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Retention of Folate, Carotenoids, and Other Quality Characteristics in Commercially Packaged Fresh Spinach



The effect of storage temperature (4 °C, 10 °C, and 20 °C) on retention of folate, carotenoids, and other quality characteristics in commercially packaged fresh spinach were determined. Based on visual color and appearance, spinach was unacceptable after 8 d, 6 d, and 4 d at 4 °C, 10 °C, and 20 °C, respectively. Color differences (AE), chlorophyll degradation, fresh weight loss, and microbial populations increased at all storage temperatures and occurred more rapidly at higher temperatures. Peroxidase activity increased but was not significantly (P > 0.05) affected by storage temperature. Lipoxygenase activity was unaffected by storage time or temperature. Substantial losses of nutrients occurred at each storage temperature. Only 53% of folate in packaged spinach was retained after 8 d, 6 d, and 4 d at 4 °C, 10 °C, and 20 °C, respectively. Carotenoid losses increased with temperature with only 54%, 61%, and 44%, respectively, of initial detected levels remaining. Vitamin and quality changes were unaffected by presence or absence of packaging.
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C: Food Chemistry & Toxicology
JFS C: Food Chemistry and Toxicology
Retention of Folate, Carotenoids, and
Other Quality Characteristics in
Commercially Packaged Fresh Spinach
ABSTRACT: The effect of storage temperature (4 °C, 10 °C, and 20 °C) on retention of folate, carotenoids, and
other quality characteristics in commercially packaged fresh spinach were determined. Based on visual color
and appearance, spinach was unacceptable after 8 d, 6 d, and 4 d at 4 °C, 10 °C, and 20 °C, respectively. Color
differences (
E), chlorophyll degradation, fresh weight loss, and microbial populations increased at all storage
temperatures and occurred more rapidly at higher temperatures. Peroxidase activity increased but was not
significantly (P > 0.05) affected by storage temperature. Lipoxygenase activity was unaffected by storage time or
temperature. Substantial losses of nutrients occurred at each storage temperature. Only 53% of folate in pack-
aged spinach was retained after 8 d, 6 d, and 4 d at 4 °C, 10 °C, and 20 °C, respectively. Carotenoid losses
increased with temperature with only 54%, 61%, and 44%, respectively, of initial detected levels remaining.
Vitamin and quality changes were unaffected by presence or absence of packaging.
Keywords: carotenoids, folate, temperature, spinach
MS 20030474 Submitted 8/20/03, Revised 9/17/03, Accepted 8/10/04. Au-
thor Pandrangi is with Dept. of Food Science and Technology, Ohio State
Univ., Columbus, Ohio. Author LaBorde is with Dept. of Food Science, Penn-
sylvania State Univ., Univ. Park, PA 16802. Direct inquiries to author
LaBorde (E-mail:
Among fresh leafy greens, spinach is an important source of nu-
trients in the diet ranking 2nd behind kale in total carotenoids
and folate (Holden and others 1999; USDA 2003) and 3rd in total
antioxidant capacity behind only garlic and kale (Cao and others
1996). A single 30-g serving of fresh spinach containing 58 g of
folate and 2015 IU of vitamin A is equivalent to 29% and 20% of the
daily value for each respective vitamin (NAS 1989).
Adequate intake of folate is an important factor in the prevention
of neural tube defects such as spina bifida and anencephaly, cor-
onary artery disease, and colorectal cancer (Herbert 1999). Caro-
tenoids in the diet are essential for normal growth, reproduction
and resistance to infection, and deficiencies have been linked to
blindness and increased risk of several types of cancers (Tee 1992).
Previous studies have demonstrated that the nutrient content
of fresh vegetables decreases during storage (Rodriquez-Amaya
1993; Buescher and others 1999). However, there are few studies on
folate degradation in fresh produce during storage. Chen and oth-
ers (1983) reported that folate in fresh spinach decreased by 26%
and 27% after holding for 7 d at 4 °C or 10 h at 20 °C. Gami and Chen
(1985) held Swiss chard at several temperatures and reported
folate decreases of 12% after 10 d at 4 °C and 43% after 6 h at 4 °C.
However, Mullin and others (1982) reported that folate levels in
fresh spinach remained unchanged after storing at 4 °C for 14 d.
Ezell and Wilcox (1962) reported minimal losses of beta-carotene
in kale and collard at 0 °C. However, losses increased to up to 67%
after 4 d of storage at 21 °C. Little or no decreases in carotenoids
have been reported in refrigerated broccoli and green beans
(Wu and others 1992; Paradis and others 1996). However, Barth and
Zhuang (1996) reported that total carotenoids in broccoli decreased
by 42% to 57% after 6 d at 5 °C, and Howard and others (1999) re-
ported a 64% decrease in broccoli after 21 d at 4 °C. In fresh spin-
ach, Simonetti and others (1991) reported a 10% decrease in beta-
carotene after 21 d at 4 to 6 °C. Kopas-Lane and Warthesen (1995)
reported beta-carotene losses of up to 18% after 8 d at 4 °C although
no changes were reported for the xanthophylls neoxanthin, violax-
anthin, and lutein.
Harvested spinach leaves are transported over long distances in
refrigerated trucks to the processing facility where they are sorted,
washed, centrifuged to remove surface moisture, and packaged in
plastic bags. Because of their high respiration rate, packages are
usually ventilated to maintain aerobic conditions inside the bag.
The shelf life of spinach is less than 14 d after harvest (Kader
2002). However, quality decline may be accelerated by structural
degradation, membrane lipid loss, increased respiration, and eth-
ylene production and is sometimes accompanied by increased en-
zymatic activity. Strategies to increase shelf life include reducing
physical damage during processing and storing at lower tempera-
tures in modified atmospheres (Price and Floros 1993). In this study,
the effects of storage temperature and time on retention of folate,
carotenoids, and other quality attributes in commercially pack-
aged fresh spinach were determined.
Materials and Methods
Sample preparation and treatments
Spinach (Spinacia oleracea L., var. Unipack 151) was obtained
from a fresh-cut processor where it had been sorted, washed, and
packed into polyethylene plastic bags (284-g capacity). Each bag
contained approximately 2.5 perforations (1-mm dia) per cm2 of
package surface. After heat-sealing, the bags were immediately
placed in refrigerated rooms until shipped. Bags of spinach were
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Folate and carotenoids in fresh spinach…
transported in insulated boxes to the Pennsylvania State Univ.
Dept. of Food Science within 12 h of packaging.
Spinach in the sealed bags and spinach leaves removed from
bags were placed in temperature-controlled stainless-steel cham-
bers (Lunaire Limited, Williamsport, Pa., U.S.A.) for storage at 4 °C,
10 °C, or 20 °C. The temperature was continuously monitored using
a digital temperature logger (Model HH2002AL, Omega Engineer-
ing Inc, Stamford, Conn., U.S.A.). Relative humidity inside the
chamber was monitored using a Digital Humidity/Temperature
Meter (FisherbrandTM, Fisher Scientific, Pittsburgh, Pa., U.S.A.).
Experiments were performed in duplicate, and for each experiment,
2 random samples were removed for each assayed parameter. 20 °C
samples removed for analysis at 1-d intervals and 4 °C and 10 °C
samples at 2-d intervals. All absolute values are expressed on a wet
weight basis to facilitate comparison to literature, whereas compar-
isons over time were made on dry weight basis because of signifi-
cant weight loss observed during storage.
Surface color of spinach leaves was measured using a spectro-
photometer (Model CM 3500d Minolta Corp., Ramsey, N.J., U.S.A.)
calibrated with a green standard tile (L* = 63.6, a* = –30.09,
b* = 8.92) as recommended by Shewfelt and others (1984). Dupli-
cate samples of 6 to 7 randomly selected leaves were placed above
the instrument so that the 30-mm aperture was completely cov-
ered. The change in total color that occurred during storage (E)
was calculated using the formula:
E = [(L*)2 + (a*)2 + (b*)2]1/2
where L*, a*, and b* are differences in spinach color between
day 0 and the sampling day (Shewfelt and others 1984).
Weight loss
At each sampling interval, spinach bags and spinach leaves were
removed from the chamber and weighed using a top loading bal-
ance (Mettler PM 4600, Mettler-Toledo, Columbus, Ohio, U.S.A.).
Average weight loss per sample was expressed as a percentage of
initial fresh weight.
Microbial populations
Mesophilic and psychrotrophic bacterial populations were de-
termined using the method of Garg and others (1990) with minor
modifications. Spinach samples were removed from the bags, and
5 g of leaves were immediately homogenized for 1 min in 45 mL of
commercial buffered peptone water (BPW, Difco, Sparks, Md.,
U.S.A.). Decimal dilutions were made in BPW and pour plated using
plate count agar (Difco). The plates were incubated for 2 d at 30 °C
for determination of mesophilic bacteria and for 14 d at 3.3 °C for
psychrotrophic bacteria.
Enzyme activity
Lipoxygenase. The method of Chen and Whitaker (1986) as
modified by Theerakulkait and Barrett (1995) was used. Twenty
grams of spinach leaves and 2 g of polyvinylpolypyrrolidone (PVPP)
were homogenized for 1 min in 40 mL of cold (4 °C) extraction buffer
(0.05 M K2HPO4, 0.05 M citric acid, 0.86 M NaCl, adjusted to pH 6.4
with 2.5 M KOH). The homogenate was filtered through 2 layers of
cheesecloth and centrifuged (4 °C) at 27000 g for 30 min. The
supernatant was kept on ice until analyzed.
For preparation of enzyme substrate, linoleic acid (157.2 L)
(Sigma-Aldrich Milwaukee, Wis., U.S.A.) was mixed with an equal
volume of Tween-20 and 10 mL of distilled water. The mixture was
clarified by adding 1.0 mL of 0.1 N NaOH and brought to volume
with 0.2 M phosphate buffer (pH 7.0) in a 200-mL volumetric flask.
This solution had a final concentration of 2.5 mM linoleic acid. The
substrate solution was allowed to equilibrate to 25 °C for 10 min,
flushed with O2 for 2 min, and 0.9 mL was mixed with 0.1 mL of
enzyme extract. A unit of enzyme activity is defined as that amount
of enzyme that produces a change in absorbance of 0.001/min at
234 nm under the assay conditions.
Peroxidase. The previously described enzyme extract that was
prepared was used for measuring peroxidase activity according to
the procedure of Shue and Chen (1991) with minor modifications.
The substrate was prepared by mixing 558 L of guaiacol (Sigma-
Aldrich) with 194.4 L of 30% H2O2 (Sigma-Aldrich) and brought to
volume with 0.2 M sodium phosphate buffer (pH 6.0) in a 100-mL
volumetric flask to obtain a final concentration of 0.05 M guaiacol
and 0.2 M H2O2. Three milliliters of substrate was mixed with 25 L
of enzyme extract and the reaction at 25 °C was monitored for 3 min
at 420 nm. A unit of enzyme activity is defined as the amount of
enzyme that produces a change in absorbance of 1.0/min at 420 nm
under assay conditions.
Gas composition
O2 and CO2 concentrations in packages of spinach were mea-
sured using Mocon PAC CHECK 450 and 550, respectively, gas an-
alyzers (Modern Controls Inc., Minneapolis, Minn., U.S.A.). Gas
samples (5 or 8 cc, respectively) were withdrawn from a single per-
foration on each package using the automatic sampler mode. Mea-
surements were made at 3 different perforations on each package
and an average value was determined. O2 and CO2 concentrations
in the storage chambers were also measured.
Folate analysis
Total folate in spinach samples was determined by enzymatic
digestion of the tissue matrix to release bound folate vitamers fol-
lowed by microbiological assay using the 96-well microplate proce-
dure described by Tamura (1990) and modified by Pandrangi and
LaBorde (2004). The microbiological method for vitamin quantifica-
tion uses the growth response of folate-dependent Lactobacillus
rhamnosus in food sample extracts that have been enzymatically
treated to release the bound vitamin. Spinach leaves (10 0.01 g)
were homogenized in a blender with 50 mL of 0.1 M phosphate buff-
er containing 114 mM ascorbic acid (Sigma-Aldrich) (final pH 4.1).
The homogenate was heated in a water bath at 100 °C for 10 min and
immediately cooled for storage at –70 °C. Folate was enzymatically
released from the tissue matrix by combining 250 L of the homoge-
nate with an equal volume of 0.3 M citric acid buffer (pH
4.0) and 500
L of protease (20 mg/mL) and then incubating at 37 °C
for 8 h. At the
end of incubation period, protease was denatured by heating the
sample at 100 °C for 5 min in a water bath. After cooling to room tem-
perature (approximately 23 °C), 200 L of protease-treated sample
was mixed with 950 L of 0.3 M phosphate buffer (pH 7.0) and 50 L
of conjugase (from rat serum, Harlan Bioproducts, Indianapolis, Ind.,
U.S.A.) and then incubated at 37 °C for 3 h. Total folate in each en-
zyme-treated sample was determined by microbiological assay us-
ing L. rhamnosus and 5-formyl tetrahydrofolate (5-HCO-H4PteGlu,
calcium salt) as the folic acid standard. Turbidimetric growth after 18
h at 37 °C was compared by measuring absorbance at 490 nm using
a 96-well microplate reader (Model 315, Bio-tek Instruments, Her-
cules, Calif., U.S.A.). Pooled human blood plasma was used as an
internal standard and assay validity was confirmed by determining
the folate content of triplicate samples of infant formula obtained
from the Natl. Inst. of Standards and Technology (standard reference
material nr 1846, NIST, Gaithersburg, Md., U.S.A.).
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Folate and carotenoids in fresh spinach…
Carotenoid and chlorophyll analysis
Sample extraction and high-performance liquid chromatogra-
phy (HPLC) analysis were achieved using the method of Bushway
(1986) modified by Kopas-Lane and Warthesen (1995). Spinach
extracts were prepared by adding 60 mL of cold (4 °C) methanol/tet-
rahydrofuran (1:1 vol/vol) containing 20 g of sodium sulfate and
1 g of magnesium carbonate to 10 g of spinach leaves and homog-
enizing for 1 min in a Brinkmann Polytron (Model PT 10/35, Brink-
mann Instruments Inc., Westbury, N.Y., U.S.A.) at a speed setting of
6. The homogenate was filtered through a Whatman nr 42 filter and
the residue was re-extracted twice. The filtrate was transferred to a
200-mL volumetric flask and diluted to volume with the homoge-
nizing solvent. A 5-mL aliquot was dried under nitrogen, and the
residue was dissolved in 1 mL of methanol. The solubilized pig-
ments were filtered through 0.45-mm Gelman membrane filter
before HPLC injection.
The reverse-phase HPLC system consisted of Waters 510 series
pumps connected to a Waters 717 autosampler (Waters Inc, Milford,
Conn., U.S.A.). The gradient solvent system consisted of 90% ace-
tonitrile/water/methanol (90/5/5, vol/vol) at a flow rate of 1.5 mL/
min reaching 100% methanol in 15 min. Pigments were separated
on a C-18 column (Model 218TP54, Grace Vydac Inc, Hesperia,
Calif., U.S.A.), and spectra were obtained using a Waters 996 pho-
todiode array detector for peak identification at 436 nm. Spectra and
retention times of chlorophyll a and b and trans beta-carotene were
compared with standards (Sigma Aldrich, Milwaukee, Wis., U.S.A.).
All other identifications were based on published spectral informa-
tion (Braumann and Grimme 1981; Quackenbush 1987). Quantities
of total carotenoids and xanthophylls in treated spinach samples
were compared by calculating changes in peak areas for each of the
identified compounds.
Statistical analysis
The data were analyzed within individual temperatures using
Analysis of Covariance. Differences between packaged and un-
packaged samples were analyzed using Dunnett’s test (Minitab,
Minitab Inc., State College, Pa., U.S.A.). Differences at the maxi-
mum storage time at each temperature were compared using 1-way
analysis of variance (ANOVA).
Results and Discussion
Spinach characteristics
Leaves were considered unacceptable for commercial sale when
they became noticeably wilted and curled along the edges with
approximately 5% of the leaves showing signs of yellowing. Shelf life
parameters used in this study are similar to those used by Piagentini
and others (2002) to describe loss of quality in fresh-cut spinach
during refrigerated storage. Based on preliminary visual observa-
tions, spinach was considered commercially unacceptable after 8
d, 6 d, and 4 d at 4 °C, 10 °C, and 20 °C, respectively. These respective
storage times were designated as shelf life values for each temper-
ature and were used as maximum storage times in subsequent ex-
Changes in visual quality were confirmed by objective measure-
ments. Color differences (E) increased at all storage temperatures
and were most rapid at higher temperatures (Figure 1). However,
after 8 d, 6 d, and 4 d at 4 °C, 10 °C, and 20 °C, respectively, E values
did not significantly (P > 0.05) differ from each other. Gnanasekha-
ran and others (1992) similarly reported that E values for fresh
spinach increased more rapidly under temperature abuse condi-
tions compared with refrigeration.
The total amount of chlorophyll initially contained in spinach
was 332.8 17 and 65.5 5 g/g, respectively. The ratio of chloro-
phyll a to b remained approximately 5:1 throughout the study.
Chlorophyll decreased (P 0.05) with increasing storage time and
degradation was more rapid at higher temperatures (Figure 2). Only
75%, 69%, and 58% of the initial amount remained after storing for
8 d, 6 d, or 4 d at 4 °C, 10 °C, or 20 °C. Results from chlorophyll de-
termination and color change (Figure 2) indicate that chlorophyll
levels at the end of each spinach shelf life at 4 °C, 10 °C, and 20 °C
do not correlate with E values.
Weight loss, caused by evaporative loss of moisture, significant-
ly (P 0.05) increased with storage time and temperature (Figure
3). After 8 d, 6 d, and 4 d at 4 °C, 10 °C, and 20 °C, fresh weights did
not differ significantly (P > 0.05) from each other. Loss of water
4C 10C
Figure 1 Change in total color (E) of packaged spinach
stored at 4, 10, and 20 °C for up to 8, 6, and 4 d, respec-
tively. Each value represents the mean of 4 determinations
at each temperature SE.
4C 10C
Figure 2 Retention of chlorophyll (%) in packaged spin-
ach stored at 4, 10, and 20 °C for up to 8, 6, and 4 d, re-
spectively. Each value represents the mean of 4 determi-
nations each temperature SE. Initial total chlorophyll con-
tent was 399 18 g/g.
4C 10C
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Folate and carotenoids in fresh spinach…
Figure 3 Fresh weight loss in packaged spinach stored
at 4, 10, and 20 °C for up to 8, 6, and 4 d, respectively.
Each value represents the mean of 10 determinations at
each temperature SE.
through transpiration can be attributed to differences in internal
relative humidity of the tissue with that of the storage chamber,
which remained within a narrow relative humidity range of 55% to
58% at all temperatures. The approximately equal loss of moisture
at the end of the shelf life for each temperature is consistent with
the equally wilted appearance of the leaves at the same storage
times and temperatures.
Microbial populations
Mesophilic and psychrotrophic bacteria populations in stored
spinach are shown in Table 1. The amount of mesophilic bacteria
populations initially contained in packaged spinach varied between
5.0 and 6.0 logs. Garg and others (1990) reported a similar range for
microbial populations in salad vegetables in the packinghouse.
Microbial populations in spinach stored at 4 °C, 10 °C, and 20 °C
increased by 1.6, 1.1, and 2.3 logs, respectively, with final levels
reaching 7.1 to 7.5 logs.
Psychrotrophic populations similarly increased from 3.6 to 5.9
logs initially to a maximum of 6.9 to 7.5 logs. Other studies have
reported similar increases in mesophilic and psychrotrophic bac-
teria on spinach leaves under refrigerated (5 °C to 7 °C) and temper-
ature abuse (10 °C) conditions (Garg and others 1990; Babic and
others 1996; Piagentini and others 2003).
Enzyme activity
Lipoxygenase activity did not significantly (P > 0.05) change
during storage at each of the temperatures studied (data not
shown). However, activity tended to be higher in spinach leaves
stored at 20 °C compared with 10 °C or 4 °C. In contrast, peroxidase
activity increased with storage time for spinach leaves stored at 4 °C
and 10 °C (Figure 4). Increases in peroxidase activity in stored spin-
ach have been reported (Baardseth and von Elbe 1989) although
both enzymes may participate in degradation reactions (Rodriquez-
Amaya 1993).
Gas composition of packages
Concentrations of oxygen and carbon dioxide inside packages
were not significantly (P > 0.05) affected by storage time or temper-
ature (data not shown). Mean O2 and CO2 levels inside the packages
were 20.1% and 0.03%, respectively, and did not significantly
(P > 0.05) differ from air inside the chamber that surrounded the
packages. Fresh vegetables respire during storage and, in sealed
packages, can result in depletion of oxygen and accumulation of car-
bon dioxide (Price and Floros 1993). These results suggest that the
perforated packaging film provided little resistance to diffusion of
gases between the respiring tissue and the atmosphere surround-
ing the bags.
Folate and carotenoid loss
Total folate in packaged spinach samples taken over the entire
experiment ranged between 84 and 225 g/100 g with a mean value
of 160 42 g/100 g. Previous studies have reported folate values
for fresh spinach from 161 to 410 g/100 g (Klein an others 1981;
Mullin and others 1982; Aiso and Tamura 1998; Lin and Lin 1999;
Shrestha and others 2000; Iwatani and others 2003; USDA 2003;
Pandrangi and LaBorde 2004). Lower folate concentrations in fresh
spinach before storage in this study may be the result of intrinsic
differences between spinach cultivars, growing and handling con-
ditions, or by vitamin degradation during minimal processing and
storage (Mullin and others 1982).
Folate levels decreased (P0.05) with increasing storage time
at approximately the same rate for each temperature (Figure 5). Af-
ter 8 d, 6 d, or 4 d at 4 °C, 10 °C, or 20 °C, folate remaining at each
temperature was an average of 53% of the initial amount. Chen and
others (1983) reported that fresh spinach held in a refrigerator (4 °C)
for 7 d showed a 26%, reduction in folate.
All-trans beta-carotene, 9-cis beta-carotene, and the xantho-
phylls neoxanthin, violaxanthin, and lutein were detected in spin-
ach samples (Figure 6). The same carotenoids were reported by
Kopas-Lane and Warthesen (1995). Values for beta-carotene in fresh
spinach before storage ranged from 54 to 127 g/g with a mean
value of 89.7 23 g/g. These values are comparable to other re-
ported ranges of 30 to 82 g/g (Quakenbush 1987; Masrizal and
others 1997; Holden and others 1999).
Total carotenoids in spinach samples, compared at each time by
adding individual HPLC peak areas, decreased (P 0.05) as stor-
4C 10C
Figure 4 Peroxidase (POD) activity of packaged spinach
stored at 4, 10, and 20 °C for up to 8, 6, and 4 d, respec-
tively. Each value represents the mean of 4 determinations
at each temperature SE.
4C 10C
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Folate and carotenoids in fresh spinach…
Table 1 Growth of mesophilic and psychrotrophic bacteria in packaged spinach stored at 4, 10, and 20 °C for up to
8, 6, and 4 d, respectively.
C 1C 2C
Storage Mesophiles Psychrotrophs Storage Mesophiles Psychrotrophs Storage Mesophiles Psychrotrophs
Time Time Time
(d) Log10 CFU/g (d) Log10 CFU/g (d) Log10 CFU/g
0 5.9 A15.5 A 0 6.0 A 5.9 A 0 5.00 A 3.6 A
2 6.4 A 5.5 A 2 7.0 B 7.0 B 1 7.3 B 7.2 B
4 5.7 A 5.3 A 4 7.2 B 7.2 B 2 7.4 B 7.4 B
6 7.5 B 7.4 B 6 7.1 B 7.0 B 3 7.5 B 7.4 B
8 7.5 B 7.5 B 4 7.3 B 6.9 B
1 Values in a column followed by different letters are significantly different (
bles compared to tissues that are not wilted or damaged (Ezell and
Wilcox 1962; Yang 1985; Price and Floros 1993).
Packaging effect
There were no significant (P > 0.05) differences for all parameters
studied between packaged and unpackaged spinach (data not
Figure 5 Retention of total folate in packaged spinach
stored at 4, 10, and 20 °C for up to 8, 6, and 4 d, respec-
tively. Each value represents the mean of 4 determinations
at each temperature SE.
4C 10C
Figure 7 — Retention of total carotenoids in packaged spin-
ach stored at 4, 10, and 20 °C for up to 8, 6, and 4 d, re-
spectively. Each value represents the mean of 4 deter-
minations at each temperature SE.
4C 10C
Figure 6 Chromatogram of spinach pigments.
age time increased and degraded more rapidly at higher tempera-
tures (Figure 7). After 8 d, 6 d, and 4 d of storage at 4 °C, 10 °C, and
20 °C, respectively, total carotenoids retained were 54%, 61%, and
44% of initial detected levels. All-trans beta-carotene levels ranged
from 84% to 34% of initial levels after storage between 4 °C and 20
°C, respectively (Table 2) and this isomer was more stable than the
9 –cis form. Retention of xanthophylls, determined by comparing
HPLC peak areas, was also enhanced at lower storage tempera-
tures. Mean retention values for the 3 compounds were 44% after 4
d at 20 °C, 59% after 6 d at 10 °C, and 65% after 8 d at 4 °C. Kopas-
Lane and Warthesen (1995) reported comparable losses of beta-car-
otene in spinach stored at 4 °C. However, they did not observe sig-
nificant losses of neoxanthin, violaxanthin, or lutein.
Lower losses of folate (Mullin and others 1982; Chen and others
1983) and carotenoids (Simonetti and others 1991; Kopas-Lane and
Warthesen 1995) have been reported by others using similarly
stored fresh spinach. In these studies, store-bought or field-grown
spinach was used that was sorted immediately before experiments
began to remove damaged or discolored leaves. In the present
study, the entire content of packages of spinach was used and may
have included leaves that had already lost moisture to evaporation
or had been damaged during washing, centrifuging, and packag-
ing. The greater loss of nutrients in this study is consistent with
reports of more rapid quality decline in wilted or wounded vegeta-
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Folate and carotenoids in fresh spinach…
Table 2 — Retention of carotenoids in packaged spinach stored at 4, 10, or 20 °C for 8, 6, or 4 d, respectively.
Carotenoids remaining (%)
Storage All-trans 9-cis
conditions beta-carotene beta-carotene Neoxanthin Violaxanthin Lutein
4 °C / 8 d 84.3 A 49.9 A 65.1 A 69.1 A 61.0 A
10 °C / 6 d 40.9 B 52.4 AB 57.5 A 55.4 AB 65.0 A
20 °C / 4 d 34.3 B 33.4 B 41.5 B 41.3 B 48.3 B
1 Values in a column followed by different letters are significantly different (
shown). Diffusion of moisture and gasses through the bag perfora-
tions is apparently unimpeded as evidenced by identical fresh
weight losses and gas compositions in packaged and unpackaged
samples. Gas composition in minimally processed vegetables is
known to strongly influence the retention of vitamins and other
quality characteristics during storage (McGill and others 1966; Barth
and others 1993; Barth and Zhuang 1996; Howard and Hernandez-
Brenes 1998). The absence of differences in microbial populations,
chlorophyll loss, color, enzyme activity, and vitamin retention be-
tween packaged and nonpackaged spinach in this study is consis-
tent with the identical fresh weight losses and gas compositions
Minimally processed vegetables are attractive to consumers
because of their convenience and nutritional value. Howev-
er, substantial losses of folate and carotenoids occurred in pack-
aged spinach under both refrigerated and temperature abuse con-
ditions. Published values for these nutrients in fresh spinach may,
therefore, not always accurately reflect levels that are consumed. It
is, therefore, essential that growers, packers, fresh-cut processors,
and retailers maintain storage temperatures as low as possible to
minimize vitamin losses in fresh spinach. Consumers should keep
fresh spinach refrigerated and use the product as close as possible
to the time at which it was purchased.
Aiso K, Tamura T. 1998. Trienzyme treatment for food folate analysis: optimal
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taminol 44:361–70.
Baardseth P, von Elbe JH. 1989. Effect of ethylene, free fatty acid, and some enzyme
systems on chlorophyll degradation. J Food Sci 54(5):1361–3.
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... All batches had the same final average value of 28.53 (± 6.56) μg/100 g, which coincided with the report of 39.4 μg/100 g by Irías-Mata et al. (2018). The loss of carotenoids in horticultural products during storage postharvest has been previously reported (Pandrangi and Laborde, 2004). In the present work, fruits that were not at yellow maturity experienced a decrease in the content of carotenoids; however, once they reached the mature stage, concentrations of these compounds remained approximately constant. ...
... On the other hand, the highest carotenoid contents were found at 15°C, and the lowest at 25°C ( Figure 3B). Temperature is a factor that affects carotenoid stability, and a higher thermal condition causes higher loss of these compounds during storage (Pandrangi and Laborde, 2004). ...
Nance (Byrsonima crassifolia) fruit is harvested when natural abscission from the plant occurs. At this stage, the shelf life is less than 5 d in ambient conditions. The aim of the present work was thus to determine how quality attributes of nance fruits are modified as a function of ripening on the tree, physiological condition at harvest, and storage temperature. Fruits at three maturity stages (green, transient, and yellow) were harvested and stored at 15 and 25°C. As fruits ripened, the hue angle turned to yellow, and lightness and chroma increased, but carotenoid content decreased. The contents of total soluble solids, total sugars, and reducing sugars increased; however, the total soluble phenols, flavonoids, and antioxidant activity decreased. It was possible to harvest at a physiological stage previous to abscission maturity even though a non-climacteric pattern was identified. Handling of transient nance fruits at 15°C extended shelf life for more than 15 d, with adequate physical and compositional attributes including high concentration of bioactive compounds and antioxidant activity. Content of total soluble solids was identified as an attribute suitable for developing a harvest index for nance fruits.
... Conversely, 5-CH 3 -H 4 -folate and H 4 -folate (in diglutamate form) levels slightly increased. O'Hare et al. [39] suggested a possible interconversion among folate forms, after one week of storage, and that the folate losses could be influenced by senescence processes such as moisture loss and leaf degradation (damaging and discolouring) as already observed by Pandangri and LaBorde [40] for spinach. This last observation seemed to be partly confirmed in the present study, where a marked loss of leaves integrity and turgidity in unprocessed/ unpackaged leaves after the third day of storage, as highlighted by sensory analysis, seemed to be associated with the marked reduction on the total folate content. ...
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Nutritional and sensory quality of wild rocket leaves (Diplotaxis tenuifolia L., cv. Grazia) subjected to commercial processing and packaging were monitored for up to 7 days of cold storage and compared with the quality of unprocessed and unpackaged leaves from the same harvest. Nutritional quality was assessed by determining total phenolics, vitamin C and folate content. Sensory quality was assessed by quantitative descriptive analysis, combined with analysis of volatile organic compounds (VOCs). No change in vitamin C and folate content was observed in processed rocket leaves up to 7 days of cold storage, whereas unprocessed leaves showed a marked loss of both of them at the end of the storage time (52% and 71%, respectively). Total phenol content decreased during cold storage in both rocket leaves samples, but at a lower extent in the processed leaves. While most sensory attributes showed significant degradation during storage in both processed and unprocessed leaves, at the end of the storage time processed leaves had better retained some appearance (colour, turgidity, integrity), flavour (bitterness and bitter persistence) and texture (firmness and chewing consistency) attributes of the fresh leaves. A marked decrease during storage of some potent key odorants, such as (Z)-3-hexenal, (E)-3-hexenal, as well as 1-penten-3-one, corresponded to the weakening of fresh herbaceous/green sensory notes and pungency in both rocket leaves samples. Commercial processing and packaging operations proved to be effective at better preserving nutritional and sensory quality of wild rocket leaves and, thus, at prolonging their shelf life.
... The possible reason for this trend might be related to the water loss through transpiration. These results are in association with Pandrangi et al. [27] who reported the weight loss in spinach leaves with increase in temperature. Effect of storage duration was also observed on moisture content and negative significant linear effect (p < 0.0001) was observed which demonstrate that moisture content of leaves get decreases with increase in storage time. ...
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Research on waste valorization and utilization of food by-products are increasing day-by-day. This trend not only protect the environment from pollution but also gives good value of food industries wastes and by-products. In this study, optimization of harvesting stage and storage conditions of radish leaves has been done by using response surface methodology. Different packaging materials such as paper, cling film and LDPE (low density polyethylene) were tried for storage. Among all, better retention of all quality parameters was observed in the leaves stored in LDPE. Maximum quality retention in terms of moisture content (94.44%), ascorbic acid (35.37 mg/100 g), DPPH scavenging activity (45.32%), total phenols (684.025 mgGAE/100 g), total flavonoids (1041.066 mg quercetin/100 g) and chlorophyll (48.795 mg/100 g) was observed with 9.55 cm length of leaves and 4 °C storage temperature on 24 h. The applied model was found suitable for current study with 94% of anticipated values. Graphical Abstract
... carotenoids, flavonoids); the contents of bioactive phytochemicals, however, can vary over time, and many studies focus on the analysis of post-harvest composition of bioactive compounds in plant-based food. Levels of flavonoids, for example, have been found to be stable during storage in spinach 14 ; for glucosinolates in broccoli, even an increase was identified during short-time storage 15 ; whereas losses of carotenoids and folate contents were reported for packaged spinach 16 . High quantities of phyllobilins have been detected in stored plant-based foods 17,18 , indicating the biochemical chlorophyll degradation program to be active post-harvest. ...
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Consumers often throw away faded greens, because taste and appearance are less appealing compared to fresh ones. We report here a family of antioxidants, the phyllobilins, which increase during storage in iceberg lettuce and cucumber. We show that informing consumers about rising levels of phyllobilins leads to a longer willingness to consume faded lettuce and to an improved health and safety perception.
... As is reported in previous studies [48,49], the ∆E* value was the parameter that best described the loss of colour when compared to hue, chroma or L*a*b* values individually (data not shown). Colour differences found here were lower than in those studies, probably due to a very slight weight loss (ca 0.44% for all the treatments). ...
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Spinach is rich in minerals, vitamins, phytochemicals and bioactive compounds with health-beneficial effects; however, this plant also tends to accumulate oxalates and nitrates in their leaves. Apart from genotype, nutrition is the pre-harvest factor that mostly affects quality attributes at harvest. Particularly, the application of compost extracts (CE) may induce resistance against soil-borne diseases and favour secondary metabolism, increasing antioxidant capacity. The objective of this study was to evaluate the effects of different types of fertilization with or without the addition of CE, on harvest quality and shelf life of minimally processed spinach (Spinacia oleracea, var. Shrike RZ) stored during 12 days at 4 °C. A compost extract (CE) was prepared by mixing a compost from agri-food wastes (vine pruning, leek waste and olive mill waste) with deionized water. CE foliar applications were done from days 28 and 56 after sowing. The treatments applied were: Control; Control + CE; NPK (inorganic NPK fertilizer 15-15-15); NPK + CE; DMPP (ENTEC Nitrofoska® plus the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP)) and DMPP + CE. After harvest, spinach leaves were minimally processed and packaged to generate a passive modified atmosphere. Nitrate content in the control treatment was reduced by the addition of CE, although in the rest of the treatments, CE addition did not produce any effect. For nitrite contents, the lowest value was obtained for the Control + CE. Moreover, the oxalate content was the lowest for the control treatment with a decreasing trend throughout the storage. The treatment Control + CE also showed the highest initial total phenolic contents, with very similar values at the end of shelf life to those observed at harvest for all the treatments. The highest differences in color as regards the initial values were detected for DMPP. Microbial loads increased for all the treatments without differences between them. The atmosphere reached at the end of the cold storage was the same for all the cases, with CO2 and O2 around 10 kPa for each one of them. After 12 days at 4 °C, all the treatments were above the limit of usability, with the spinach leaves acceptable for consumption. The results found in this study indicate that the addition of CE might be convenient for obtaining spinach rich in bioactive compounds and with low concentrations of antinutritional factors, without affecting the microbial load of the final product.
... p0985 Fresh vegetables are usually stored before selling or after selling and before processing. Pandrangi and LaBorde (2004) encountered losses of 47% of folate in packaged spinach after 8 days of storage at 4 C, while Chen et al. (1983) reported a loss of 26% after 7 days in a refrigerator. p0990 The majority of foods, of plant and animal origin, is consumed after some type of processing. ...
This chapter covers the chemical changes food processing and storage can produce on vitamins. The four fat-soluble vitamins and nine classically recognized water-soluble vitamins have been systematically treated featuring their chemical characteristics, their nutritional and biochemical functions, the human body dependence on these micronutrients, and very importantly, the chemical mechanisms by which they are degraded during food processing and storage. Although one of the main concerns of food processors is to avoid or minimize the destruction of these organic substances so important to human health, losses are still inevitable and one of the purposes of this chapter is to lay down through schemes, figures, and concise explanations the reactions and the influencing factors of the degradative process in an attempt to guide food technologists, food processors, and health scientists.
... Dark green spinach leaf color is directly associated with freshness (Martínez-Sánchez et al. 2019). In spinach, leaf chlorophyll breakdown and color change have been directly related to reactive oxygen species (ROS)-regulated senescence (Yamauchi and Watada 1991) and all of these phenomena are accelerated by non-optimum storage temperature (Pandrangi and LaBorde 2004). The spinach produced in the high tunnels had lower respiration rates during storage. ...
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Fresh produce constitutes 44% by weight of the global food losses and waste. Postharvest losses of fresh produce are closely related to the preharvest field conditions during growth. In the Central US, many small acreage vegetable growers are utilizing high tunnels, which have been successful in increasing the yield of several crops. Little is known about the effect of this production system on the postharvest losses. This study tested the hypothesis that the production system will affect the postharvest losses of organic spinach (Spinacia oleracea, “Corvair”) when stored at 3 or 13 °C. Comparative open field and high tunnel trials were conducted from 2015 to 2017. Postharvest losses were evaluated with regard to the spinach quality characteristics and shelf life. During storage at 3 °C, there were no major quality differences between the spinach grown in the two production systems. During storage at 13 °C, in both years, spinach grown in the high tunnels had 1.2 to 2.3% higher water content than spinach grown in the open field. In the second year, high tunnel spinach stored at 13 °C had a lower respiration rate (P < 0.05) and a slower rate of yellowing as indicated by higher chlorophyll content (P < 0.001), significantly lower lightness values, and significantly higher hue values than open field spinach. The high tunnel spinach demonstrated longer shelf life in year 1 and higher quality towards the end of storage in year two when compared to open field spinach when stored at 13 °C. This is the first study to examine the effect of the high tunnel production system on the postharvest quality and losses of spinach. Our results indicate that high tunnels can reduce the postharvest food losses of spinach when stored at 13 °C, due to increased water content and decreased senescence rate.
The objective of this study was to explore the potentiality and mechanism of visible and near-infrared (Vis-NIR) spectroscopy in estimating the freshness of komatsuna. We monitored the cumulative CO2 production of komatsuna stored under different conditions as a freshness indicator and measured the Vis-NIR spectra of komatsuna as the predictor. Using the informative wavelengths (IW) selected using the stepwise selectivity ratio method, we constructed an accurate freshness prediction model through PLSR analysis. The IW in the visible region were attributed to pigments such as chlorophyll. In the NIR region, ten amino acids were identified as directly or indirectly contributing to the IW and were highly related to freshness. They were confirmed on the basis of the strong correlations between the informative NIR signals and NMR signals, which were determined using statistical heterospectroscopy. The results demonstrate the feasibility of Vis-NIR spectroscopy in estimating the freshness of komatsuna using the IW.
Vegetables lose quality after harvest mainly due to water-related weight loss. In households, vegetables are usually stored in refrigerators, typically providing temperatures from 0 – 14°C and relative humidity ranges from 20 – 100 %. Weight loss is related to the storage humidity conditions. As a consequence, storage systems providing humidity-controlled conditions are decisive for the evaluation of the freshness performance of refrigerators. To evaluate the impact of storage zones on the fresh weight loss of stored goods, the international standard IEC 63169:2020 “Electrical household and similar cooling and freezing appliances – Food preservation” has been released. It uses a cellulose based food simulant, overcoming influences of cultivar and post-harvest handling conditions of real vegetables. This publication compares the weight loss of spinach and the food simulant in chilled storage conditions at three ranges of relative humidity. Different cultivars and pre-handling modifications of the spinach were used to determine the impact of product parameters on weight loss. A round robin test was performed to analyse the applicability of the IEC 63169:2020. The results prove the applicability of the standard to analyse the impact of humidity controlled storage on weight loss: It is shown that pre-handling has an impact on spinach's weight loss, which is overcome by the food simulant. For all tested conditions, the weight loss behaviour from spinach and the food simulant is correlating in test durations up to 72 h. The round robin test shows that the test method of IEC 63169:2020 provides repeatable and reproducible results.
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Developing more nutrient‐rich, sustainable food supply chains aligns with the co‐benefits of tackling malnutrition and minimizing food loss and waste. While food waste and nutrient loss as a function of food waste and processing have separately been a topic of much previous research, nutrient loss as a function of both processing and food waste from farm to fork has not been addressed. This critical analysis was motivated by the: challenge of nourishing a growing population, the economic impact of food waste, the societal costs of malnutrition, and the overall need to extend produce shelf life sustainably. Both food and nutrient loss and waste can occur simultaneously at various levels throughout the value chain as a function of different processing methods. Combined effects of food waste and nutrient availability/losses were determined through a systematic analysis of the available peer‐reviewed research data during thermal, nonthermal, and minimal processing for tomatoes, spinach, and kidney beans. The waste and loss datasets were derived from the USDA, the FAO, and the US EPA databases. This work presents a justification for more research to reduce nutrient loss and food waste to obtain a more sustainable supply of nutrients in the food industry. Practical Applications This analysis serves as a guide for food industry stakeholders concerned with nutrient retention as a result of processing and food waste in the food value chain. It also assesses the combined impact of processing and food waste on nutrient loss from farm to fork. Available nutrient retention data as a function of retort, microwave, high pressure, aseptic and fresh processing, and food waste were employed. To our knowledge, there has not been a study on food waste as a function of processing that considers nutrient retention and loss as a function of food waste within the entire value chain. A summary of specific research needs for a holistic view on nutrient retention affecting product, process, and package conditions through the value chain was presented.
Surveys were made of commercial processing lines used to prepare fresh-cut vegetables such as chopped salad ingredients, carrot sticks, and cauliflower florets. Washing and chlorinated water dips only partially removed the microorganisms that were intrinsic to the vegetables. Major sources of in-plant contamination were the shredders used to prepare chopped lettuce and coleslaw. Gram-negative rods were the predominant microflora with species of Pseudomonas being most numerous; many were psychrotrophic. Only low numbers of lactic acid bacteria and fungi were recovered. Copyright © International Association of Milk, Food and Environmental Sanitarians.