Postharvest Biology and Technology 57 (2010) 7276

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Postharvest Biology and Technology

journal homepage: www.elsevier.com/locate/postharvbio

Effect of nitric oxide on browning and lignication of peeled bamboo shoots

Huqing Yang , Cunshan Zhou, Fenghua Wu, Jiyu Cheng
School of Agriculture and Food Science, Zhejiang Forestry University, 88 North Circular Road, Linan, Zhejiang 311300, PR China

a r t i c l e

i n f o

Article history:
Received 5 January 2010
Accepted 13 February 2010
Keywords:
Bamboo shoot
Nitric oxide
Lignication
Browning

a b s t r a c t
The effects of nitric oxide (NO) on browning and lignication of bamboo shoots were investigated. Bamboo
shoots were dipped for 1 h in 0.5 mM sodium nitroprusside (SNP), a nitric oxide donor, then packed in
0.01 mm thick polyethylene bags, and stored for 10 days at 10 C. SNP treatment inhibited activities of PPO,
POD and PAL and maintained high total phenol contents, thus delaying external browning during storage.
Furthermore, SNP treatment showed a signicant inhibition of the synthesis of lignin and cellulose and
delayed tissue lignication, indicating that application of NO may be a promising method for extending
shelf-life and maintaining quality of peeled bamboo shoots.
2010 Elsevier B.V. All rights reserved.

1. Introduction
Bamboo shoots are immature, expanding culms that emerge
from nodes of the rhizome of the plants. While they are commonly
consumed as a vegetable, various degradative changes are induced
during posthavrest handling, including peeling and cutting. An
important factor causing quality deterioration is the development
of browning on the shoot surface. Browning reactions are generally
assumed to be a direct consequence of polyphenol oxidase (PPO)
and peroxidase (POD) action on polyphenols to form quinones,
which ultimately polymerize to produce the browning appearance in fresh-cut fruit and vegetable products (DeglInnocenti et
al., 2005). Another property of peeled bamboo shoots during storage is an unusual increase in rmness and toughness of the esh
from the cut-end towards the tip due to the tissue wounding, and
this increase in rmness may be the result of lignication (Luo et al.,
2008a). Therefore, delaying or reducing enzymatic browning and
lignication should be a way to extend storage life and maintain
quality of peeled shoots.
Nitric oxide (NO) is a highly reactive free radical gas known to
be involved in response to stress and senescence of horticultural
products. Short-term exposure to a low concentration of NO gas
or its donor compounds has been shown to extend the postharvest
life of various fruit and vegetables (Wills et al., 2000, 2007; Zhu and
Zhou, 2007). It has also been shown that they delayed the onset of
browning of apple slices (Pristijono et al., 2006), cut lettuce (Wills et
al., 2008) and longan (Duan et al., 2007). However, there have been
no reports on the effect of NO on peeled bamboo shoots during
storage.

Corresponding author. Tel.: +86 571 63748155; fax: +86 571 63741276.
E-mail address: yanghuqing@sohu.com (H. Yang).
0925-5214/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.postharvbio.2010.02.004

The purpose of this work was to study the physiological and

biochemical changes of peeled bamboo shoots during postharvest
handling to test whether NO treatment could inhibit the external
browning and tissue lignication.
2. Materials and methods
2.1. Plant materials and treatments
Shoots of bamboo (Phyllostachys violascens) were harvested at
Linan (Zhejiang Province, China) in April 2008 and immediately
transported to our laboratory, where they were selected for uniformity of shape and size. The shoots were washed in tap water
and hand-peeled. About 5 cm was removed from the cut-end of the
shoots with a sharp knife and the remainder was washed again
with 0.04% sodium hypochlorite solution. A total of 720 bamboo
shoots were divided into two equal groups, comprising three replicates of 120 in each group. In a preliminary study, the shoots were
stored at 10 C for 12 days after treatment with sodium nitroprusside (SNP, NO donor) at concentrations of between 0.1 and 1.0 mM.
It was found that treatment with SNP at 0.5 mM resulted in better
esh quality and this concentration therefore was used. Shoots of
one group were dipped in 0.5 mM SNP aqueous solutions for 1 h
at 25 C, and the other group was treated with water as the control. All shoots were then surface-dried with cold air. Each stack
of 40 bamboo shoots were packed in 0.01 mm thick polyethylene
bags (long 90 cm, wide 45 cm) with several holes for ventilation and
stored at 10 0.5 C, RH 90%. Shoot rmness, browning index and
ethylene production were assessed every 2 days. Tissue samples
(about 100 g from each replicate) were frozen in liquid nitrogen
and stored at 80 C until used for the determination of total
phenols, lignin and cellulose contents and activities of PPO, POD
and PAL.

H. Yang et al. / Postharvest Biology and Technology 57 (2010) 7276

2.2. Browning index

Surface browning was assessed by measuring the extent of the
total browned area on each shoot of the 10 in each replicate according to a 5 grade scale: 0, none; 1, browning area <10%; 2, browning
area 1025%; 3, browning area 2550%; 4, browning area >50%.
The browning index was calculated as (browning level number
of shoots with that browning level)/(total number of shoots).
2.3. Determination of ethylene production
A sample of 3 shoots from each replicate (3 3 per treatment)
was sealed in a 1 L jar for 1 h at 20 C and then a 1 mL gas sample
was collected from the jar with a syringe. The ethylene concentrations were determined using a SP-6890 gas chromatograph (Lunan
Chemical Industrial Instruments Co., Shandong, China) with a ame
ionization detector and a GDX-502 column held at 90 C. Rates of
ethylene production were expressed as L ethylene kg1 h1 FW.

73

50 mM sodium borate buffer at pH 8.7 for peroxidase (POD). All

extract procedures were conducted at 4 C. The extracts were then
homogenised and centrifuged at 12,000 g for 30 min at 4 C. The
supernatants were used for the enzyme assays.
PAL was assayed by the method described by Zucker (1968).
One unit of PAL activity was dened as the amount of enzyme
that causes the increase in absorbance of 0.01 at 290 nm in 1 h
under the specied conditions. PPO activity was assayed following the method of Murr and Morris (1974). One unit of PPO activity
was dened as the amount of enzyme that causes the increase in
absorbance of 0.01 at 410 nm in 1 min under the specied conditions. POD activity was assayed using guaiacol as a donor and H2 O2
as a substrate according to the method of Kochba et al. (1977). One
unit of POD activity was dened as an increase of 0.01 in absorbance
per minute at 460 nm under the assay conditions.
Protein content was determined according to the method of
Bradford (1976), with bovine serum as the standard.
2.8. Statistical analysis

2.4. Texture measurement

Texture was measured on 10 individual shoots per treatment,
comprising 3, 3, and 4 shoots from each of the three replicates.
Firmness (N) was tested at the middle of the shoot using a TAXT2i texture analyzer (Stable Micro Systems, England) tted with
a 5 mm diameter probe. The penetration rate was 1 mm/s with a
nal penetration depth of 10 mm and data were expressed in N.
2.5. Determination of total phenols
Total phenols were extracted by homogenizing 3 g of frozen
tissue powder with 12 mL of 80% ethanol and centrifuging the
homogenate at 12,000 g for 20 min. Total phenols were estimated
on the supernatant by a colorimetric assay based on procedures
described by Singleton and Rossi (1965). Absorbance at 725 nm was
measured using gallic acid as a standard. The phenol contents were
expressed as gallic acid equivalents in milligrams on a fresh weight
(FW) basis.
2.6. Determination of lignin and cellulose contents
About 5 g of frozen samples were used for lignin analysis. Lignin
was gravimetrically determined according the method of Luo et al.
(2008a) and the results expressed as g lignin per 100 g fresh weight.
Cellulose was extracted and measured by the method of Oomena
et al. (2004) with modications. For the isolation of cell wall material (CWM), 10 g of frozen tissue powder was extracted in a 50 mM
Tris:HCl, pH 7.2 solution containing 1% SDS for 3 h at room temperature with continuous shaking. The extract was centrifuged at
16,000 g for 20 min. The residue was washed with water, ethanol,
and acetone and air-dried. Fifty milligrams of CWM was incubated
for 90 min at 120 C in 5 mL 2 M triuoroacetic acid. The remaining
cellulose was pelleted and washed with water and ethanol. The pellet was solubilized in 67% (v/v) H2 SO4 at 37 C for 60 min and diluted
appropriately for the determination of cellulose content colorimetrically at 620 nm using anthrone as coloring agent and glucose as
the quantication standard.

Each experiment was repeated three times and the data were
processed by analysis of variance (ANOVA). Treatments were compared at P = 0.05 according to least signicant difference (LSD) test.
3. Results
3.1. Effect of NO treatment on ethylene production
Ethylene production from control shoots increased rapidly and
achieved a climacteric peak on the 4th day, decreasing gradually
(Fig. 1). The shoots treated with 0.5 mM SNP also exhibited a climacteric rise after 4 days similar to control shoots, but the level
was lower. SNP treatment markedly suppressed ethylene production at 10 C. On the 4th day, ethylene production in treated shoots
was signicantly lower than that of the control shoots (P < 0.05).
3.2. Effect of NO treatment on browning index
The browning index of the control increased sharply after the
rst two days, reaching 3 at the fourth day and 3.8 at the sixth
day, with serious browning rendering the product inedible (Fig. 2).
The SNP treatment showed only minor browning with a browning
index of 0.4 in the rst four days. At the end of the storage period,
the browning index of treated shoots reached 2, but at that level the
shoots still had commercial value and were edible. Compared to the
control, SNP treatment signicantly (P < 0.01) inhibited browning.

2.7. Assays of enzyme activities

Phenylalamine ammonia lyase (PAL) was extracted from 5 g
frozen tissue with 0.2 M sodium borate buffer at pH 8.8 containing 40 g/L polyvinylpyrrolidone, 2 mM EDTA and 5 mM of
-mercaptoethanol. Five grams of frozen tissue was ground with
25 mL of 0.2 M sodium phosphate buffer (pH 6.5) containing 1%
polyvinylpyrrolidone (PVP) for polyphenol oxidase (PPO), or with

Fig. 1. Effect of NO treatment on ethylene production of bamboo shoots stored at

10 C. Each value is presented as the mean standard error (n = 3).

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H. Yang et al. / Postharvest Biology and Technology 57 (2010) 7276

Fig. 2. Browning index of bamboo shoots stored at 10 C. Each value is presented as

the mean standard error (n = 3).

3.3. Effect of NO treatment on rmness

Shoot rmness increased steadily, with about a 50% increase
over the 10 day storage period at 10 C (Fig. 3). Shoots treated with
SNP showed a similar trend, but the rmness increase was retarded
(P < 0.05), which corresponded to about a 30% increase over the
same period. It is unlikely that weight loss was responsible for these
differences in rmness, since there were no differences in weight
loss among the treatments (data not shown).
3.4. Total phenol content
As shown in Fig. 4a, the total phenol content of the shoots
decreased gradually during storage. In SNP-treated shoots this was
signicantly greater than that of the control (P < 0.05), demonstrating a delayed decrease of total phenol by the SNP treatment.
3.5. Effect of NO on lignin and cellulose contents
As shown in Fig. 4b, lignin content of shoots from both control and SNP treatments increased during storage. The change in
cellulose content was similar to that of lignin (Fig. 4c). SNP treatment markedly delayed the increase in lignin and cellulose contents

Fig. 4. Effects of NO on total phenol (a), lignin (b) and cellulose (c) contents of bamboo shoots during storage at 10 C. Each value is presented as the mean standard
error (n = 3).

(P < 0.05). After 10 days of storage, the lignin and cellulose contents
in control shoots were 1.39% and 1.23%, as compared with 0.90%
and 0.96% in SNP-treated shoots, respectively.
3.6. Activities of PAL, PPO and POD

Fig. 3. Firmness of bamboo shoots stored at 10 C. Each value is presented as the

mean standard error (n = 10).

Activities of PAL, PPO and POD increased with storage time

(Fig. 5ac). SNP treatment signicantly (P < 0.05) inhibited the
increase in activities of these three enzymes. At the end of storage, the activities of PAL, PPO and POD in SNP-treated shoots

H. Yang et al. / Postharvest Biology and Technology 57 (2010) 7276

Fig. 5. Effects of NO on activities of PAL (a), PPO (b) and POD (c) of bamboo shoots
during storage at 10 C. Each value is presented as the mean standard error (n = 3).

were 31.9%, 27.2% and 21.6%, respectively, lower than those in the
control.
4. Discussion
The bamboo shoots are not easy to store due to a rapid loss
of tenderness mostly resulting from lignication. Lignin synthesis is the result of coordinated action of many related enzymes
including PAL, PPO and POD. PAL is a key enzyme that catalyses the
conversion of phenylalanine to trans-cinnamic acid in the phenylpropanoid pathway, which is the rst step in the biosynthesis of
lignin in plants. POD catalyses the polymerisation of monolignol
to form lignin. In addition, PPO can use as substrates those phenolic compounds which are precursors of lignin synthesis (Boudet et
al., 2003). The accumulation of lignin in bamboo shoots observed

75

during 10 days storage was associated with increased activities of

PAL, PPO and POD. Treatments such as 1-methylcyclopropene and
modied atmosphere packaging that inhibit lignication of bamboo shoots have been shown to reduce the activities of PAL, PPO and
POD (Luo et al., 2007, 2008b; Shen et al., 2006). In the present study,
treatment with SNP signicantly reduced the activities of PAL, PPO
and POD (Fig. 5ac). These results suggest that the effect of SNP in
reducing the development of lignication of bamboo shoots was
associated with inhibited activities of these enzymes involved in
lignin synthesis.
Bamboo shoots readily brown, especially when peeled or cut.
This process is enzymatic and can be linked to lignication through
the common involvement of the three major enzymes PPO, POD and
PAL. All of the three enzymes have been shown to be responsible
for browning reactions during postharvest handling, storage and
processing of fruit and vegetables (Peiser et al., 1998). In this study,
increasing PAL, PPO and POD activities were found to correspond
with an increase in the external browning index in bamboo shoots
during storage. SNP treatment signicantly inhibited the activities of the three enzymes in association with higher total phenolic
contents (Fig. 4a), thus resulting in reduction of the browning reaction and a lower external browning index (Fig. 2). Similar results
with inhibition of browning by NO have been reported in apples
(Pristijono et al., 2006), longans (Duan et al., 2007) and cut lettuce
(Wills et al., 2008).
While our results show that SNP was effective in reducing browning and lignication, the mechanism of NO action is
unknown. Leshem (2000) proposed that the oxidative properties
of NO are important mediating factors and that NO or its reaction products can oxidatively inactivate enzyme co-factors, such as
ascorbate and ferrous ion. The possible mechanism of NO in modulating PAL, PPO and POD in bamboo shoots and other materials
needs further study.
We observed a strong inhibition of ethylene production in
SNP-treated bamboo shoots (Fig. 1). The inhibition of ethylene
biosynthesis has also been reported in NO-fumigated kiwifruit,
peach, strawberry, and tomato (Eum et al., 2009; Flores et al., 2008;
Zhu et al., 2008; Zhu and Zhou, 2007). Anti-senescent action of NO
in plant tissues has been proposed to take place via the inhibition
of ethylene biosynthesis (Leshem et al., 1998; Zhu et al., 2006). The
possible association between inhibition of ethylene production and
reduction of expression of PAL, PPO and POD by NO should also be
part of any further exploration of mechanisms of NO action.
In conclusion, treatment with SNP effectively inhibited external
browning and tissue lignication of peeled bamboo shoots during
storage. The inhibition of quality deterioration by NO was related to
reduce activities of PPO, POD and PAL as well as reduced biosynthesis of ethylene. Thus, it is suggested that application of NO may be a
promising method for extending shelf-life and maintaining quality
of peeled bamboo shoots.
Acknowledgements
We appreciate the kind review of the manuscript by Professor
Tiejin Ying at the Department of Food Science and Nutrition at Zhejiang University. We also thank Dr. Qingpo Liu, Zhejiang Forestry
University, China, for good advice about revision of the paper.
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