Accepted Manuscript Short communication Effect of chitosan and its derivatives as antifungal and preservative agents on postharvest green asparagus Miao Qiu, Chu Wu, Gerui Ren, Xinle Liang, Xiangyang Wang, Jianying Huang PII: DOI: Reference:
S0308-8146(14)00047-8 http://dx.doi.org/10.1016/j.foodchem.2014.01.026 FOCH 15251
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
1 July 2013 7 December 2013 13 January 2014
Please cite this article as: Qiu, M., Wu, C., Ren, G., Liang, X., Wang, X., Huang, J., Effect of chitosan and its derivatives as antifungal and preservative agents on postharvest green asparagus, Food Chemistry (2014), doi: http:// dx.doi.org/10.1016/j.foodchem.2014.01.026
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1
Effect of chitosan and its derivatives as antifungal and preservative
2
agents on postharvest green asparagus
3 4
Miao Qiu, Chu Wu, Gerui Ren, Xinle Liang, Xiangyang Wang*, Jianying Huang*
5
Key Laboratory of Fruits and Vegetables Postharvest and Processing Technology
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Research of Zhejiang Province, College of Food Science and Biotechnology, Zhejiang
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Gongshang University, Hangzhou, 310018, PR China
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ABSTRACT
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The antifungal activity and effect of high-molecular weight chitosan (H-chitosan), low-
10
molecular weight chitosan (L-chitosan) and carboxymethyl chitosan (C-chitosan)
11
coatings on postharvest green asparagus were evaluated. L-chitosan and H-chitosan
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efficiently inhibited the radial growth of Fusarium concentricum separated from
13
postharvest green asparagus at 4 mg/ml, which appeared to be more effective in
14
inhibiting spore germination and germ tube elongation than that of C-chitosan. Notably,
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spore germination was totally inhibited by L-chitosan and H-chitosan at 0.05 mg/ml.
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Coated asparagus did not show any apparent sign of phytotoxicity and maintained good
17
quality over 28 days of cold storage, according to the weight loss and general quality
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aspects. Present results inferred that chitosan could act as an attractive preservative agent
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for postharvest green asparagus owing to its antifungal activity and its ability to stimulate
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some defense responses during storage.
21
* *
Corresponding author: Tel: 86-571-28877777, E-mail:
[email protected]. Corresponding author: Tel: 86-571-28877777, E-mail:
[email protected] 22
Keywords: Chitosan; asparagus; Fusarium concentricum; antifungal activity; spore
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germination.
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1. Introduction
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Green asparagus (Asparagus officinalis L.) is eaten worldwide as a popular fresh
27
vegetable due to its high nutritional values. However, its respiration rate makes it
28
particularly susceptible to postharvest perish. Moreover, asparagus is easily infected by
29
the Fusarium species causing stem and crown blight (Logrieco, Doko, Moretti, Frisullo
30
& Visconti, 1998), resulting in the decay of postharvest asparagus. Application of
31
chemical fungicides, such as benomyl, is controversial in many countries due to health
32
risk concerns, although it is by far the most effective method to control postharvest
33
diseases.
34
A common method to extend the storage life of asparagus is atmospheric
35
modification and cold storage (McKenzie, Greer, Heyes & Hurst, 2004). Gradually, other
36
reagents including aqueous ozone (An, Zhang & Lu, 2007), 6-benzylaminopurine (Wei &
37
Ye, 2011), salicylic acid (Wei, Liu, Su, Liu & Ye, 2011) and silver nanoparticles-PVP
38
(An, Zhang, Wang & Tang, 2008) have been used combined with optimal conditions,
39
such as cold storage or modified atmospheres packaging (MAP), to prevent or lessen
40
postharvest asparagus changes in physicochemical and chemical composition.
41
At a high relative humidity, most fruit and vegetable can retain good quality.
42
However, they are particularly susceptible to postharvest fungal infection under such
43
conditions. The common storage method, refrigeration, is uneconomical and removes
44
moisture, though it can reduce the respiration rate of asparagus. Thus, there is an ongoing
45
challenge to develop efficient and safe approaches for the control of postharvest diseases
46
and maintenance of postharvest green asparagus quality.
47
Chitosan has emerged not only as a promising and economic source for efficient and
48
versatile antimicrobial material, but also as a biocompatible and biodegradable polymer
49
with various applications (Dutta, Dutta & Tripathi, 2004; Bansal, Sharma, Sharma, Pal &
50
Malviya, 2011; Zhang, Geng, Jiang, Li & Huang, 2012; Geisberger, Gyenge, Hinger,
51
Kach, Maake & Patzke, 2013). It has been extensively used as edible coatings to preserve
52
the quality of many foods (Chien, Sheu & Yang, 2007; Mohan, Ravishankar & Lalitha,
53
2012; Huang, Chen, Qiu & Li, 2012; Dutta, Tripathi, Mehrotra & Dutta, 2009) and as a
54
bioactive fungicide (EI Ghaouth, Arul, Grenier & Asselin, 1992).
55
Herein, Fusarium concentricum was isolated from postharvest asparagus, which was
56
stored at 28 °C, 70% RH, subsequently identified by internal transcribed spacer (ITS)
57
sequences in combination with the morphological characters. The antifungal activity of
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three different chitosan derivatives against F. concentricum was evaluated. Moreover, in
59
order to assess the potential of chitosan as a natural food preservative, the effect of those
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coatings on postharvest green asparagus quality during cold storage was investigated, to
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determine if chitosan coatings directly affect fungal growth and induce lignification.
62 63
2. Materials and methods
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2.1. Plant material
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Green asparagus spears were harvested from a production site located in Hangzhou
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(China). After harvesting, the samples were rapidly placed in crushed ice and transported
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to the laboratory within 2 hours. Spears selected for the study were straight, undamaged,
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16–20 mm in diameter and ~30 cm in length with closed bracts and no visible signs of
69
injury.
70
2.2. Fungal isolation
71
Diseased spear stems were surface-sterilized in 0.1% potassium permanganate for 3-
72
5 min, then excised with a sterile scalpel and plated on antibiotic potato-dextrose agar
73
(PDA). The plates were incubated at 28 °C in the dark and examined daily for 7 days
74
(Karolewski et al., 2011).
75
2.3. Morphological identification
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Identification was achieved by placing a drop of sterile water on a clean slide with
77
the aid of a mounting needle, where a small portion of the mycelium from the fungal
78
cultures was removed. The mycelium was spread very well on the slide with the aid of
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the needle. A cover slip was gently applied with little pressure to eliminate air bubbles.
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The slide was then mounted and observed with a × 10 eyepiece and × 40, × 100 objective
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lenses, respectively. Cultural characters were assessed by eye, further detailed
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microscopic examinations of the isolate was performed using an Olympus BX51
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polarizing microscope. The species encountered were identified in accordance with the
84
reference (Nirenberg & O’ Donnell, 1998).
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2.4. Molecular identification
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Species identification of the collections studied was based on the analysis of the ITS
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sequences in combination with the morphological characters. DNA was extracted and
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purified using the phenol-chloroform-isoamyl alcohol extraction method (Murray &
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Thompson, 1980). ITS was amplified with the primer sets ITS1/ITS4 (Takaiwa, Kiyoharu
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& Masahiro, 1985). PCR was performed in an Applied Bio systems 2620 thermocycler.
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The amplification was confirmed on 1% agarose electrophoresis gels stained with
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ethidium bromide (Stöger, Schaffer & Ruppitsch, 2006). DNA sequencing was performed
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at Beijing Inovogen Tech. Co..
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2.5. Antifungal ability assays
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Antifungal assays were performed based on the method of Jasso de Rodríguez,
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Hernández-Castillo, & Rodríguez-García (2005). The L-chitosan, with a viscosity of 20
97
mPa.s and deacetylated degree of 96.1% (Haixin Biological Products Co., Ltd., Zhejiang,
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China), and C-chitosan, with a viscosity of 92.5 mPa.s and deacetylated degree of 91.0%
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(Qingdao Honghai Biotechnology Company, Shandong, China) to be tested was
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dissolved in deionized water, while H-chitosan, with a viscosity of 423 mPa.s and
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deacetylated degree of 95.6%, was dissolved in 0.1% acetic acid, and the pH was then
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adjusted to 5.6 with 1 M NaOH solution. Potato dextrose agar (PDA) media including 0.5,
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1.0, 2.0, 4.0 mg/ml chitosans solutions were sterilized at 121 °C for 20 min and then
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poured onto sterile petri dishes (9-cm diameter). The plates were inoculated with 3-mm-
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diameter plugs taken from the margins of 7 days old colonies on PDA. Control plates
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were also inoculated with the fungus. Subsequently, all plates were incubated in the dark
107
at 28 °C. Three replicates for each sample concentration were used. When the mycelium
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of fungi reached the edges of the control plate (without the added samples), the antifungal
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index was calculated as follows:
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Antifungal index (%) = (1 – Da/Db) × 100%
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Where Da is the diameter of the growth zone in the test plates and Db is the diameter of
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growth zone in the control plate.
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2.6. Spore germination of conidia
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Aliquots of 25 ml of different chitosan solutions (0.01, 0.02, 0.03, 0.05 mg/ml)
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approximately containing 1 × 106 conidia/ml were placed at the centre of multi-well
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microscope slides each containing 10 wells. After being incubated in the dark at 28 °C for
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8 h, the numbers of germinated and non-germinated conidia were recorded and the
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percentage germination was calculated (Tikhonov et al., 2006). The length of spores were
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measured after 8 h of incubation at 400 × with the Image-Pro Plus 6.3 analyzing software
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(Media Cybernetics, Bethesda, MD, USA).
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2.7. Pathogenicity test
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A spore suspension (50 µl) containing a concentration 1 × 106 colony-forming units
123
per ml (cfu/ml) was inserted into the 5 mm deep puncture wounds made by a sterile cork
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borer (3 mm diameter) in the top of fresh asparagus. Asparagus inoculated with sterile
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water was used as the control. Then they were placed in polythene bags, tightly sealed,
126
and incubated in the dark at 28 °C for 4 days. After incubation, the crown was cut in half
127
longitudinally through the inoculation site with a sterile knife and examined for the extent
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of soft rot. The pathogenicity test was conducted twice.
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2.8. Treatment and storage of asparagus samples
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Coating was carried out at room temperature by dipping the asparagus spears for 10
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min in 1%, 0.25% (w/v) H-chitosan including 0.5% acetic acid suspensions, 1%, 0.25%
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(w/v) C-chitosan and L-chitosan suspensions, and deionized water as a control,
133
respectively. After slowly drying at ambient conditions (22 °C, 70% RH) for about 2 h,
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the treatments were stored at 2 °C with 95% RH for subsequent quality assessments at 0,
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7, 14, 21, 28 and 35 days throughout the storage.
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2.9. Visual appearance
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The sensory evaluation of asparagus was determined by a sensory panel of 10
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trained assessors. A four-point score based on a hedonic scale was employed (1, very
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good; 2, good; 3, acceptable; 4, unacceptable). A value of three was considered as the
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commercial acceptability threshold (Villanueva, Tenorio, Sagardoy, Redondo & Saco,
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2005).
142
2.10. Weight loss
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The weight loss was determined by periodical weighing, and the results were
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expressed as the percentage loss from the initial weight (Sothornvit & Kiatchanapaibul,
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2009).
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2.11. Lignin determination
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The lignin content of the different parts of the asparagus spears was determined with
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the thioacidoglycolysis method, according to the literature (Bruce & West, 1989).
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2.12. Statistical analysis
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The data were analyzed using analysis of variance (ANOVA) (P < 0.05). The mean
151
differences were established via the Duncan’s multiple range tests. The data were
152
analyzed by SPSS software (Version 18.0, SPSS Inc., Chicago, IL).
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3. Results and discussion
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3.1. Identification of fungi
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The ITS sequence obtained has been deposited into the NCBI GenBank database
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and matched against the nucleotide–nucleotide database through Blast for final
157
identification. Representative sequences with high similarity were aligned using ClustalX
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1.81, and then a phylogenetic tree was constructed by mega 5.0 on the basis of a UPGMA
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analysis with 1000 bootstrap replications (Fig. 1). The resultant dendrogram were well-
160
fitted with all the bootstrap values higher than 98. Cluster analysis clearly revealed the
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strain sample belonging to the species of Fusarium. oxysporum or Fusarium.
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concentricum.
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The colony grew very rapidly (0.87–0.94 cm/day) on PDA at the optimum
164
temperature of 28 °C. As shown in Fig. 2, the mycelium on PDA was floccose, whitish
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(Fig. 2A). The pigment on the reverse plate on PDA varied from greyish-rose to carmine
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red (Fig. 2B). The conidia borne in the aerial mycelium were obovoid or oval to allantoid.
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Conidiophores of the mycelium were sparsely branched with only a few polyphialides,
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short and wide (Fig. 2D). Based on the shape of the conidiophore and the characteristics
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described in previous reports (Nirenberg & O’ Donnell, 1998), the strain sample possibly
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attributed to the species of F. concentricum.
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3.2. In vitro antifungal effect
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In the case of F. concentricum, considerable differences were observed in the
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treatments of L-chitosan and H-chitosan with respect to the C-chitosan. As shown in
174
Fig. 3, the antifungal activity of chitosan was concentration-dependent. When using a
175
concentration of 4 mg/ml, there was a marked fungus radial growth inhibition in
176
evaluations of L-chitosan and H-chitosan. The antifungal indexes for L-chitosan and H-
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chitosan were 89% and 74%, respectively. Whereas, the antifungal index of C-chitosan
178
was only 10%.
179
It has been well speculated that the negatively charged residues of chitosan can
180
interfere with the fungal cell surface to change the permeability of the plasma membrane,
181
thereby inhibiting the growth of several phytopathogenic fungi (Leuba & Stossel, 1986).
182
In the present study, the interactions between different chitosan derivatives and the fungal
183
cell surface are different. L-Chitosan can easily approach the fungal cells to give a target
184
inhibition by disturbing fungi metabolism due to its relatively small and flexible structure
185
compared to H-Chitosan. Previous studies have reported that chitosan possessing proper
186
molecular weight at proper lower concentration had preferable inhibition ability against
187
Aspergillus niger (Li, Feng, Yang, Wang & Su, 2008). Our study also showed that L-
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chitosan exhibited significantly (P < 0.05) better inhibition ability against F.
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concentricum than that of H-chitosan. However, C-chitosan was observed to have lower
190
antifungal activity, probably due to its amphipathic structure hampering the interaction of
191
the negatively charged residues with the phospholipid groups present in the fungal cells.
192
3.3. Spore germination
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Spores were clearly more sensitive to chitosan than hyphae. The effect of the
194
chitosans on the germination of conidia of F. concentricum was studied at a concentration
195
range from 0.01 to 0.05 mg/ml (Table 1). As shown in Table 1, L-chitosan and H-chitosan
196
markedly reduced the spore germination and germ tube elongation of F. concentricum. At
197
the concentration of 0.05 mg/ml, spore germination was almost totally inhibited by L-
198
chitosan and H-chitosan (P < 0.05). However, C-chitosan showed no effect on the
199
germination of F. concentricum even exposed at a concentration of 0.05 mg/ml. In this
200
study, the inhibitory effect of L-chitosan and H-chitosan was dose-dependent, and L-
201
chitosan exhibited a slightly higher inhibitory activity than H-chitosan. Since the pattern
202
associated with the molecular weight of chitosan and its effect on spore germination is
203
not clear (Bautista-Banos, Hernandez-Lopez, Hernandez-Lauzardo, Trejo-Espino,
204
Bautista-Ceron & Melo-Giorgana, 2005), it is necessary to carry out more studies about
205
the mode of action of the physicochemical properties of chitosan on F. concentricum.
206
The inhibitory effects of the three chitosans on germ tube elongation were also
207
different. Germ tube elongation of F. concentricum was significantly inhibited when the
208
H-chitosan and L-chitosan concentration were higher than 0.02 mg/ml (P < 0.05). The
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shortest germ tubes of F. concentricum were observed with H-chitosan and L-chitosan at
210
0.05 mg/ml. The longest germ tubes were observed for C-chitosan.
211
3.4. Pathogenicity test
212
Symptoms are the visible reactions including wilt, rot, vascular discoloration, as
213
well as brown lesions on root and stem surfaces, when asparagus are infected and
214
colonized by Fusarium species (Corpas-Hervias, Melero-Vara, Zurera-Muñoz &
215
Basallote-Ureba, 2006), ultimately resulting in disease.
216
The brown lesions on spears were obviously observed, and the growth of mycelia
217
was visible from the 4th day of storage at 28 °C. This causal pathogen was re-isolated
218
from the infected asparagus, and the diagnosis was confirmed by the morphological
219
changes and molecular identification previously developed for F. concentricum. This
220
study has fulfilled Koch’s postulates for F. concentricum as a causative pathogen fungus
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of green asparagus.
222
3.4. Visual appearance
223
Although sensory attributes are an objective parameter, it is a fundamental criterion
224
for determining the shelf life of the product. The changes of visual characteristics of all
225
asparagus samples were presented in Table S1. The control batches showed longitudinal
226
striation (from no striation up to strong striation), dryness, and colour changes (from
227
bright green to dull olive green) earlier than those chitosan treated samples. The visual
228
characteristics changes were easily noticeable after 28 days for the control, and the
229
asparagus was no longer acceptable for selling. In the case of the chitosan treated batches,
230
the overall shelf life was relatively prolonged compared to the control by the above
231
sensory quality. At the 35th day, 0.25% of the C-chitosan and 0.25% of the L-chitosan
232
treated batches could be acceptable for the public, and a shelf-life extension of 7 days
233
was achieved (Table S1).
234
3.5. Weight loss
235
Weight loss gives direct information about the quality of asparagus, which is crucial
236
and valuable, due to the fact that every loss in weight leads to an economical loss. As
237
shown in Fig. 4, the losses of all chitosan-treated batches were relatively lower than that
238
of control during the entire storage time. At the 21st day, the losses of weight were 4.57%
239
(1% C-chitosan treated), 3.73% (0.25% C-chitosan treated), 5.93% (1% L-chitosan
240
treated), 6.52% (0.25% L-chitosan treated), 6.83% (1% H-chitosan treated) and 6.78%
241
(0.25% H-chitosan treated) respectively, whereas, that of the control was 8.68%. The
242
chitosan-treated batches significantly (P < 0.05) reduced the weight loss of asparagus
243
during storage. Among those chitosan-treated batches, asparagus treated with 0.25% C-
244
chitosan apparently exhibited the best moisture maintenance ability and contributed to a
245
better quality of asparagus during the cold storage. Chitosan can be used as a protective
246
barrier due to its film-forming property, thus allowing control of the biochemical changes
247
in the metabolism of asparagus by preventing water loss or creating a modified
248
atmosphere surrounding the asparagus. However, it also has the properties of moisture
249
absorption, and will somewhat absorb water from the asparagus texture in case of high
250
viscosity and high concentration conditions, leading to a lesser effect on the weight loss
251
of asparagus. It was further inferred that chitosan edible coatings could create a physical
252
barrier to moisture loss comparable to MAP in terms of retarding dehydration and
253
shriveling, while different concentrations and different chitosan derivatives exhibited
254
different effects on postharvest asparagus.
255
3.6. Lignin content
256
The extent of lignification is another important factor in the quality of asparagus
257
because it can also decrease the fresh market value. As shown in Fig. 5, the lignin
258
concentration of all asparagus samples increased rapidly over the first 7 days, and there
259
was no significant (P > 0.05) difference in the lignin content of the control and those
260
treated. Augmented lignification was observed in the H-chitosan-treated batches till the
261
14th day of storage. After that, the lignification deposition of all chitosan-treated batches
262
increased gradually during the later storage excluding the control batches. At the 35th day
263
of storage, the lignin content of 0.25% treated C-chitosan and 0.25% treated L-chitosan
264
sample were 4.81 mg/100 g FW and 4.74 mg/100 g FW, respectively, while that of the
265
control was 5.74 mg/100 g FW. Previous studies have reported that the lignin deposition
266
in postharvest was correlated with different enzymes, in which phenylalanine ammonia-
267
lyase (PAL) was supposed to be a key enzyme for the biosynthetic pathway of lignin
268
(Flores, Oosterhaven, Martínez-Madrid & Romojaro, 2005; Tzoumaki, Biliaderis &
269
Vasilakakis, 2009; Chen & McClure, 2000). These data suggested that the application of
270
exogenous chitosan, especially H-chitosan, can potentiate responses, enhancing PAL
271
activity and lignin biosynthesis in the initial storage period. This may result in the
272
reinforcement of the cell wall and the formation of an efficient physical barrier to restrict
273
subsequent fungal penetration and infection. Later, the irritable effect of chitosan on
274
PAL turned to inhibition, thereby reducing the biosynthesis of lignin and maintaining the
275
asparagus quality.
276 277
4. Conclusions
278
Fusarium concentricum isolated from postharvest green asparagus was identified,
279
and the fungal inhibition of chitosan derivatives, as well as quality effect of chitosan
280
derivatives coatings on postharvest green asparagus was evaluated. The chitosan
281
derivative coating was effective in reducing the decay of green asparagus caused by F.
282
concentricum. As tested in vitro, L-chitosan and H-chitosan inhibited the radial growth of
283
F. concentricum, with a remarkable effect at a concentration of 4 mg/ml, and totally
284
inhibited spore germination at a concentration of 0.05 mg/ml, indicating that chitosan
285
derivatives were either fungistatic or fungicidal. The observed inhibition of decay and
286
augmented lignification was attributed to the fungistatic activity of chitosan and its ability
287
to induce a defense reaction.
288
It was concluded that chitosan was worthy of further study as a natural preservative
289
for green asparagus and other foods prone to fungal spoilage. However, the successful
290
application of any novel antimicrobial agent in food preservation depends on a number of
291
factors. Adequate control of microbial growth in foods using chitosan requires extensive
292
knowledge of the factors that determine chitosan performance.
293 294
Acknowledgments
295
This study was supported by the Natural Science Foundation of the Technology
296
Planning Project of Zhejiang Province of China (2011C12031), and the National Science
297
Foundation of China (21102129). We thank to Beijing Inovogen Tech. Co. for DNA
298
sequencing.
299 300
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401
of
chitin
and
chitosan
with
the
[(2-Hydroxy-3-
402 403 404
Fig. 1. UPGMA tree based on sequence analysis of internal transcribed spacer sequences. Values on
405
the branches indicate percent bootstrap condidence values.
406
407 408
A
B
C
D
409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424
Fig. 2. Asparagus with symptoms of rot caused by F.concentricum. A, observe colony
425
cultures on PDA after incubation at 28 °C for 7 days; B, reverse colony cultures on PDA
426
after incubation at 28 °C for 7 days; C, F. concentricum mold (× 40); D, Detail of
427
conidiophores (× 100).
428
429
100
C-chitosan
L-chitosan
H-chitosan
90
Antifungal index (%)
80 70 60 50 40 30 20 10 0 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Concentration (mg/mL)
430 431
Fig. 3. Effect of C-chitosan, L-chitosan and H-chitosan on antifungal index of
432
F. concentricum. Vertical bars represent standard deviation of means.
433
434
16
C-chitosan 1% C-chitosan 0.25% L-chitosan 1% L-chitosan 0.25% H-chitosan 1% H-chitosan 0.25% Control
14
Weight loss (%)
12 10
a b
a
8
a
6
b b bb
4 2
a b
c
a a b
c
b c b
ccc
b
c
c c dd
d e
d
c c cb d
0 7
14
21
28
35
Storage time (days)
435 436
Fig. 4. Changes in weight loss of asparagus stored at 2 °C for 35 days. Each data point is
437
the mean of three replicate samples. Vertical bars represent standard deviation of means.
438
439 440
6.0
5.5
Lignin (mg/100 g FW)
5.0
4.5
4.0
3.5
a C-chitosan 1% C-chitosan 0.25% a L-chitosan 1% b bb b L-chitosan 0.25% a cd c H-chitosan 1% b dd H-chitosan 0.25% c cd de a Control e c ab c f bc dd cd de a de ab e abc abc bc cd d jj jj jj j
3.0
2.5 0
7
14
21
28
35
Storage time (days)
441 442
Fig. 5. Changes in lignin content of asparagus stored at 2 °C for 35 days. Each data point
443
is the mean of three replicate samples. Vertical bars represent standard deviation of means.
444 445 446
447
Table 1
448
Effect of C-chitosan, L-chitosan and H-chitosan on spore germination and germ tube
449
length of F.concentricum.
450 451 452
0
Germination,% (mean±standard error) 100
Germ tube length, µm 23.6
C-chitosan
0.05 0.03 0.02 0.01
100 100 100 100
21.5 21.6 22.2 22.9
L-chitosan
0.05 0.03 0.02 0.01
0 42.96±2.15 63.22±0.43 84.44±2.36
7.70 9.27 15.8 16.1
H-chitosan
0.05 0.03 0.02 0.01
0 47.57±4.17 71.23±3.48 95.92±3.76
7.69 9.67 13.9 23.2
Type of chitosan
Concentration, mg/ml
Control
453
The potential for using chitosan as a natural food preservative was assessed.
454
Antifungal ability and spore germination inhibition of chitosan were investigated.
455
Effect of chitosan derivatives coatings on postharvest green asparagus was evaluated.
456 457