全 文 ：Received 30 Mar. 2004 Accepted 6 Jul. 2004
Supported by the Key Programs for Science and Technology Development of Shanghai Science Committee (023912063).
* Author for correspondence. Tel (Fax): +86 (0)21 64783085; E-mail: .
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Acta Botanica Sinica
植 物 学 报 2004, 46 (10): 1249-1255
Quantitative Roughness Analysis of Post-harvest Agaricus bisporus
by Atomic Force Microscopy
YANG Hong-Shun1, 2, FENG Guo-Ping2, AN Hong-Jie3, LI Yun-Fei2*
(1. Institute of Refrigeration and Cryogenics Engineering, School of Mechanical and Power Engineering,
Shanghai Jiao Tong University, Shanghai 200030, China;
2. Department of Food Science and Technology, School of Agriculture and Biology, Shanghai Jiao Tong
University, Shanghai 201101, China;
3. Nanobiology Laboratory, School of Life Science and Technology, Shanghai Jiao Tong University, Shanghai 200030, China)
Abstract: The moisture loss degree is important in determining the quality of post-harvest mushroom
(Agaricus bisporus (Lange) Sing). Quantitative roughness analyzed by atomic force microscopy (AFM) was
proposed to denote the degree of shrinkage, with arithmetic average roughness (Ra) and root mean square
roughness (Rq) as parameters. The initial value of Ra was (30.035±1.839) nm, while those of 2 ℃, 25 ℃
and dynamic temperature on the 2nd day were (40.139±3.359) nm, (54.393±13.534) nm and (41.197±
6.555) nm, respectively. There is a similar tendency for the results of Ra and Rq. Both values of roughness
increased in duration of storage and with increasing temperatures. The three-dimensional profile of the
pileus epicutis could signify the process of water evaporation intuitionally. The tendency was in accor-
dance with the roughness results, especially for the earlier stage of the storage (0-2 d). The outcome of
roughness analysis could signify the differences of storage conditions. It was shown that the roughness
measured by atomic force microscopy effectively reflected the moisture loss degree of the mushroom
pileus epicutis during post-harvest storage.
Key words: mushroom; roughness degree; atomic force microscopy (AFM); moisture loss; modified
atmosphere
Appearance is a primary factor in quality judgment of
fruits and vegetables (Veraverbeke et al., 2003a). A loss in
weight of about 5% often causes produce to lose fresh-
ness and appear wilted (Kang and Lee, 1998). High relative
humidity (RH) could help minimize the shriveling, but too
high RH will result in fungal decay during storage
(Veraverbeke et al., 2003b). It is inevitable that shriveling of
produce happens.
The traditional criteria used to determine the degree of
shriveling are sensory evaluation and weight loss. Weight
loss is not uniform in different parts of the fruit body.
However, consumers are concerned more about the ap-
pearance of the skin of produce.
Instrumental measurements are often preferred to sen-
sory evaluations in research and commercial situations
because they reduce variances in judgment among indi-
viduals and can provide a common language.
Most recently emphasis has been placed on develop-
ing sensors for real-time, non-destructive sorting (Abott,
1999). Neither light microscopy (LM) nor scanning elec-
tron microscopy (SEM) produces qualitative topographi-
cal data in a straightforward manner (Wan and Tian, 2002;
Wang and Huang, 2003). The structures of the different
parts of the mushroom coatings were studied by SEM but
without the roughness data of the coatings (Hershko and
Nussinovitch, 1998a). Veraverbeke et al. (2003a) investi-
gated fruit surface layers with confocal laser scanning mi-
croscopy (CLSM) and environmental scanning electron
microscopy (ESEM). Staining was needed and CLSM was
not sufficient because this technique only detects fluores-
cence and reflection results in a much blurred image (Liu et
al., 2003).
Gibbs and Bishop (1996) proposed using a geostatistical
technique to describe bio-film surface roughness. Height
measurements are accurate to 1 mm or less, which is a low
resolution for investigating the changes of roughness.
Burdon and Clark (2001) had monitored the impact of water
loss using serial quantitative proton magnetic resonance
imaging; however, these results were not intuitional.
The way in advance of biology and material field on
nanometer scale in the past decades has been found in
application with atomic force microscopy (AFM). The ad-
vantage of high resolution allows precise topography im-
aging in a controlled way and Pico Newton magnitude force
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041250
measurement. AFM could image kinds of samples, such as
unstained and uncoated structures, in air or fluid, permit-
ting direct observation of native specimens and ongoing
processes under native or near-native conditions. The to-
pology of plant materials has been studied by AFM (Wilbert
et al., 1998; Round et al., 2000). Hershko and Nussinovitch
(1998b) compared the roughness between the onion skin
surface and the chloroform-cleaned onion surface. Also,
there are some reports about qualitative roughness analy-
sis with AFM for plastic films and metal surfaces (Darrot et
al., 1995; Reed et al., 1998; Lindseth and Bardal, 1999).
However, to our best knowledge, there is no report about
the changes of the roughness of produce during storage.
The main objective of this paper is to propose that the
roughness of mushroom pileus epicutis could be used as
one of the evaluating criteria for signifying the appearance
during post-harvest. AFM was applied to make quantita-
tive and accurate measurements of the topography of the
mushroom surfaces.
1 Materials and Methods
1.1 Materials
Mushrooms (Agaricus bisporus (Lange) Sing), 4 h after
harvested, were bought from Qibao supermarket in
Shanghai, then pre-cooled at 2 ℃ for 12 h. After being
sorted out and wiped off the stalk, mushrooms were washed
and dipped in 0.1 mol/L NaCl to inactivate the polyphenol
oxidase. Then mushrooms were drained (2 000 Pa, 20 min)
by the vacuum evaporation machine (Shanghai Yiheng Sci-
ence and Technology Co. Ltd.) to remove water on the
surfaces and packed in 0.035 mm low density polyethylene
(LDPE) packages, and then divided into three groups and
from now on the time was recorded as 0 d. Groups 1 and 2
were stored at 2 ℃ and 25 ℃, respectively, through all the
test. However, group 3 was stored at different tempera-
tures for different stages to simulate the cold chain in com-
mercial distribution, which is shown below:
25 ℃, 2 h → 2 ℃, 6 h → 15 ℃, 0.5 h → 4 ℃, 72 h → 15
℃, 0.5 h→2 ℃, 5 h→25 ℃, 0.5 h→2 ℃, 48 h→25 ℃
1.2 Atomic force microscopy (AFM) measurement and
roughness analysis
Samples were cut into thin pieces which fit into AFM
imaging, and were stuck onto mica surface by double-sided
tape, mounted onto the sample stage, then scanned with
Tapping mode at the scan speed of about 1-2 Hz.
Tapping mode was carried out using a multimode
NanoScope IIIa AFM (digital instruments Santa Barbara,
CA, USA) equipped with a Si3N4 cantilevered scanner with
a 12 mm×12 mm scan size and a 4-mm vertical range. It has
a high resolution with about 0.1 nm for vertical range and
1-2 nm for lateral (Darrot et al., 1995).
To make the results comparable, the images were ob-
tained from the center area of each surface. Since the area
was much small, it did not contain any vasculature. Before
imaging each sample, the integrity of the AFM tip was veri-
fied by imaging a reference standard with a known rough-
ness of 5-7 nm (Reed et al., 1998).
Atomic force microscopes are generally limited to small
scanned areas. Three images of different zones were exam-
ined and offline analyzed with version 5.12 software on
each specimen in order to average the roughness value
(Darrot et al., 1995).
Height measurements were performed on small (from
2.0 mm× 2.0 mm to 5.0 mm× 5.0 mm) surfaces of mush-
room pileus epicutis after different storage conditions. Only
selected representative images of each type are presented
in this paper.
The height variation is represented by a color scale in
which pink is high and purple is low for all images. Different
scales are used in the vertical and horizontal scale.
Two amplitude parameters have been used. The arith-
metic average roughness, Ra, and the root mean square
(RMS) roughness, Rq, are given by:
Ra = ∑ ∑ |Z(i, j)-Zave| (1)
Rq = (2)
Where Z (i, j) denotes the topography data for the sur-
face after specimen tilt-correction, Zave is the average sur-
face height, i and j correspond to pixels in the x and the y
directions, and the maximum number of pixels in the two
directions are given by nx and ny.
All parameters used are well known from surface
metrology. The calculations were made using tilt-corrected
topography data (Lindseth and Bardal, 1999).
1.3 Statistical analysis
Statistical analysis of results through ANOVA (P < 0.05)
and Duncan’s multiple range test were performed using
SAS 8.0.
2 Results
Figures 1 and 2 show the plane and three-dimensional
profiles of the mushroom pileus epicutis before being di-
vided into three groups, respectively. The Ra was 31.367
nm (shown in Fig.3), other two parallel samples were 30.801
∑ ∑
1
nxny
nx ny
i=1j=1
nxny
nx ny
i=1j=1
[Z(i, j)-Zave]2
YANG Hong-Shun et al.: Quantitative Roughness Analysis of Post-harvest Agaricus bisporus by Atomic Force Microscopy 1251
nm and 27.937 nm (not shown in the paper). And the statis-
tical result was (34.033 ± 5.116) nm (Table 1).
Figures 4-9 show the plane and three-dimensional pro-
files of mushroom pileus epicutis of group 1 to group 3
Figs.1-8. Plane and three-dimensional (3-D) profiles of mushroom pileus epicutis at different storage stages by atomic force
microscopy (AFM). 1. Plane, 0 d. 2. 3-D, 0 d. 3. Roughness analysis of the pileus epicutis at 0 d. 4. Plane, 2 ℃, 2 d. 5. 3-D, 2 ℃, 2 d.
6. Plane, 25 ℃, 2 d. 7. Plane, 25 ℃, 2 d. 8. Plane, dynamic temperature, 2 d.
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041252
Figs.9-16. Plane and three-dimensional (3-D) profiles of mushroom pileus epicutis at different storage stages by atomic force
microscopy (AFM). 9. 3-D, dynamic temperature, 2 d. 10. Plane, 2 ℃, 4 d. 11. 3-D, 2 ℃, 4 d. 12. Plane, 25 ℃, 4 d. 13. 3-D, 25 ℃, 4
d. 14. Plane, dynamic temperature, 4 d. 15. Plane, dynamic temperature, 4 d. 16. Plane, 2 ℃, 6 d.
YANG Hong-Shun et al.: Quantitative Roughness Analysis of Post-harvest Agaricus bisporus by Atomic Force Microscopy 1253
after two-day storage, and Figs.10-15 show the plane and
three-dimensional profiles after four-day storage, and Figs.
16 and 19 show the plane and three-dimensional profiles of
groups 1 and 3 after six-day storage. The group 2 was not
assayed on the 6th day storage because the samples had
rotted.
Table 1 shows the statistical results of Ra for all the
groups. And Table 2 shows Rq after the similar process for
these groups.
3 Discussion
It was clear from Table 1 that the roughness was differ-
ent with different storage conditions and the roughness
values of group 1 to group 3 increased with the time.
Figures 1-19 show the intuitional process of the moisture
evaporation from mushroom pileus epicutis. For example,
the three-dimensional profile of the mushroom pileus
epicutis before storage was much smooth (shown in Fig.2),
but fluctuated after two-day storage (from Fig.4 to Fig.9).
The higher the temperature was, the more rough in profile.
This tendency was similar from the 2nd to the 6th day
storage. However, the trend from the beginning to the 2nd
day was more obvious than that from the 2nd to the 6th
day, which was a great help to know the quality of mush-
room after post-harvest (Hershko et al., 1998). Ra and Rq
have the similar tendency for these groups.
For preserving the integrity of samples, the tapping
mode, which was developed specifically for soft materials,
was used. With this technique, the probe oscillated verti-
cally so that it could lightly tap the sample during imaging
rather than slide over the surface, which virtually elimi-
nated lateral shearing and sample damage for the majority
of specimens and improved the lateral resolution of the
AFM image (Yang et al., 2003).
Hershko and Nussinovitch (1998a) had reported that
the Ra of fresh and dry mushroom were (2.1 ± 0.8) mm and
(2.2 ± 0.5) mm, respectively. The values are larger than in
our experiments. However, the maximum height of onion
skin is only 628 nm, which showed the maximum was near
our experimental value. The scanned size was greatly smaller
than Hershko and Nussinovitch (1998b) used. It is impor-
tant to remark that the roughness value depends on the
scanned area and on the number of data points; roughness
decreases in line with the scanned area because of the sur-
face statistics, i.e. the fractal behavior (Darrot et al., 1995).
AFM allows imaging at ambient conditions but is useful
only for flat surfaces. Height differences of a specific sur-
face exceeding the z-piezo range can cause damage of the
tip, and steep or rough structures result in unrealistic im-
ages because of the limited aspect ratio of the tip (Fig.7).
But it is an appropriate method for investigating the fine
structures of individual areas. The wax platelet assayed 7-
10 nm (Ensikat et al., 2000). And unpolished stainless steel
was (75 ± 29) nm of Ra (Verran et al., 2000). Compared with
these values, the results of mushroom in this paper were
credible.
AFM are generally limited to small scanned areas. Im-
ages from these microscopes might be helpful when
Figs.17-19. Plane and three-dimensional (3-D) profiles of
mushroom pileus epicutis at different storage stages by atomic
force microscopy (AFM). 17. Plane, 2 ℃, 6 d. 18. Plane, dy-
namic temperature, 6 d. 19. Plane, dynamic temperature, 6 d.
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041254
Table 1 Effect of storage conditions on the arithmetic average roughness (Ra) of mushroom pileus epicutis/nm
Group
Storage time (d)
0 2 4 6
1 30.035±1.839 a 40.139±3.359 a 58.593±12.330 b 98.480±7.804 b
2 30.035±1.839 a 54.393±13.534 a 164.81±26.956 a na
3 30.035±1.839 a 41.197±6.555 a 88.825±8.843 b 141.07±18.477 a
Values shown are mean ± SD where n = 3. Values in the same column with the same superscript letters (a or b) indicate no significant differences
by the Duncan’s multiple range test (P < 0.05). na, not analysis because of out of marketing value.
Table 2 Effect of storage conditions on the root mean square roughness (Rq) of mushroom pileus epicutis/nm
Group
Storage time (d)
0 2 4 6
1 36.656±3.475 a 49.308±4.549 a 75.055±17.038 c 123.613±14.888 b
2 36.656±3.475 a 68.581±17.489 a 200.51±15.123 a na
3 36.656±3.475 a 50.213±7.800 a 116.327±10.015 b 171.213±24.311 a
Values shown are mean ± SD where n = 3. Values in the same column with the same superscript letters (a or b) indicate no significant differences
by the Duncan’s multiple range test (P < 0.05). na, not analysis because of out of marketing value.
evaluating topographic data, since these are well estab-
lished methods for characterizing surfaces, and also con-
trast mechanism of LM and SEM are well known (Lindseth
and Bardal, 1999). To appreciate the quality of homogene-
ity of the samples, on a large scale, it is also advised to use
the SEM in combination with the AFM, each technique has
its own pertinent scale of investigation and thus they prove
very complementary (Darrot et al., 1995).
The present work is not only important for characteriza-
tion of mushroom surfaces. It should be relevant for any
problem requiring topography measurements of strongly
reflecting produce surfaces, having topographic features
from around 10 mm and down well into the sub-micrometre
range.
4 Conclusion
The roughness analysis gained from AFM was effec-
tive in determining the shrinkage degree of the mushroom
during storage. Both values of Ra and Rq increased in dura-
tion of storage and with increasing temperatures. The three-
dimensional profile of the pileus epicutis could intuitionally
signify the process of water evaporation. And the surface
would become rougher with the increment of time and rise
of temperature. It is appropriate for Ra or Rq roughness
measured by AFM to denote the appearance quality of
mushroom, especially for the early stage of storage. It was
shown that the roughness measured by AFM effectively
signified the moisture loss of the mushroom pileus epicutis
during post-harvest.
Acknowledgements: The authors would like to thank Dr.
Ian B. Ferguson (The Horticulture and Food Research
Institute, New Zealand) for his useful suggestions and Dr.
LAN Yu-Bin (Fort Valley State University, USA) for his
careful reading on this manuscript.
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