy vegetable whichs rich in health promoting compounds such as vitamin A and C, andavonoids (Conte et al., 2008). Spinach has a relatively high respi-ation and water-loss rates, and is prone to tissue decay (Hodgesnd Forney, 2003), microbial growth (Conte et al., 2008), andoss of nutrients (Bergquist et al., 2006; Pandrangi and Laaborde,004), all of which lead to a low storage potential. Cold storaget ∼7.5 ◦C with a high relative humidity (RH) (>90%) can sig-ificantly improve the shelf life of spinach (Kader, 2002). ANEay induce specific, systemic physiological responses, including
licitation of the phenyl-propanoid and flavonoid pathways ofpinach leaves, thus leading to promotion of nutritional qualitynd anti-radical capacity (Fan et al., 2010). This study was under-aken to investigate the effect of pre-harvest ANE-treatment onhe post-harvest senescence, the retention of vitamin C and the
aintenance of other quality attributes in spinach during stor-ge.
. Materials and methods
.1. Plant culture and treatment
Seeds of spinach (Spinacia oleracea L., var. Unipack 12) wereurchased from Stokes Seeds Co, Thorold, Ontario, Canada. Solubleowder of Ascophyllum nodosum alkaline extract (ANE) (Acadian®)as obtained from Acadian Seaplants Limited, Dartmouth, Nova
cotia, Canada. The organic and inorganic compositions of ANE areresented in the supplementary information section of our previ-us publication (Rayirath et al., 2009). Spinach seeds were plantedt 1.5 cm deep in plastic pots (12.7 cm in diameter; 3 seeds perot and later thinned to one plant per pot) containing sweet soil (1
imestone: 100 peat-moss: 100 perlite: 30 sand, w/v/v/v). Pots werelaced in a growth chamber maintained at 18 ◦C and 95% relativeumidity (RH) with a photo period of 10 h light/14 h dark under
ight from fluorescent tubes and incandescent bulbs of approx-mately 350–400 �mol m−2 s−1. Fertilization with water-soluble0N-20P2O5-20K2O, at a concentration of 200 ppm was initiated
weeks after sowing and then applied every 4 days at a rate of00 mL per pot. Plants were grown for 6 weeks.
Solutions of ANE were prepared by dissolving soluble powdern 1.0 L distilled water with continuous stirring for 10 min. Spinachlants were root drenched with various ANE solutions (i.e. 0, 0.1,.0, or 5.0 g L−1) at the rate of 50 mL per plant on days 21, 28 and5 after planting.
Plants in growth chambers were arranged using randomized
lock design. Twenty spinach plants were used in each treatment,nd the experiments were repeated 4 times. Control plants wererrigated with 50 ml distilled water in place of the ANE treat-
ent.
turae 170 (2014) 70–74 71
2.2. Sample preparation and storage
On day 42 after sowing, leaves from the 5–7th positions countedfrom the bottom were manually cut using a pair of sharp scissorsat a petiole length of three-fourths of the leaf blade length. Thesewere placed in plastic bags in a refrigerated cooler (4 ◦C) within 1 hof harvest.
Unipack 12 is considered as a relative tough cultivar that isnot prone to deteriorate as other spinach cultivars, thus 10 ◦C waschosen as storage temperature for this study. For the studies onchanges during post-harvest storage, randomly selected spinachleaves from each treatment were placed into perforated plasticbags (50 g per bag), sealed and then stored in a dark, controlledstorage room maintained at 10 ◦C with a relative humidity (RH)of ≥95%. Samples were removed from storage on day 0, 7, 14, 21,28, and 35 post-harvest. The leaves were de-veined and choppedinto small pieces (≈0.5–1 cm2). Pieces were thoroughly mixed andsub-samples immediately used for total ascorbate, malondialde-hyde (MDA) and total chlorophyll assays, or bagged, flash-frozen inliquid nitrogen and lyophilized to be stored in sealed plastic bagsat −80 ◦C until use.
2.3. Weight loss measurement
To determine the weight loss during storage, the perforatedplastic bags containing spinach leaves were weighed on day 0, andthe leaves were weighed on the day of removal. Fresh weight losswas expressed as a percentage of the initial fresh weight.
To determine changes in dry weight (DW) during storage, oneach sampling day, de-veined leaves were dried in an oven at 85 ◦Cfor 48 h and weighed.
3. Visual quality analysis
Leaf quality (color and turgor) was assessed in fresh tissuesbased on the visual quality scale developed by Agriculture andAgri-Food Canada (Supplementary Fig. 1).
3.1. Determination of total chlorophyll
All extraction procedures were carried out in dim light on ice.Chlorophyll content was analyzed. Fresh leaf tissue (0.8 g) wasimmediately ground using a mortar and a pestle with cold methanolin the presence of a small amount of inert sand. After centrifug-ing for 10 min at 10,000 × g at 4 ◦C, the pellets were re-extractedin methanol until all color was removed. The volume of combinedextracts was made up to 50 mL in a volumetric flask. Absorbancewas measured at 652, 665, and 750 nm using a spectrophotometer(UV-1700 PharmaSpec, Shimadzu, Tokyo, Japan). The total chloro-phyll content was calculated according to Ritchie (Ritchie, 2008).
3.2. Determination of total ascorbate
Total ascorbate was analyzed following the method of Hodgesand Lester (2006) using l-ascorbic acid as a standard. Fresh choppedleaves (5 g) were immediately ground in a mortar and pestlewith inert sand and 15 mL ice-cold, freshly prepared 5% (w/v) m-phosphoric acid. The homogenate was centrifuged at 10,000 × g for15 min at 4 ◦C. Total ascorbate was determined by initially incubat-ing 100 �L supernatant, 500 �L 150 mM KH2PO4 buffer (pH 7.4)containing 5 mM ethylene diamine tetraacetic acid (EDTA), and
100 �L 10 mM dithiothreitol (DTT) at room temperature for 50 min.Following then 100 �L of 0.5% (w/v) N-ethylmaleimide (NEM) wasadded to remove excess DTT. In order to develop color in reactionmixtures, reagent solutions were added in the order of 400 �L 10%
72 D. Fan et al. / Scientia Horticulturae 170 (2014) 70–74
Days after harvesting7 14 21 28 35
Wei
ght l
oss
(%)
0
10
20
30
Control 0.1 g L-1
1.0 g L-1
5.0 g L-1
a
ab
b
ab
a
a
b
ab
a
b b b
Fig. 1. The effect of Ascophyllum nodosum extract (ANE) on fresh weight losses ofsBd
(dwat
3
usbntt0twamc66B
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5
5
wtoai
0 7 14 21 28 35
Col
or
0
2
4
6
8
10 Control
0.1 g L-1
1.0 g L-1
5.0 g L-1
aa a
ba
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A
Days after harvesting
0 7 14 21 28 35
Turg
or
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2
4
6Control0.1 g L-1
1.0 g L-1
5.0 g L-1
a
bc c
B
pinach leaves held over 35 d post-detachment at 10 ◦C in the dark and RH ≥ 95%.ars indicate mean ± standard error. Values with the same letters on each samplingay are not significantly different (P < 0.05).
w/v) TCA, 400 �L 44% o-phosphoric acid, 400 �L 4% (w/v) �-�1-ipyridyl, and 200 �L 30 g L−1 ferric chloride. The reaction mixturesere incubated at 40 ◦C for 60 min in a shaking water bath, and
bsorbance was taken at 525 nm. The results were calculated fromhe l-ascorbic acid standard curve and expressed as �mol g−1 FW.
.3. Lipid peroxidation analysis
The malondialdehyde (MDA) concentration was measuredsing a modified method after Hodges et al. (2006). Fresh leafamples (2 g) were homogenized in 8 mL 80% ethanol, followedy centrifugation at 3,000 × g at 4 ◦C for 10 min. The super-atant (100 �L) and 900 �L distilled water were added to aest tube with 1 mL of either (i)–TBA (thiobarbituric acid) solu-ion which comprised 20% (w/v) trichloroacetic acid (TCA) and.01% (w/v) butylated hydroxytoluene (BHT), or (ii) +TBA solu-ion containing the above plus 0.65% (w/v) TBA. The mixtureas vortexed, heated at 95 ◦C in a dry bath for 25 min, cooled
nd then centrifuged at 3,000 × g for 10 min. Absorbance waseasured at 440, 532, and 600 nm. MDA equivalents were cal-
ulated using the following formula: (1) [(Abs 532+TBA) − (Abs00+TBA) − (Abs 532-TBA − Abs600-TBA)] = A, (2) [(Abs 440+TBA − Abs00+TBA) 0.0571] = B, (3) MDA equivalents (nmol mL−1) = 106 × [(A-)/157 000].
. Statistical analysis
The effects of treatment were analyzed by ANOVA and dif-erences were considered statistically significant at P ≤ 0.05 usingukeys Honestly Significant Differences (HSD) test of the COSTAT®
tatistical software. Regression analysis was conducted for visualuality with MDA and ascorbate content, respectively.
. Results
.1. Weight loss
The loss of fresh weight increased with storage time (Fig. 1). Theeight loss of the leaves from the control plants was higher than
hat of the leaves from ANE treated spinach. Pre-harvest treatmentf spinach with 1.0 g L−1 ANE significantly reduced the weight losss compared with control and other treatments after day 21 dur-ng the 35-day storage. An average daily loss of 0.6% was observed
Fig. 2. The effect of Ascophyllum nodosum extract (ANE) on (A) color and (B) turgorof spinach leaves held over 35 d post-detachment at 10 ◦C in the dark with RH ≥ 95%.Bars indicate mean ± standard error.
in control plants while the loss from 1.0 g L−1 ANE treatment was0.4%. However, the dry matter content of the leaves from differenttreatments was about 110 g kg−1 and did not change significantlyduring storage or between treatments and the control (data notshown).
5.2. Visual quality
Stored spinach leaves were examined for visual quality (colorand turgor) over a 35-day period. The freshly harvested leavesscored the maximum 10 for visual quality and 5 for turgor. Thevisual quality gradually decreased during storage in all the treat-ments (data not shown).
Control leaves started to curl and yellow after day 7 and becamenotably wilted after day 14 with approximately 25% of the leavesshowing signs of yellowing. The loss of turgor also increased duringstorage in all leaves but was significantly less in 1.0 g L−1 ANE-treated leaves on day 14, 21, and 35 (Fig. 2A). Moreover, 1.0 g L−1
treatment significantly better maintained the color of spinachleaves during the 35-day storage period except on day 35 (Fig. 2B).
5.3. Chlorophyll content
The total chlorophyll content of spinach leaves was 7 mg g−1 DWand there was no significant difference between ANE-treated anduntreated leaves. Little change in chlorophyll content was observed
D. Fan et al. / Scientia Horticul
Days after harvesting0 7 14 21 28 35
Mal
ondi
alde
hyde
(nm
ol g
-1 F
W)
0
2
4
6
8
10
12
14 Control 0.1 g L-1
1.0 g L-1
5.0 g L-1
a
b
a
ab
a
b
a a
b
b
a
a
b
ab
abab
Fig. 3. The effect of Ascophyllum nodosum extract (ANE) on malondialdehyde (MDA)c ◦
we
ucoa(
6
tlswmpt1d1lhMr
7
iFttNw
7
tt(lc
ontent levels of spinach leaves held over 35 d post-detachment at 10 C in the darkith RH ≥ 95%. Bars indicate mean ± standard error. Values with the same letters on
ach sampling day are not significantly different (P < 0.05).
ntil after 14 days of storage. No significant difference in the loss ofhlorophyll was found between control and treated leaves at eachf the sampling time during the post-harvest storage. The aver-ge loss of total chlorophyll was roughly 85 �g g−1 DW per daySupplementary Fig. 2).
. Lipid peroxidation
Oxidative stress, as estimated by malondialdehyde (MDA) con-ent, significantly increased after day 7 in the detached spinacheaves from all treatments (Fig. 3). The initial levels of MDA were notignificantly different between control and treated leaves, whichas 2–3 nmol g−1 FW. As compared with control and 0.1 g L−1 treat-ent, ANE treatment at 1.0 and 5.0 g L−1 significantly reduced lipid
eroxidation on day 14, 21, and 35. The concentration of MDA inhe control leaves increased to over 8 nmol g−1 FW compared with.0 g L−1 treatment that recorded values below 5 nmol g−1 FW onay 21, and over 13 nmol g−1 FW in controls compared with below0 nmol g−1 FW in ANE-treated leaves at day 35. Overall, control
eaves showed a significant increase after day 14 and exhibited theighest level of lipid peroxidation throughout the storage period.DA content was significantly higher in all leaves at the end of
emoval versus the start.
. Ascorbate content
There were no significant differences in total ascorbate contentn leaves between control and treatments on day 0 (Supplementaryig. 3). There was a rapid decrease in ascorbate content in all thereatments during the first 21 days storage. The total ascorbate con-ent declined from 3.6 to 1.3 �mol g−1 FW during 35 days of storage.o differences in total ascorbate between control and treated leavesere noted throughout the post-harvest storage period.
.1. Linear regression analysis
Using regression analysis high correlations were found betweenhe visual quality with MDA levels (R2 = 0.811) and ascorbate con-
2
ent (R = 0.820), respectively, during the 35-day storage periodSupplementary Fig. 4). The higher visual quality of the spinacheaves correlated with a lower level of lipid peroxidation or a higheroncentration of ascorbate.
turae 170 (2014) 70–74 73
8. Discussion
The results presented in this paper demonstrated that the pre-harvest treatment of spinach with ANE (Rayirath et al., 2009)improves the post-harvest storage quality. ANE was applied as rootirrigation which resulted in an enhanced storage quality of cutleaves. This suggests that the organic and inorganic componentspresent in ANE (Rayirath et al., 2009) might have induced certainsystemic physiological changes in the plant and lead to improvedpost-harvest shelf life of spinach leaves.
Spinach is an important leafy vegetable in regard to nutritionalquality and ranks third in total antioxidant capacity, behind garlic(Allium sativum) and kale (Brassica oleracea) (Cao et al., 1996). Thegenotype and growth conditions impact plant metabolism, whichin turn can affect crop quality at harvest and post-harvest storage(Weston and Barth, 1997). Commercially, spinach leaves are storedin polypropylene bags at low temperatures close to 0 ◦C (Bergquistet al., 2006) but often they are kept in the range of 4–10 ◦C. Post-harvest changes in spinach may include losses in visual quality andincrease in microbial populations concomitant with reduction innutrient content (Conte et al., 2008).
Weight loss and visible signs of deterioration are two majorconcerns in the storage of leafy vegetables. Fresh weight loss wassignificantly reduced in ANE-treatment (1.0 g L−1) as compared tothe control after day 21 during storage. The dry weight (DW) ofspinach leaves from all treatments was however not affected. Onepossibility is that the application of ANE may lead to reducedactivities of lipoxygenase and peroxidase which have been shownto accelerate the degradation processes (Pandrangi and Laaborde,2004).
Changes in leaf visual quality were analyzed using two parame-ters, color and turgor. Leaves harvested from untreated plant turnedyellow and wilted early during storage as compared to the leavesharvested from ANE-treated plant. ANE-treated (1.0 g L−1) leavesretained turgor better than the control or other concentrationsof ANE tested. The shriveled appearance of spinach leaves dur-ing storage correlated with the loss of fresh weight in each of thetreatment. In order to understand the extent chlorophyll contentmay affect visual quality of stored spinach leaves, the changes oftotal chlorophyll during storage were studied. The total chlorophyllcontent decreased during storage regardless of the treatment, anddegradation rate increased after day 14 of storage, which was inconcurrence with previous report (Hodges and Forney, 2003). Thissuggests that the changes in chlorophyll levels may not directlycorrelate with visual quality of stored spinach leaves. One possiblereason is that the loss of chlorophyll in leaves tends to be distributedunequally (Bergquist et al., 2006).
Changes in color of the harvested leaves have been shown tobe related to senescence (YamauchiN and Watada, 1991) whichis largely regulated by reactive oxygen species (ROS). Increasedlipid peroxidation is one of the major characteristics of leaf senes-cence and malondialdehyde (MDA) is the major by-product oflipid peroxidation. Zhang and Ervin (2008) reported that ANEapplication significantly reduced lipid peroxidation, accompaniedwith notable increase in leaf superoxide dismutase activity increeping bentgrass (Agrostis stolonifera L.) under heat stress. Pre-harvest ANE treatments did not affect the MDA content of leavesat the time of harvest. Although the MDA content increasedduring storage in all treatments, ANE treatments at 1.0 and5.0 g L−1 significantly reduced the rate of increase after day 7, sug-gesting that pre-harvest treatment of spinach with ANE mightreduce ROS production through stimulation of antioxidant sys-
tems. The high level of polyunsaturated fatty acids (PUFA) inspinach has been shown to be associated with high MDA con-tent; hence ANE application to plants prior to-harvest may alsolead to direct effects on the reduced activities of enzymes that are
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ssociated with phospholipids degradation and subsequent senes-ence (Barclay and McKersie, 1994).
Vitamin C is a key component of the plant defense systemsHodges and Forney, 2003). Higher initial ascorbic acid content andnitial dry matter content in baby spinach resulted in better visualuality during storage (Bergquist et al., 2006). Ascorbate contentecreased significantly during post-harvest storage and no dif-erence was observed between ANE-treated leaves and untreatedontrols. The correlation between DW and visual quality in maturepinach leaves during storage was weak (R2 = 0.304). However,trong correlations were found between MDA or ascorbate con-ent with the visual quality of spinach leaves during storage. This
ay be attributed to the higher antioxidant levels in the leaves andn increased ability to detoxify ROS during post-harvest storage,eading to a retarded senescence rate (Zhang and Ervin, 2008).
In our earlier study (Fan et al., 2011) pre-harvest treatmentf ANE increased the antioxidant content of spinach. Hence theeduced MDA might be due to the elevated anti-oxidative capac-ty in spinach leaves. Differences in the changes in fresh weight,hlorophyll content and MDA levels between ANE-treated leavesnd control during post-harvest storage suggest that pre-harvestreatment of spinach with ANE, especially at 1.0 g L−1, improve thetorage quality of spinach by regulating the rate of senescence androbably are independent of ascorbate content.
. Conclusions
The effect of root drench pre-harvest treatment of spinach withscophyllum nodosum extract (ANE) on post-harvest storage anduality was studied. Treatment of spinach with ANE improveduality of fresh-cut spinach. The treatment significantly reducedhe loss of fresh weight and improved visual quality and turgorver the period of storage. ANE-treatment reduced lipid perox-dation in cut spinach leaves during storage suggesting the ANEeduced oxidative stress in spinach. The total chlorophyll contentnd the ascorbate concentration were however not affected byNE-treatment over a 35-day storage period. The results suggest
he potential of ANE to improve shelf life of spinach.
cknowledgements
BP’s lab is supported by grants from the Natural Sciences andngineering Research Council of Canada (NSERC), Nova Scotiaepartment of Agriculture & Marketing (NSDAF) and Acadian Sea-lants Limited. DMH lab is supported by AAFC operating funds.
ppendix A. Supplementary data
Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.scienta.014.02.038.
turae 170 (2014) 70–74
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