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Solubleproteincontentinminimallyprocessed
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Soluble Protein Content in Minimally Processed Vegetables during Storage
Lata Masiha, Hubert Roginskia, Robert Premierb, Bruce Tomkinsband Said Ajlounia*
a
Gilbert Chandler College, Department of Agriculture and Food Systems, The University of
Melbourne, Werribee, Victoria, 3030, Australia
b
Institute for Horticultural Development, Agriculture Victoria, Private Bag 15, South
Eastern Mail Centre, Victoria 3176, Australia
a*
Correspondence should be addressed to: Dr. Said Ajlouni, Gilbert Chandler College, The University
of Melbourne, Sneydes Road, Werribee 3030, Victoria, Australia; Telephone: +61 3 9217 5206; Fax:
+61 3 9741 9396; Email: [email protected]
Abstract
Soluble protein content (SPC) and electrophoretic protein profile in minimally processed Broccoli,
Dutch carrot and Cos lettuce were determined after 0, 3, 5, 7, 10 and 12 days of storage at 12ºC and 95
± 2% relative humidity. An increase in SPC in broccoli tissues (florets, stems and whole) was observed
on day 3, followed by a slight fluctuation thereafter. Similar observations were recorded in shredded
carrot, which showed a significant (p<0.05) increase in SPC after 3 days at 12ºC. However, changes in
SPC in Cos lettuce were different from broccoli and carrot, and showed a significant (p<0.05) decrease
after 3 days. The SDS-PAGE profile revealed a continuous decrease in the band intensity of soluble
proteins from broccoli, Dutch carrot and Cos lettuce throughout the storage period. Complete
disappearance of some bands was observed in Cos lettuce leaves and shredded carrot after 12 days of
storage at 12ºC.
Keywords: minimal processing; handling; soluble protein content; packaged; storage; shelf life;
electrophoresis
1. Introduction
Vegetables are perishable products with an active metabolism, which continues after harvest. The
quality of vegetables offered to consumers depends primarily upon cultivars, growing conditions and
methods of preparation, storage and handling after harvest.
During the last decade, consumers’ desire for convenience and their ever increasing concern over
eating healthy foods along with improvements in packaging technology, have given rise to a new
category of vegetable products that are called “minimally processed vegetables” (MPV) (Klassen,
1994; Saracino, Pensa & Spieze, 1991; Yildez, 1994).
Minimal processing of vegetables refers to the process of converting harvested fresh vegetables into a
convenient peeled, cored, chopped or sliced, washed and packaged product that is 100% edible and has
retained quality attributes to a high degree (Bolin and Huxsoll 1991a; Powrie & Skura, 1991). The
range of MPV encompasses a wide variety of fresh produce including packs of broccoli, shredded
carrots, cauliflower, shredded lettuce, and salad mixes (O'Connor and Skarshshewski 1992). As tissues
of MPV are subjected to mechanical handling during processing (cutting, shredding, washing and
packaging), these products become more perishable than their whole counterparts (Shewfelt, 1987;
McDonald, Risse and Barmore, 1990; Barriga and Trachy, 1991). Cut and damaged surfaces in MPV
release nutrients and some intracellular enzymes, such as polyphenol oxidase, and provide good
conditions for some enzymatic activities and possible microbial spoilage. Senescence, and microbial
deterioration in MPV continues and shortens their shelf life even under the recommended chilling
storage conditions (0-8°C). O’Connor and Skarshshewski (1992) reported that the shelf life of some
MPV products is less than 5 days at 4°C.
Most recent studies of fresh produce have focussed on disinfection and shelf life extension of
minimally processed products. However, the nutritional value of MPV has been neglected or poorly
investigated. This study examined changes in soluble protein content and monitored developments in
the protein profile during storage. It is expected that results of this study will provide an indirect
indication about the nutritional value of MPV and to reveal some changes in the protein profiles that
can be used as markers for shelf life evaluation.
2. Material and methods
2.1. Sample Preparation
Fresh broccoli samples (Brassica oleracea L. var. italica) were obtained from Fresh Select (Aust.) Pty
Ltd and Cos lettuce and Dutch carrot samples were bought from a local Safeway supermarket,
Werribee, Victoria, Australia within 24 hours of harvest. The broccoli samples were cut into florets and
stems, the Cos lettuce was used as intact leaves (whole leaves), leaves without stem (leaves only),
2
and Cos stem (Stem only), and the carrot samples were taken from the middle portion of the carrot and
treated as whole and shredded. All samples were washed for 2 min in chlorinated water (50 ppm as
calcium hypochlorite) at 4ºC and spun dried using a household model spin drier for 2mins before
packaging. Within each product, the prepared tissues were mixed, and weighed (approximately 100 g)
in perforated plastic bags. All packaged samples were stored in a randomized complete block design in
a Humiditherm refrigerator (Thermoline Scientific Equipment Pty. Ltd., NSW Australia) at 12ºC and
95 ± 2% relative humidity prior to analysis after storage for 0,3,5,7,10 and 12 days. The entire
experiment was repeated three times and duplicate samples were analyzed within each replicate.
2.1.1. Freeze-drying
Due to the large number of samples and the extensive time required for the chemical analysis on each
day of measurement, all samples were visually examined, freeze-dried, and stored at –800C for future
analysis. Before freezing about 100 g of the vegetables under examination were chopped into small
pieces and homogenized with 50 mL distilled water using a high speed Waring blender (Waring
Commercial, Waring Service Centre, New Hartford, CT 06057). The homogenate was transferred into
a round bottom flask and kept in the freezer at -20oC overnight. The frozen samples in the flasks were
attached to the manifold of the freeze-drier (Dynavac Engineering Pty.Ltd. Australia) and dried at 40oC. Freeze-dried samples were weighed, transferred into sample bottles and kept at -80oC until
analysis.
2.2. Soluble Protein Extraction
A freeze-dried sample (0.1 gram) was mixed with 2 mL distilled water and vortexed for 5 minutes at
room temperature. The tubes were centrifuged for 20 minutes at 5000 rpm (Heraeus, Labofuge A, Foss
Electric Aust. Pty Ltd.). The supernatant was collected in eppendorf tubes and stored at 4oC for 2 hr
before analyzed.
2.3. Measurement of Soluble Protein Content
A modified Lowry protein assay (Lowry et al., 1951) was used to determine the concentration of
soluble proteins in the extracted samples. The tests were carried out in triplicate. Protein extract was
diluted 1:50 in distilled water, and 0.5 mL of the diluted sample was mixed with 0.5 mL of Lowry
3
reagent and vortexed for a minute at room temperature. The samples were then incubated in a water
bath at 37oC for exactly 10 minutes, followed by the addition of 1.5 mL of Folin/Ciocalteu (F/C)
reagent and incubation for another 20 minutes at 52oC. Absorbance was measured at 680 nm using a
Diode Array Spectrophotometer (8452A Hewlett Packard Vectra CS). Standards containing 0 to 100
µg/mL of 5% bovine serum albumin (BSA) solution were prepared following the same procedures. The
Spectrophotometer was calibrated using a standard solution of 500 µL of deionised water, 500 µL of
Lowry reagent and 1500 µL of F/C reagent.
2.4. Polyacrylamide Gel Electrophoresis (SDS-PAGE)
One dimensional sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of proteins was
performed using a vertical slab system (BioRad's PROTEANII xi cell and Modular Mini-PROTEAN
II electrophoresis system, Bio-Rad Laboratories, Hercules, CA 94547) in a discontinuous buffer system
of 12.5% gel buffered by 1.875 M tris-HCl at pH 8.8. A tris-Glycine electrode buffer was used to run
the gel. A small proportion (50 µL) of soluble protein extract was mixed with 100 µL of sample buffer
and 5 µL of mercapto-ethanol and heated at 100oC for 5 minutes. A microsyringe was used to load
exactly 50 µL of the protein solution onto the gel. A standard broad range protein marker (BioRad) was
run alongside the samples and electrophoresis was carried out at a constant current of 20 mA and 200
V. The protein bands were visualized by a fixative-stain solution of Coomassie blue (R250) and then
destained using a 7% acetic acid and 5% methanol wash.
2.5. Statistical Analysis
Analysis of variance (ANOVA) was performed on data from soluble protein evaluation, using SPSS
6.1 (SPSS Inc. Chicago, Illinois, USA) and the means were separated using least significant difference
(LSD) at 95% confidence level.
3. Results and Discussion
Proteins in minimally processed vegetables (MPV) are mostly functional proteins, present in the form
of enzymes (Wills, McGlasson, Graham & Joyce, 1998). This observation may explain the very small
contents of proteins in MPVs such as iceberg lettuce, celery, broccoli, carrot and tomatoes, which
contain 1.3, 0.7, 3, 1 and 0.9% protein, respectively, based on fresh weight (Chin & Dudek, 1988). In
the present study, the average soluble protein contents in Dutch carrot, Cos lettuce and broccoli on the
4
day of minimal processing were 0.53±0.04, 1.91±0.02 and 26.37±1.62 mg/g dry weight (d.wt),
respectively. Due to these initial variations in soluble protein contents and because each of these
studied vegetables has different physical and chemical characteristics, it was expected that their
response to minimal processing would be different. Consequently, changes in soluble protein contents
and in the protein profile in each of these minimally processed vegetables were examined separately.
3.1.1.
Dutch carrot
The initial soluble protein content (SPC) in shredded carrot (0.43±0.02 mg/g d.wt) was significantly
lower than in whole carrot (0.53±0.03 mg/g d.wt). However, SPC in shredded carrot increased by 37%
after 3 days of storage at 12oC and retained that level of SPC until day 7 when SPC in shredded tissue
dropped significantly (p<0.05) to reach its lowest value (0.39±0.05 mg/g d.wt) throughout the storage
period (Fig. 1).
Unlike the shredded tissues, whole carrot had a stable SPC during the first 3 days of storage, followed
by a significant decrease on day 5. A sudden increase in SPC in whole carrot was noted between day 10
and 12 of storage. The increment in soluble protein during the last few days of storage may be
attributed to the formation of some stress proteins, or to the senescence, degradation and enzymatic
activities that soften the texture and lead to the formation of more soluble proteins. The decline in SPC
between days 5 and 10 could be related to the utilization of soluble proteins in some metabolic
activities, such as a substrate for respiration, when the carbohydrate sources become very limited.
Similar conclusion was reported by King, Woollard, Irving & Borst, (1990), who indicated that more
CO2 was released from asparagus spears over 3-5 days than could be accounted for by carbohydrates
loss. They suggested the use of alternative substrate (e.g. protein or protein breakdown) for respiration
in asparagus. Hill, Tomkins & Mespel (1994) also cited the utilisation of protein as a metabolic
substrate in asparagus spears as reported by Platenius (1942); Lill, King & Donoghue (1990) and
Liptone (1990).
Results from the polyacrylamide gel electrophoresis showed that the molecular weights of soluble
proteins isolated from whole and shredded carrot ranged from 31 to 97 kDa (Fig. 2). All protein bands
from the whole carrot soluble protein extract looked more intense than those isolated from shredded
samples. These observations do not agree with the results of protein quantitative analysis (Fig.1), which
may be related to some variation in the volume of protein extract loaded onto the gel. The other
5
important observation from the protein profile is the disappearance of bands at molecular weights 31
and 97 kDa from both whole and shredded sample extracts after 3 days of storage at 12oC (Fig. 2).
Such findings should be further investigated for possible use as shelf life markers.
3.1.2. Cos Lettuce
Soluble protein content in whole Cos lettuce was 1.91±0.02 mg/g d.wt. and ranged from
1.76±0.01mg/g d.wt. in the stem to 2.24±0.01 mg/g d.wt. in the leaves (Fig 3). Changes in SPC in Cos
lettuce were different from those observed in carrot (Fig. 1) and in broccoli (Fig. 5). A significant
(p<0.05) decline in soluble protein contents in Cos lettuce was observed after 3 days of storage at 12oC,
followed by an immediate recovery and increase on day 5 (Fig. 3). The sudden decrease in SPC in the
cut Cos lettuce tissues after 3 days of storage could be attributed to the leaching and loss of soluble
proteins owing to the damaged cellular structure. Such conclusion is in agreement with that reported by
Klein (1987), who linked nutrient loss with the destruction of cellular compartmentation in fruits and
vegetables. However, the decline in SPC in the intact whole Cos lettuce tissues is more related to the
various metabolic activities in these fresh living tissues. It may be possible that high rate of metabolic
activity in whole Cos lettuce immediately after processing cause the breakdown of soluble protein as an
additional substrate for respiration. Similar observations were noted by Phan (1987), who reported a
decline in the essential amino acid contents in lettuce after harvest.
The increment in the SPC in Cos lettuce after 5 days of storage could also be stress related as with
Dutch carrot. The detected fluctuation in the soluble protein contents in minimally processed Cos
lettuce suggests that this produce can maintain its nutritional value (soluble protein) even after 12 days
of storage at 12oC.
The SDS-PAGE separation of soluble proteins isolated from Cos lettuce indicated that most of the
soluble proteins were in the molecular weight range of 45-50 kDa (Fig. 4). The band pattern of Cos
lettuce soluble protein extract illustrated good correlation between band intensity and soluble protein
contents. The decline in SPC after 3 days of storage (Fig. 3) was clearly indicated by low band
intensity (lines 4, 5, and 6 in Fig. 4), while the recovery and the increase of SPC after day 5 was also
reflected by stronger band intensities in these samples (lines 8-13).
3.1.3. Broccoli
6
Whole and dissected tissues (florets and stems) of minimally processed broccoli had different levels of
soluble protein on the day of processing (Fig. 5). The SPC in floret (29.9±0.62 mg/g d. wt) was
significantly (p<0.05) higher than the whole broccoli (26.37±1.62 mg/g d. wt) and in the stems
(22.73±1.24 mg/g d.wt). The SPC increased significantly (p<0.05) in all broccoli tissues after three
days of storage, and remained stable until day 5. However, the SPC declined on day 7 and reached
amounts comparable to those reported on day zero and day 12 of storage (Fig. 5). The largest quantities
of soluble proteins in all broccoli tissues were detected on day 10 of storage at 12oC.
Although results of soluble protein analysis in broccoli revealed a significant decrease in SPC on days
7 and 12 of storage, the amounts of soluble proteins measured on these days were still similar or even
higher than the initial concentrations at day zero. These observations strongly indicate that minimal
processing of broccoli can increase the content of soluble proteins. The exact mechanisms of soluble
protein synthesis in minimally processed broccoli are not fully understood. However, it can be
speculated that similar to other fresh produce tissues (Yoshimura, Yabuta, Ishikawa & Shigeoka,
2000), steps in minimal processing (cutting, shredding, washing with chemicals, packaging and
refrigeration) may stress the produce and activate some metabolic systems that trigger the formation of
soluble protein (stress protein). Stress proteins are induced in plants in response to various factors, such
as temperature, oxygen, water, salt and pH. Chung, Vercellotti & Sanders (1998) showed that a number
of dehydrin-related stress proteins were detected in peanut seeds of different maturity and curing
stages. The same authors indicated that stress proteins occur in peanut seeds during maturation and
curing because these processes are known to be associated with water deficit and anaerobic
metabolism. Another study by Yoshimura, Yabuta, Ishikawa & Shigeoka (2000) revealed that
ascorbate peroxidase isoenzyme was remarkably increased in spinach leaves in response to high-light
stress and methyl viologen treatment.
The electrophoretic patterns of soluble protein profiles from broccoli floret and stem showed some
significant changes over storage time (Fig. 6). The majority of soluble proteins were in the lower
molecular weight range (31-66 kDa). Certain bands (55 kDa) from floret and stem soluble protein
extract were very intense at the start of storage, and declined gradually toward the end of storage time.
Low molecular weight bands (33 kDa) vanished after 5 days of storage, while new bands of higher
molecular weight (50 kDa) appeared on day 12 (Fig. 6). Protein bands (55 kDa) in floret soluble
protein extract were more intense than those in the stem. Such observation may indicate the presence of
7
more soluble protein in the floret. Data from the soluble protein quantification using Lowry method are
in full agreement with that conclusion (Fig. 5).
4. Conclusion
Minimally processed vegetables are not considered good sources of proteins because of the small
amounts they contain. However, the significant increase in SPC in broccoli and Dutch carrot
immediately after processing may improve the nutritional value of these MPVs during the initial days
of storage (5-7 days). The recovery from the initial decline and the subsequent increase in SPC of Cos
lettuce after day 5 may indicate that Cos lettuce will maintain its nutritional value, with regard to
protein content until 10-12 days of storage at 12oC.
Additionally, the disappearance of some bands from the protein profiles should be examined further for
possible application of that phenomenon as an indicator of senescence and shelf life of MPVs. It is
clear that data from SDS-PAGE can provide significant information about changes in soluble protein of
minimally processed vegetables during storage. Some of these changes could be utilized as markers to
estimate quality and shelf life of fresh produce during storage. However, more investigation is needed
to measure the intensity of various bands, and to identify the amino acid profile within each isolated
protein.
Acknowledgement
The authors gratefully acknowledge the financial support from Cooperative Research Centre for
International Food Manufacture & Packaging Science, Australia.
8
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Protein Content [mg/g dry weight]
0.7
0.6
0.5
0.4
0.3
0.2
Whole Carrot
0.1
Shredded Carrot
0
0
3
5
7
10
12
Storage time [days]
Figure 1: Soluble protein content in whole and shredded Dutch carrot during postharvest
storage. Each value represents the mean of six measurements ± standard deviation.
200
116
97
66
45
31
21
14
65
1
2
3
4
5
6
7
8
9
10 11
12 13
Figure 2: Electrophoretic separation of soluble proteins from Dutch carrot
1-D0W; 2-D0S; 3-D3W; 4-D3S; 5-D5W; 6-D5S; 7-standard; 8-D7W; 9-D7S;
10-D10W; 11-D10S; 12-D12W; 13-D12S.
D-Day; W-whole; S-shredded.
11
3.00
Protein content
[mg/g dry weight]
2.50
2.00
1.50
1.00
Whole Lettuce
0.50
Cos Leaves
Cos Stem
0.00
0
3
5
7
10
12
Storage time [days]
Figure 3: Soluble protein content in whole Cos lettuce, leaves and stems during
postharvest storage. Each value represents the mean of six measurements ± standard
deviation.
200
97
66
45
31
1
2
3
4
5
6
7
8
9
10 11 12 13
Figure 4: Electrophoretic separation of soluble proteins present
in Cos lettuce. 1-D0W; 2-D0L; 3-D0S; 4-D3W; 5-D3L; 6-D3S;
7- Std; 8-D7W; 9-D7L; 10-D7S; 11-D10W; 12-D10L; 13-D10S.
D-Day; W-Whole; L-leaves; S-Stem.
12
Protein Content [mg/g dry weight]
45
40
35
30
25
20
15
Whole Broccoli
10
Broccoli Floret
5
Broccoli Stem
0
0
3
5
7
10
12
Storage time [days]
Figure 5: Soluble protein content in whole broccoli, floret and stems during
postharvest storage. Each value represents the mean of six measurements ± standard
deviation.
A
C
B
200
97
66
45
31
0
3
5
7
10 12
0 3
Storage time [days]
5
7
10
12
Figure 6: Electrophoretic separation of soluble proteins from floret
and stem of broccoli. A: Floret; B: Stem; C: Broad range protein marker.
13