Journal of Food Engineering 183 (2016) 9e15

Contents lists available at ScienceDirect

Journal of Food Engineering

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

Novel 2D and 3D imaging of internal aerated structure of

ultrasonically treated foams and cakes using X-ray tomography and
X-ray microtomography
M.C. Tan a, N.L. Chin a, *, Y.A. Yusof a, J. Abdullah b
a
b

Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
Plant Assessment Technology Group, Malaysian Nuclear Agency (Nuclear Malaysia), Bangi, 43000 Kajang, Selangor, Malaysia

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 20 August 2015
Received in revised form
5 March 2016
Accepted 20 March 2016
Available online 21 March 2016

The aerated structure of ultrasound treated foams and its resulted cake structures were examined using
X-ray tomography and X-ray microtomography, leading to highly contrasted two-dimensional (2D) and
three-dimensional (3D) images. Through these imaging techniques and software approaches, the effect
of ultrasound treatment on the bubble size distribution was distinguished clearly. Microbubbles in foam
which were in the size range of 0e0.00125 mm3 and in cakes which were in the range of 0e1 mm2
increased by 48% and 29% respectively after ultrasonic treatment at a frequency of 20 kHz.
2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords:
X-ray microtomography
X-ray tomography
2D image
3D image
Aerated foam
Cake

1. Introduction
Imaging is one of the direct methods to visualize and analyze
bubbles or cells matrix in food products. Traditionally, bubble sizes
in cake batter have been examined by taking photographs of
aerated batter spread out on a thin lm using microscope-linked
camera (Niranjan and Silva, 2008; Sahi and Alava, 2003) or tinted
cross sections of cake samples at 5 mm thickness with black oilbased ink delineating pore walls using a digital camera (Barrett
and Ross, 1990; Kocer et al., 2007; Lange et al., 1994; Smolarz
et al., 1989). The photographs were analyzed using imaging software. The three-dimensional structure was then drawn based on
the assumed symmetrical two-dimensional images. This imaging
technique is a destructive method and it also arises problems in
generating a sharp image due to inaccurate measurement of the
walls and curvature effects (Niranjan and Sahu, 2009; Niranjan and
Silva, 2007). As such, bubble size distribution is difcult to be
measured and analyzed (Niranjan and Silva, 2007), especially with
the traditional imaging method. The poorly understood aerated

* Corresponding author.
E-mail address: chinnl@upm.edu.my (N.L. Chin).

foods' structures, however, did not stop the evolutionary food

manufacturers from seeking to exploit the versatility of bubbles as a
food ingredient (Campbell and Mougeot, 1999). Air is an important
ingredient in aerated foods such as beverages, baked, dairy, egg and
confectionery products to enhance the appearance, texture, and
digestibility of the food products (Campbell and Mougeot, 1999).
For bakery products, air attribution has a signicant inuence on
the quality of the nal product because the cells in the nal products are originated as bubbles in the batter during the mixing stage,
and that integrity of air cells would determine the volume of the
nal products (Edoura-Gaena et al., 2007).
The rapid developments in technology have improved those
traditional imaging methods into more effective and accurate ones.
Today, information about microstructure of food products and ingredients can be obtained using various imaging techniques such as
bright-eld, polarizing and uorescence light microscopy, confocal
scanning laser microscopy and electron microscopy. A relatively
new technique, X-ray tomography and more advanced X-ray
microtomography can probe the microstructure of samples noninvasively up to a few millimeters and down to few micrometers
of resolution, the advantages also include observations under
environmental conditions without sample-disturbing preparations
which occur in the uorescence light microscopy and electron

http://dx.doi.org/10.1016/j.jfoodeng.2016.03.008
0260-8774/ 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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M.C. Tan et al. / Journal of Food Engineering 183 (2016) 9e15

microscopy techniques (van Dalen et al., 2003). The X-ray tomography and X-ray microtomography techniques have emerged as
important and useful non-invasive imaging tools to measure the
internal microstructure of cellular food products. X-ray microtomography has been attempted for use to generate the 3D images
of porous rice kernels and whipped cream (van Dalen et al., 2003),
aerated chocolate, mousse, marshmallow and mufns (Lim and
Barigou, 2004), bread crumb (Falcone et al., 2005; Moussawi
et al., 2014), extruded rye bran (Alam et al., 2013), dough (Bellido
et al., 2006), biscuit, breadstick, emulsion and coffee (Laverse
et al., 2012), biopolymer foams (Trater et al., 2005), ice crystal
formation in strawberry (Nurzahida et al., 2010), banana slices

onard et al., 2008), apple tissue (Mendoza et al., 2007), mango
(Le
during ripening (Cantre et al., 2014) and processed meat (Frisullo
et al., 2009) while X-ray tomography has been used to generate
images of extruded starch (Babin et al., 2007), bread crumbs
(Lassoued et al., 2007), cornakes (Chaunier et al., 2007) and fruits
(Rogge et al., 2013).
The objective of this study is to investigate the effects of ultrasound treatment in protein suspensions for making foams to be
used in the baking of cake. The highly aerated foam and baked cake
structure were then assessed via imaging using X-ray microtomography and X-ray tomography techniques. The collected X-ray
microtomography and X-ray tomography images were then
reconstructed into 2D and 3D images for a complete evaluation of
the internal aerated structures.
2. Materials and methods
2.1. Materials
The cake making process uses protein suspensions from protein
powder, i.e. whey protein concentrate (Textrion PROGEL 800, DMV
International, BA Veghel, Netherlands) because it is a protein with
the highest nutritional value (Garca-Garibay et al., 2008) and
contains all of the essential amino acids in higher concentration
compared to vegetable proteins such as soy, corn and wheat gluten
(Recio et al., 2008). The typical composition of the whey protein
concentrate is 80% protein, 6.5% lactose, 6.3% fat, 4.4% ash and 4.9%
moisture with 6.5 pH.
2.2. Cake preparation from ultrasonically treated foam
The aqueous suspensions of whey protein powder at 20% (w/w)
concentration were prepared by dispersing 50 g of dry matter into
200 g of distilled water in a 500 mL beaker and stirrer using a
mechanical stirrer (RW20 DZM.n S2, IKA Works (Asia) Sendirian
Berhad, Malaysia) at 355 rpm for 20 min until homogenous suspensions were obtained. The solution was sonicated with
20 kHze400 W high intensity ultrasound probe (Digital Sonier
Model 450, Branson Ultrasonics Corporation, Danbury, Connecticut,
USA) at 60% amplitude for 25 min. The control sample has no ultrasound treatment and is known as an untreated sample. 250 g of
the treated or untreated whey protein suspension was whipped
into foam at room temperature in a mixer (5K5SSS, Kitchen Aid Inc.,
St. Joseph, Michigan, USA) at 330 rpm for 15 min.
The whipped foam was used for cake making following the
Angel food cake recipe with an adapted formulation given in Table 1
(Yamazaki and Lord, 1971). Sugar was added into the foam during
the extended mixing of time 4 min. Mixing continued for another
4 min at a reduced speed of 160 rpm when pre-mixed salt with
our was added to obtain cake batter. The speed of mixing the cake
batter was increased to 330 rpm during the nal 15 s. 450 g of cake
batter was poured into the cake tin with dimensions showed in
Fig. 1 and baked at 170
C with oven heat levels setting of 20% (top):

Table 1
Angel food cake formulations.
Ingredients

Mass of cake loading (g)

Flour
Sugar
Salt
Protein suspension

100
250
3
250

Total mass

603

Fig. 1. Schematic diagram of hollow baking tin with diameters given at the top, center
and bottom sections.

30% (side): 10% (bottom) for 40 min in an electronic baking oven

(ST-02, Salva Industrial, Spain). The baked cakes were inverted on a
wire rack immediately and cooled for 5 min before removing them
from the tins. The cakes were left in ambient for 1 h for further

mez et al., 2008; Tan et al., 2011) before cake imaging
cooling (Go
analyses.
2.3. Preparation of X-ray microtomography sample holder
Fig. 2a is the schematic diagram of the sample holder designed
specically for the loading of the foam sample with minimal
disturbance of its aerated structure. It was made with Perspex
(polymethyl metacrylate) and placed inside the chamber of the Xray microtomography system. The entire sample holder is separated into three parts, namely the cap, body and stand to ease
sample loading and for cleaning purposes. The cap and stand are
sealed hermetically using O-rings to tight up the closure. For
sample loading, the body with 11 mm inner diameter and 1 mm
wall thickness was gently pressed into the aerated foam, then
plugged on the stand and closed with the cap as showed in Fig. 2b.
2.4. X-ray microtomography and X-ray tomography techniques
Fig. 3 shows the Skyscan 1172 desktop microtomography system
used for scanning images of foam and cake samples. The X-ray
microtomography system includes a chamber where it is kept
closed by a lead shield when the experiment is running to prevent
X-ray beams escaping from the chamber (Nurzahida et al., 2010).
The foam sample loaded in the sample holder was xed securely to
the right position in the chamber. For cake imaging, a small cake
sample was cut into a cube in the dimension of
1.5 cm  1.5 cm  1.5 cm and placed on the 1.5 cm diameter
specimen holder platform. The lead shield was closed and the
scanning operation started after the desired settings were set. The
X-ray beams from the X-ray tube produced by its X-ray source
penetrated into the sample. The sample was rotated on its vertical
axis by a rotational stage over 180
with a step size of 1
to provide
different sampling directions. While the dataset is massively undersampled, there are still insights about the structure which can be

M.C. Tan et al. / Journal of Food Engineering 183 (2016) 9e15

11

Fig. 2. (a) Schematic diagram of X-ray microtomography sample holder and (b) Sample holder with foam lled-in.

Fig. 3. Schematic diagram of X-ray microtomography system.

gained. The images data of line integrals were collected from many
angles by a charge-coupled device (CCD) camera and conveyed to
the computer for construction into a two dimensional image,
known as the tomography process. The settings for foam and cake
samples were same, i.e. with the X-ray tube set at a power of 10 W,
80 kV of voltage and 124 mA of current source. The total acquisition
of images and read-out time was about 11 min for one projection.

The image pixel size was set at 17.2 mm resolution and the camera
pixel size used was 1280  1024 pixels with no lter. A set of at
cross sections of 2D images was obtained after tomographical
reconstruction by using the NRecon software of the Skyscan
system.
The concept of X-ray tomography is similar to the X-ray
microtomography and it was used for larger cake samples (Tan

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M.C. Tan et al. / Journal of Food Engineering 183 (2016) 9e15

Fig. 4. (a) 2D and (b) 3D microtomography images of foam whipped with (1) untreated and (2) treated whey protein suspension.

et al., 2014). The whole cake sample was placed on an adjustable

rotational stage within the X-ray source and detector with a 54 cm
distance between the X-ray source and cake sample and 58 cm
distance between the cake sample and the detector. The cake images were taken every 0.5
around a 360
path. The X-ray tube was
set with 100 kV of voltage and 2 mA of current source. The detector
size was 230  1310 pixels, corresponding to a eld of view of
61  347 mm. Each cake was scanned at the top, center and bottom
layers with 1.5 cm from the center to the top or bottom.
2.5. Reconstruction of 2D and 3D images analysis of foam and cake
samples
The collected images from the X-ray microtomography were
reconstructed into 2D images by NRecon software of Skyscan system and 3D images by using VGStudio MAX 2.2 software. The region desired for volume reconstruct can be set by keying in the
range of top and bottom slices or by dragging the lines on the image
stack displayed at the start page. A middle slice of the image stack
was chosen as a previewing slice together with the data displayed
in a histogram. It is important to choose a slice which goes through
the dense parts of the object to avoid truncation of higher values in
the nal image. The dynamic range of the displayed histogram in
the output page in NRecon software was set as 0.01 to 0.08 to obtain
a contrast image.
VGStudio MAX is the high-end software from the Volume
Graphics range of products for the visualization and analysis of
computer tomography data and it has become a virtual standard
for industrial voxel data analysis (Anonymous, 2013). The 2D

image stack reconstructed by NRecon software was imported

through the directory in VGStudio MAX 2.2 software and mapped
to unsigned 8 bit in ramp mode with X, Y and Z in resolution of
17.2 mm. The region of interest to reconstruct the 3D image was
tuned from the top, right and front projections in the preview
window. The contrast in the image reconstructed was then
adjusted by tuning in the opacity manipulation area, and the
image was set to display in rendering setting of volume renderer
(Scatter HQ). The 2D and 3D images were constructed in a similar
way for both the foam and cake. For whole cake imaging, the X-ray
tomography images of the cake at the top, center and bottom
layers were constructed as 2D images using the Backprojection
software (Tan et al., 2014).
An analysis of bubbles size distribution of foam was performed
on the constructed 3D images from x-ray microtomography using
voids analysis with default algorithm and defect analysis tool by the
VGStudio MAX 2.2 software. The probability threshold was set at
0.5. The number of bubbles at three different size ranges,
0e0.0025 mm3, 0.00251e0.005 mm3 and 0.0051e0.01 mm3 was
averaged from three randomly selected areas of 155  155  155
voxels in each foam sample. An analysis of bubbles size distribution
in the cake was performed on the constructed 2D images from xray tomography using ImageJ software. For each layer, the number
of bubbles at different size ranges, from 0 to 1 mm2, consecutively
ranged at every 1 mm2 until 10 mm2 and above was analyzed at
each quarter at 200  200 pixels. The average was taken from three
layers of cake. A statistical comparison of analysis of variance and
Bonferroni correction post-hoc test on the number of bubbles at
every size range due to ultrasound effect was performed.

M.C. Tan et al. / Journal of Food Engineering 183 (2016) 9e15

13

foam. A layer of reconstructed 2D image is presented in Fig. 4a,

while Fig. 4b shows the constructed 3D images. It is obvious that
the bubbles in the foam formed by treated whey protein suspension in Fig. 4-a2 and 4-b2 are smaller in size and more evenly
distributed than the untreated whey protein foam in Fig. 4-a1 and
4-b1.
An analysis of the 3D images of foam using the defect detection
analysis shows that the overall total number of bubbles in the size
range of 0e0.01 mm3 has increased 45% averagely from 1296 to
1883 after treatment with P < 0.0005 (Fig. 5a). Zooming into that
range, bubbles in the size range of 0e0.00125 mm3 was found to
be the biggest population, which occupied 89% and 91% of the
total bubbles respectively before and after treatment (Fig. 5b). The
number of this biggest population of bubbles has increased 48%
after treatment with P < 0.0005, while the number of
bigger bubbles in size ranges of 0.001251e0.0025 mm3,
0.00251e0.005 mm3 and 0.0051e0.01 mm3 has increased by 17%,
23% and 35% respectively, with P < 0.5. This shows that ultrasound
has increased the number of microbubbles in whipped whey
protein foam, where Bonferroni correction post-hoc test also
indicated that the number of bubbles in size range 0e0.01 mm3
after treatment were signicantly higher than control. The cavitation effect of ultrasonication which causes implosion of the
bigger bubbles in whey protein suspension (Suslick, 1988; Soria
and Villamiel, 2010) to implode giving a higher number of
microbubbles.

3.2. Aerated cake structure

Fig. 5. (a) Cumulative distribution and (b) frequency distribution of bubbles at various
size ranges in ultrasonically treated and untreated aerated whey protein foams.

The images of the top, center and bottom layers of an uncut cake
scanned using X-ray tomography are shown in Fig. 6 while Fig. 7 are

Fig. 6. 2D tomography images of cake baked with (a) untreated and (b) treated whey protein suspension at the (1) top, (2) center, and (3) bottom layers.

3. Results and discussion

3.1. Aerated foam structure
The X-ray microtomography technique was used to examine the
changes in the structure of bubbles in the ultrasonically treated

2D and 3D images scanned using X-ray microtomography from a

smaller cube sample. From both gures, which show the whole
cake and a section of a cake, it is clear that the bubbles in the control
cake are not as evenly distributed as the treated. The air cells seem
to coalesce and form bigger air cells. The structure of the control
cake is also coarser with ring defects throughout the cake layers

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M.C. Tan et al. / Journal of Food Engineering 183 (2016) 9e15

Fig. 7. (a) 2D and (b) 3D microtomography images of cake baked with (1) untreated and (2) treated whey protein suspension.

observed by its brighter color in Fig. 6a. This suggests that ultrasonic treatment on protein suspension has helped to minimize and
distribute the bubbles more evenly. Fig. 8 shows the analysis of
bubbles in terms of cumulative and frequency distribution at
various size ranges. The overall total number of bubbles in the size
range of 0e10 mm2 has increased 32% from 1641 to 2196 after
treatment with P < 0.005 (Fig. 8a). The bubbles in the size range of
0e1 mm2 are the largest in population, which occupied 65% and
63% of the total bubbles, respectively before and after treatment
(Fig. 8b). The average number of this group of bubbles has increased
29% after treatment with P < 0.05, while the average number of
bubbles in other size ranges of 1.001e2 mm2, 2.001e3 mm2 and
3.001e4 mm2 has increased 69% (P < 0.005), 51% (P < 0.05) and 48%
(P < 0.05) respectively. The number of bubbles with area larger than
10 mm2 has decreased 49% after treatment with P < 0.1. The Bonferroni correction post-hoc test indicated that ultrasound treatment signicantly increased the number of bubbles in size ranges
of 0e10 mm2, 0e1 mm2 and 1.001e2 mm2 when compared with
untreated sample.

4. Conclusion

Fig. 8. (a) Cumulative distribution and (b) frequency distribution of bubbles at various
size ranges in cakes formulated by whey protein foam with and without ultrasound.

X-ray microtomography can visualize and differentiate the effects of ultrasound treated whey protein foams and cakes made
from it by its aerated structure in terms of bubbles attributes, while
X-ray tomography visualized the whole cake structure nondestructively. With the defect detection analysis of VGStudio
MAX 2.2 and ImageJ software, both tomography techniques have
quantitatively indicated that ultrasound treatment has increased
the number of microbubbles in foam and cake systems with more
even distributions.

M.C. Tan et al. / Journal of Food Engineering 183 (2016) 9e15

Acknowledgements
This work was supported by the Ministry of Education (MOE),
Malaysia, through the Fundamental Research Grant Scheme (FRGS/
2/2013/TK05/UPM/02/5). The authors would like to thank Alex
Corporation (M) Sendirian Berhad, Selangor (Malaysia) for
providing VGStudio MAX 2.2 software for this research.
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