Food Hydrocolloids 30 (2013) 495e503

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

The use of microbial transglutaminase and soy protein isolate to enhance

retention of capsaicin in capsaicin-enriched layered noodles
Ling-Yun Li, Azhar Mat Easa*, Min-Tze Liong, Thuan-Chew Tan, Wan-Teck Foo
Food Technology Division, School of Industrial Technology, University Sains Malaysia, 11800 USM, Minden, Penang, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Specialty layered noodles (LN) were prepared by sandwiching a capsaicin-enriched dough (CED)
Received 24 April 2012 between two gastro-resistant dough layers made up of wheat flour, soy protein isolate (SPI) and
Accepted 26 July 2012 microbial transglutaminase (MTG) at 0.5 (0.5MTG LN), 1.0 (1.0MTG LN) and 1.5 (1.5MYG LN) g/100 g of
wheat-SPI flour. The textural, tensile and structural breakdown properties, capsaicin retention, micro-
Keywords: structures and the sensory characteristics of cooked LN were evaluated. Compared to other LN, 1.5MTG
Capsaicin retention
LN exhibited the highest textural and tensile parameters, highest capsaicin retention, densest structure
Layered noodles
and was the most difficult to breakdown. The sensory quality of all LN was acceptable, even though it was
Chili powder-enriched dough
Structure breakdown
the Control LN (prepared without SPI and MTG) that scored the highest acceptability. In general,
Microbial transglutaminase increasing the level of MTG in the sandwiching dough layers of MTG LN reduced the release of capsaicin
in simulated mouth, gastric and intestinal conditions, and these results could be due to increased protein
cross-linking.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction products that can deliver capsaicin in a controlled manner. As such,

Capsaicin is the predominate substance responsible for the hot
character of chili peppers, which is known to exert the function of
anti-obesity through increasing the energy expenditure and
decreasing the appetite. After ingestion of capsaicin, the energy
metabolism is enhanced via the activation of sympathetic nervous
system (Kang et al., 2010; Westerterp-Plantenga, Diepvens, Jossen,
Berube-Parent, & Tremblay, 2006). This causes an increased secre-
tion of catecholamine from the adrenal medulla (Yoshioka et al.,
1999) and the expression of certain fat degrading proteins (Joo,
Kim, Choi, & Yun, 2009). The feeling of satiety is promoted and
the energy intake is reduced (Reinbach, Smeets, Martinussen,
Moller, & Westerterp-Plantenga, 2009; Westerterp-Plantenga,
Smeets, & Lejeune, 2005). However, the application of capsaicin
in the food industry for treating or preventing obesity is limited
because the effective dose of capsaicin application in food for
weight control (10e30 mg) (Reinbach, Martinussen, & Moller, 2010;
Yoshioka et al., 2004; Yoshioka et al., 1999) is much higher than the
limitation of intake for normal people (1e10 mg) (Craft & Porreca,
1992; Yoshioka et al., 2004; Yoshioka, St-Pierre, Suzuki, & Trem-
blay, 1998). A product that is directly incorporated with capsaicin
may be too hot for most consumers to consume. This calls for
* Corresponding author. Tel.: þ60 4 653 3888x222; fax: þ60 4 657 3678.
E-mail address: azhar@usm.my (A.M. Easa).
some cutting-edge companies have already introduced delivery
systems that are based on encapsulation technologies using
capsules that are designed to release capsaicin in the intestine, but
not in the stomach, to reduce irritation and pain. While this
approach is suitable for nutraceutical applications, more innovation
is desirable in order to achieve similar functions in food products.
“Layered Noodle” (LN) is a specialty noodle product that is
prepared by sandwiching a capsaicin-enriched dough (CED) layer
with two gastro-resistant dough layers, which might be a good
candidate to carry the functions of capsaicin (Li, Liong, & Easa,
2011). This idea is possible because many Asian consumers
choose to slurp the noodle rather than chewing them extensively
during eating. Because a quantity of capsaicin is retained in the
middle layer of the LN, consumers should experience reduced pain
in the mouth. The release of capsaicin in the stomach and intestine
will be expected to be controlled thus reducing irritation (Li et al.,
2011).
To achieve the controlled-release properties, the overall struc-
ture of the LN should be strong and resistant to breakdown, in order
for it to retain capsaicin and release it in a controlled manner.
Microbial transglutaminase (MTG) is a thermal stable enzyme,
which is isolated from Streptoverticillium sp. and legally to be
assessed as a processing aid food enzyme. MTG has great potential
to be used in the food industry as an agent to improve firmness,
elasticity and water-holding capacity of food through the mild
enzyme reactions (Aalami & Leelavathi, 2008; Basman, Köksel, &

0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodhyd.2012.07.017
496 L.-Y. Li et al. / Food Hydrocolloids 30 (2013) 495e503
Arli, 2006). After incorporating MTG, high molecular weight poly-
mers were formed through ε-(g-Glu)Lys bonds, which reinforce the
network structure and modified the properties of the products
(Motoki & Seguro, 1998). The effects of MTG can be obtained in
various kinds of noodles and pasta, but the degree of change in the
physical properties varies with the sorts of products. By adjusting
the amount of MTG, the textural qualities of noodles/pasta can be
controlled (Kuraishi, Yamazaki, & Susa, 2001). Furthermore, since
the cross-links introduced by MTG are heat-stable, the modified
structures of noodle are retained for longer even after cooking.
Therefore, the use of MTG at different levels might be a safe and
efficient way to develop the gastro-resistant properties of LN
enriched with capsaicin.
Soy protein isolate (SPI) has been broadly used as the functional
ingredient and texture enhancer in the food industry (Tang, Wu,
Chen, & Yang, 2006). SPI that is treated with additional food-
based protein cross-linking enzyme, such as MTG, has been
shown to enhance the network structure and gastro-resistant
properties of SPI gels and noodles (Gan, Cheng, & Easa, 2008;
Gan, Ong, Wong, & Easa, 2009; Yeoh, Alkarkhi, Ramli, & Easa, 2011).
Thus, MTG treated SPI that is incorporated with LN could function
as the gastro-resistant layers that enhance the capsaicin retention
of LN.
We have described the basic preparation of the so-called layered
noodles (Li et al., 2011). Other than this, there is no report in the
literature that describes the preparation or evaluation of layered
noodles for controlling the release of capsaicin. Thus, the objective
of this study was to evaluate the textural and breakdown proper-
ties, capsaicin retention, microstructures and sensory characteris-
tics of the layered noodles as a function of MTG concentration.
2. Materials and methods
2.1. Materials
The basic ingredients for noodle preparation (i.e., wheat flour,
kansui reagent and chili powder) were purchased from a local
supermarket (Tesco Extra, Penang, Malaysia). The food grade soy
protein isolate (SPI) was purchased from Sim Company Sdn. Bhd.,
Penang. Microbial Transglutaminase (MTG) was purchased from
Ajinomoto Co., Inc. (Tokyo, Japan). The acetonitrile and capsaicin
standard reagents used for capsaicin content determination were
HPLC grade and purchased from Fluka, Switzerland. Other chem-
icals and reagents used for preparation of the artificial saliva and
analysis were of analytical grade. Xanthan gum and a-amylase
(type VI-B from Porcine Pancreas) were purchased from Sigma
Chemical Co. (St. Louis, US). Sodium hydrogen carbonate, sodium
chloride, potassium chloride, calcium chloride dehydrate and di-
potassium hydrogen phosphate (anhydrous) were purchased from
Systerm Sdn. Bhd. (Selangor, Malaysia).
2.2. Dough and layered noodle (LN) preparation
2.2.1. Dough sheets preparation
LN was prepared by sandwiching the CED sheet with two
gastro-resistant dough sheets. The CED formulation consisted of
100 g of mixed flour which including the equal parts of wheat flour
and chili powder, 40 g of distilled water, 1 g of salt and 1 g of kansui
reagent. The mixed flour used for making gastro-resistant dough
were: (a) 100 g of wheat flour (Control LN); (b) 95 g of wheat
flour þ 5 g of SPI þ 0.5 g of MTG (0.5MTG LN); (c) 95 g of wheat
flour þ 5 g of SPI þ 1.0 g of MTG (1.0MTG LN) and (d) 95 g of wheat
flour þ 5 g of SPI þ 1.5 g of MTG (1.5MTG LN). Distilled water (50 g),
Salt (1 g) and kansui reagent (1 g) were included in the formulation
for preparing the gastro-resistant dough. Powder materials for
dough preparation were initially mixed with a mixer (KitchenAid,
USA) for 2 min. Kansui reagent and salt were dissolved in distilled
water and added to the flour mixture. The suspension was mixed
with speed 1 (60 rpm) for 8 min and then sheeted on a noodle
machine (HP-150F, Nanjing Hope Co. Ltd., Jiangsu, China) with an
initial gap setting at width 1. The dough sheets were subjected to
the sheeting process for two more times, with each time folding the
dough sheet longitudinally to ensure homogeneity. Subsequently,
the dough sheets underwent sheeting process (without folding in
between gap setting) with gap setting at width from 2 to 6, until the
final sheet thickness reached around 1.7 mm.
2.2.2. LN preparation
LN making involved the sandwiching of the CED sheet with two
sandwiching dough layers, followed by another sheeting process
but without the folding step in between gap setting. The sheeting
was carried out using the same noodle machine but with gap
setting set at width 4, 5 and 6. Then the obtained dough sheets
were slit with the same noodle machine and the noodles were
incubated at 40  C for 4 h. After incubation, the layered noodles
were steamed for 2 min in a steamer and then allowed to shape and
dry at 60  C for 5 h using a hot air dryer (Afos, Model Mini,
No.CK80520, England) before being stored in a plastic bag at room
temperature prior to cooking and further analysis. Unless other-
wise stated, the layered noodles were cooked in boiling water at
a ratio of 1:50 (one part of noodle in 50 parts of water) for 7.5 min
and then cooled at room temperature before analysis.
2.3. Texture and tensile analysis
The textural properties of LN were evaluated using texture
profile analysis (TPA) and tensile tests. Texture Analyzer (TA-TX2
model, Stable Micro Systems, Surrey, UK) fitted with a 30 kg load
cell was used for both of these measurements. The calibration was
performed using a 5 kg load cell before analysis.
TPA was performed with a cylindrical probe (diameter 35 mm).
Two strands of cooked noodle samples (length 70 mm) were placed
parallel on the middle of the compression plate, and two contin-
uous compressions were carried out. The instrument setting were:
Mode, Measure force in compression; Trigger type, auto e 20 g;
Pre-test speed, 2.0 mm/s; Test speed, 0.8 mm/s; Post-test speed,
0.8 mm/s; Strain, 75%; Interval between two compression, 1 s (Lu,
Guo, & Zhang, 2009). Six textural parameters were obtained from
the force (N) e distance (mm) curve: hardness (N), adhesiveness (N.
sec), chewiness, resilience, springiness and cohesiveness.
The tensile test of cooked layered noodles was carried out by an
A/SPR probe. Cooked noodles were positioned on the rig arm slots
and the load cell was raised until the noodle strand ruptured. Rig
calibration with a return trigger path at 15 mm was performed
before the analysis. The measurement parameters were: Mode,
measure force in tension; Option, return to start; Pre-test speed,
3.0 mm/s; Test speed, 3.0 mm/s; Post-test speed, 5.0 mm/s;
Distance, 100 mm. The maximum force and breaking time were
recorded from the force (N)edisplacement (mm) curve. The tensile
strength and elasticity were calculated based on the methods
described by Gan et al. (2009).
2.4. Structural breakdown analysis
Structural breakdown analysis was processed using a multiple
extrusion cell (MEC) attached to a texture analyzer (TA-TX2 model,
Stable Micro Systems, Surrey, UK) fitted with a 30 kg load cell (Foo,
Yew, Liong, & Azhar, 2011). The MEC consists of a cylindrical inner
sample vessel (diameter 25 mm, length 105 mm, thickness 1 mm).
The sample vessel was fitted inside a water jacketed cylindrical
 L.-Y. Li et al. / Food Hydrocolloids 30 (2013) 495e503
tube attached centrally to the base of the texture analyzer. A
circular plate piston (diameter 22 mm, thickness 3 mm) with six
holes (diameter 6 mm) was attached via a thin rod to the texture
analyzer through a cell lid (screw cap). Before running each
measurement, the force was “tared” to remove the mass of the
piston. The temperature of MEC was maintained at 37  C by using
a digital heating circulator (Model Protech HC-10, Tech-Lab Scien-
tific Sdn. Bhd., Selangor, Malaysia). Twenty grams of the noodle
samples followed by 10 mL of the artificial saliva were added into
the sample vessel. The composition of the artificial saliva was
NaHCO3 (5.208 g), K2HPO4 (1.241 g), NaCl (0.877 g), KCl (0.447 g),
CaCl2$2H2O (0.441 g), xanthan gum (0.920 g), and a-amylase
(2,000,000 U) in 1 L of distilled water. The pH of the artificial saliva
was adjusted to 7.0 by 0.1 M of HCl (Boland, Buhr, Giannouli, & van
Ruth, 2004).
During the test, the geometry rod moved up and down with the
following instrument setting: Mode, measure force in compression;
Option, cycle until count; Test-Speed, 10 mm/s; Post-Test Speed,
5 mm/s; Distance, 95 mm; Count, 20 cycles; Data Acquisition Rate,
2 pps. A force (N) e time (s) graph was generated and the data was
fitted into a single exponential decay equation,
 
n
WðnÞ ¼ winf þ w1 exp
n1
where W(n) is the work during each extrusion cycle (n), winf is the
work per extrusion after a large (infinite) number of extrusions, w1
is a measure for the amount and the strength of noodle structure
that has been broken down and n1 determines the decay rate of the
work per extrusion with an increasing number of extrusions.
2.5. Determination of capsaicin content
The cooked noodle samples were mixed with a certain amount
of acetonitrile and incubated in a water bath with gentle agitation
at 82  C for 4 h. The extract aliquots were cleaned up by passing
through 4.5 mm membrane filter prior to HPLC analysis (Pino et al.,
2007). The chromatographic system was coupled with a computer
equipped with the Millenium 32 chormatograph manager software
package, a HPLC pump (Waters 2960) and a photodiode-array
detector (Waters 996 PDA detector). The sample (20 mL) was
injected onto a reverse phase column (Waters Atlantis C18 column,
3 mm, 100 mm  4.6 mm) and eluted with a mobile phase con-
taining acetonitrile/water/acetic acid (50:50:1) at a flow rate of
1.0 mL/min. The capsaicin content was detected at 280 nm based on
a calibration curve with capsaicin standard reagent (Fluka,
Switzerland).
2.6. Capsaicin retention of LN
The percentage release of capsaicin from LN or capsaicin
retention of LN was determined under simulated mouth, gastric
and intestinal conditions.
For the simulation of mouth conditions, the MEC method
described in Section 2.4 was performed to mimic human mastica-
tion. The circulated water at temperature 37  C was applied
surrounding the sample vessel. The sample was poured out of the
sample vessel after 1, 3, 5 and 7 cycles of extrusion and followed by
rinsing with 200 mL of the distilled water to remove the released
capsaicin and artificial saliva. The capsaicin content in the rinsed
noodle was extracted with 20 mL of acetonitrile.
For the simulated gastric and intestinal conditions, the cooked
layered noodles (2 g) were placed in a 50 mL capped test tube. For
gastric condition (pH 1.2), 20 mL of 0.1 N HCl (pH 1.2) solution was
added. For intestinal condition (pH 7.4), 20 mL of 0.05 M phosphate
497
buffer (pH 7.4) solution was added. All the test tubes were incu-
bated at 37  C. For each pH condition, a test tube was removed at
time intervals of 30, 60, 90, 120, 150 and 180 min. The buffer
solution was drained and the samples were rinsed with distilled
water for three times. The capsaicin contents in the rinsed noodles
were extracted with 10 mL of acetonitrile.
The concentration of capsaicin was determined using HPLC
method as described in Section 2.5. The release percentage was
expressed using the following equation:
C0  Ct
Releaseð%Þ ¼  100 (2)
C0
where C0 is the initial capsaicin content in the noodle and Ct is the
capsaicin content remaining in the noodles at designated time t.
Noodles with lower % release are considered high in capsaicin-
retention.
2.7. Scanning electron microscope
The microstructures of the LN were examined by using Scanning
Electron Microscope (Leo Supra 50, Oberkochem, Germany). The
noodle samples were dried with a freeze drier (Freezone 12, Lab-
(1)
conco Corporation, USA) before SEM testing. The dried noodle
samples were fractured by folding and followed by mounting on
a specimen stubs with fractured side faced up. The mounted noodle
samples were coated with PtePd by using a sputter coater (Polaron
SC515, Fisons Instruments, UK) and the cross-section of the layered
noodles were observed with SEM at 50x magnification.
2.8. Sensory evaluation
Four types of cooked layered noodles were prepared for sensory
evaluation by using the 9-points hedonic scale (1 ¼ Dislike
extremely, 5 ¼ Neither like or dislike, 9 ¼ Like extremely) (Yadav,
Yadav, & Kumar, 2011). Samples were cut into 5 cm pieces and
presented individually in a randomized order. The sensory panel-
ists comprised of 20 students and staffs (11 females and 9 males) of
the Food Technology Division, Universiti Sains Malaysia, Penang,
Malaysia. Pungent food was avoided before the test. Drinking water
and full cream milk were used to rinse the mouth and remove the
spicy sensation between samples. The attributes evaluated
included appearance, smoothness, odor, firmness, springiness,
chewiness, flavor and overall acceptability.
2.9. Statistical analysis
Results were expressed as mean  standard deviation of sepa-
rate determinations. Comparison of means was performed by one-
way analysis of variance (ANOVA) followed by Duncan’s test.
Statistical analyses were run using Statistical Package for Social
Science for Windows, version 17.0 (P < 0.05). (SPSS Institute Inc.,
Cary NC).
3. Results and discussion
3.1. Description of the LN samples
Typically LN was wide and straight with a flat surface. The cross-
section of cooked LN is shown in Fig. 1 in which the reddish CED
layer is sandwiched by two pale-yellow gastro-resistant dough
layers. The surfaces of all LN were smooth and the three layers
remained intact after cooking. The LN containing MTG (0.5MTG LN,
1.0MTG LN and 1.5MTG LN) were similar in appearance to the
Control LN except they were more slippery on the surface and
498 L.-Y. Li et al. / Food Hydrocolloids 30 (2013) 495e503
Fig. 1. Cross-section view of a typical LN displaying the capsaicin-enriched dough
(CED) being sandwiched by two layers of gastro-resistant dough.
harder in texture. The thickness of the noodles ranged from 1.9 to
2.3 mm, while the length of each noodle strand was longer than
20 cm, with a width between 7.5 and 8.5 mm. Such a dimension
was designed to ease handling during tensile measurement (Yeoh
et al., 2011).
From the preliminary tests, if a high amount of capsaicin (50% w/
w) was directly incorporated into a conventional noodle without
layers, the loss during preparation was almost 61%. However, the
percentage loss was reduced to almost 40% if CED is sandwiched by
two control layers made from wheat flour dough. Thus, even
without the using of gastro-resistant layers, the capsaicin
percentage loss during preparation was reduced by using the
layering system. The capsaicin content of the chili powder used for
the preparation of CED was 2.0 mg/g (w31,384 Scoville Heat Value,
SHV) and the final capsaicin content of the LN samples ranged
between 12 and 17 mg/100 g of dried LN.
3.2. The noodle textural properties
The TPA attempts to assess noodle’s general texture through
mimicking the mastication process by measuring the force
responses of two repetitive compressions on the same noodle site.
The TPA parameters of the cooked LN are shown in Table 1. The
1.5MTG LN was the hardest and may require the highest level of
force to chew. The values of hardness of the noodles were in the
order: 1.5MTG LN > 1.0MTG LN > 0.5MTG LN > Control LN.
Chewiness represents the amount of energy needed to disintegrate
food for swallowing, and the addition MTG have a positive rela-
tionship with chewiness parameter. The increase in hardness and
chewiness with MTG treatment was related to the fact that the MTG
cross-linking formed through ε-(g-Glu)Lys bonds yields a strong
and tight protein network trapped the starch granules of the
sandwiching dough layers, limiting the excessive water uptake
during cooking (Aalami & Leelavathi, 2008; Choy, Hughes, & Small,
2010; Yeoh et al., 2011) and inhibiting the softening of texture
caused by the extra swelling of starch granules. Similar results have
been reported by other authors who indicated that MTG has the
potential to increase the hardness and chewiness of noodles (Foo
et al., 2011; Wu & Corke, 2005).
Adhesiveness is more of a surface characteristic and depends on
a combined effect of adhesive and cohesive forces, viscosity and
Table 1
TPA and mechanical parameters for cooked LN.
Sample name Textural properties
Hardness (N) Adhesiveness (N s) Chewiness (kPa)
Control LN 113.86  4.43a 54.93  7.59b 59.47  2.78ab
0.5MTG LN 123.26  9.26b 38.67  6.32a 57.37  4.61a
1.0MTG LN 129.47  10.33b 41.09  12.13a 62.72  5.72bc
1.5MTG LN 138.70  8.32c 50.89  8.34b 64.63  5.22c
Results are presented as mean values  standard deviation of 10 replicates.
Different letters superscripted on the results indicate significant difference (P < 0.05) between samples for each parameter tested.
Control LN, represents LN prepared with sandwiching layers made up of wheat flour; 0.5MTG LN, 1.0MTG LN and 1.5MTG LN represent LN prepared with sandwiching dough
layers prepared with SPI, wheat flour and 0.5, 1.0 and 1.5% of MTG respectively.
viscoelasticity (Adhikari, Howes, Bhandari, & Truong, 2001). The
Control LN showed higher values of adhesiveness compared to
MTG LN. The adhesiveness values decreased when SPI was used as
the sandwiching layers, however the value significantly increased
with the additional MTG level at 1.5 g/100 g (1.5MTG LN). This
might be due to the new formed covalent cross-links produced
more compact and inflexible structures and trapped the starch
granules. Thus, less starch granules will leach out to the surface of
the layered noodle and thereby reduce the adhesiveness
(Limroongreungrat & Huang, 2007; Sissons, Aravind, & Fellows,
2010). However, a much denser protein network formed with the
increasing of MTG content, the starch granules might be squeezed
out of the network and caused the raising of adhesiveness (Basman,
Köksel, & Ng, 2002; Bonet, Cabollero, Gómez, & Rosell, 2005).
Springiness and resiliency parameters can be used to assess the
ability of the noodle to regain its original shape after compression,
while cohesiveness is the ability of a material to stick to itself. The
Control LN had significantly (P < 0.05) higher springiness
compared to MTG LN, even though other parameters were similar
in values suggesting that MTG incorporation in the sandwiching
layers had little influence on these parameters.
The incorporation of SPI and MTG in the sandwiching layers had
a positive influence on the tensile strength of LN (Table 1).
Increasing the amount of MTG caused an increase in the tensile
strength of LN. 1.0MTG and 1.5MTG LN had significantly higher
(P < 0.05) tensile strength than 0.5MTG LN, and the lowest tensile
strength was shown by the Control LN. The ability of SPI to form gel
enhanced the network formation and the MTG further improved
the network density of the noodles by introducing more cross-
linkings (Gan et al., 2009) in the protein network within the
sandwiching layers. The elasticity of the MTG LN was significantly
(P < 0.05) higher than Control LN, but was similar among the three
MTG LN samples. The Control LN structure is mainly held by
hydrated gluten, which is capable of contributing to the elastic
behavior. During cooking, the gluten network starts to degrade
progressively due to the breakdown of the intermolecular bonds of
starch molecules in the presence of water and heat (Bache &
Donald, 1998; Sozer, Dalgic, & Kaya, 2007). Since the cross-linked
LN formed a much denser network around the starch granules,
which may have limited water up-take and restricted the diffusion
of starch, they suffer least structural degradation (Basman et al.,
2006).
3.3. Structural breakdown properties
Structural breakdown characteristics are very important for the
capsaicin-enriched LN because they have a direct relationship with
the capsaicin retention. A structure that is difficult to breakdown is
expected to retain higher capsaicin content. The curve of force as
a function of time representing the breakdown of the Control LN
during MEC analysis is shown in Fig. 2a. As the piston moved
downward, the increase in resistance force was recorded as positive
Mechanical properties
Resilience Springiness Cohesiveness Tensile strength (kPa) Elasticity (kPa)
0.31  0.01b 0.89  0.02b 0.59  0.01b 65.34  4.67a 22.70  2.42a
0.29  0.01a 0.84  0.04a 0.56  0.02a 71.03  1.74b 28.61  2.02b
0.31  0.02b 0.85  0.03a 0.57  0.02b 79.82  3.24c 29.84  3.17b
0.30  0.02ab 0.84  0.02a 0.56  0.01a 79.35  3.35c 25.98  4.40b
 L.-Y. Li et al. / Food Hydrocolloids 30 (2013) 495e503 499

Fig. 2. (a) Curve of force as a function of time representing the breakdown of the control LN during MEC breakdown analysis (b) Breakdown of LN in the MEC; control LN (C),
0.5MTG LN (O), 1.0MTG LN (-) and 1.5MTG LN (,). Control LN, represents LN prepared with sandwiching layers made up of wheat flour; 0.5MTG LN, 1.0MTG LN and 1.5MTG LN
represent LN prepared with sandwiching dough layers prepared with SPI, wheat flour and 0.5, 1.0 and 1.5% of MTG respectively.

values. The change in direction of piston movement caused a sharp
decrease in the force value and the forces recorded during upward
extrusion were marked as negative values (Foo et al., 2011). These
downward and upward extrusion movements together formed one
complete extrusion cycle and 20 extrusion cycles were performed
to observe the structural breakdown patterns of the LN. The area
under each downward extrusion movement represents the amount
of work needed to breakdown the intact noodle.
The force values of each sample were fitted to a single expo-
nential decay function (Eq. (1)) and plotted (Fig. 2b). The work done
during the first extrusion cycle (w1) represents the amount of work
required to breakdown the intact noodle, was in the order: 1.5MTG
LN > 1.0MTG LN > 0.5MTG LN > Control LN. From the second
extrusion, 1.5MTG LN and 1.0MTG LN depicted similar breakdown
work that was the highest among all samples, followed by that of
0.5MTG LN and Control LN. After a number of extrusions (winf), no
500 L.-Y. Li et al. / Food Hydrocolloids 30 (2013) 495e503
structural element was further broken down. The energy was used
to force the material through the holes in the plunger, which can be
taken as the viscosity of the sample at that particular moment.
The meaningful parameters that were generated from the decay
curves are shown in Table 2. A higher value of w1 reflected more
works needed to deform and break the structures. The significant
differences (P < 0.05) existed between all the samples. The w1
results were consistent with the hardness trend of the TPA, i.e.
more work was needed to breakdown the hardest structure. The
highest winf value was indicated by Control LN, implying that the
Control LN had a sticky behavior. That might be due to the structure
of Control LN that was not as dense as the other samples containing
SPI and MTG cross-linking occurring in the sandwiching layers. The
addition of artificial saliva had accelerated the degradation of
starch. It is probable that the number of starch granules leaching
out onto the noodle’s surface and digested by the enzyme was
higher in the Control LN than in the MTG LN. However, since the
MTG LN samples exhibited similar winf values, it could be that the
MTG LN were ruptured in similar conditions regardless of the level
of MTG applied.
The parameter n1 determines the breakdown rate with
increasing number of extrusions. Lower n1 reflects a faster break-
down rate, which showed that sample was relatively easy to be
ruptured. Control LN showed significantly lower n1 value (P < 0.05)
than that of any of the MTG LN, suggesting that the breakdown
rates of MTG LN were slower than the Control LN. This might be due
to the highly dense structure of MTG LN as a result of protein cross-
linking (Bellido & Hatcher, 2011; Wu & Corke, 2005) in the sand-
wiching layers. However, there was no significant difference
(P > 0.05) in the n1 values between MTG LN samples.
3.4. Capsaicin retention
The percentage of capsaicin released from the LN was influenced
by the breakdown of the structure, the extrusion conditions and the
pH environments (Sanz & Luyten, 2006; Southon & Faulks, 2003).
Three conditions were simulated for evaluation of the capsaicin
release characteristics in the mouth, gastric and intestinal
conditions.
3.4.1. Simulated mouth condition
The % release of capsaicin as a function of time representing the
capsaicin retention in human mouth is shown in Fig. 3. This was
generated by extrusion with the average chewing speed of a typical
human (Dong, Puckett, & Dawes, 1995). After the first extrusion
cycle, the capsaicin release percentage of the cooked LN samples
was w30, 14, 5 and 0% for Control LN, 0.5MTG LN, 1.0MTG LN and
1.5MTG LN, respectively. In all cases, the capsaicin release
percentages were increased with the increasing extrusion cycles
from 1 to 7. After the 7th cycle, w40, 30, 25 and 20% of the capsaicin
Table 2
MEC parameters for different types of LN.
Noodle formulations w1 (J) winf (J) n1 (J/cycle)
Control LN 1.54  0.25a 0.25  0.02b 0.35  0.04a
0.5MTG LN 2.01  0.28b 0.11  0.02a 0.50  0.08b
1.0MTG LN 2.45  0.10c 0.10  0.01a 0.50  0.07b
1.5MTG LN 2.88  0.11d 0.09  0.07a 0.50  0.04b
Results are presented as mean values  standard deviation of 4 replicates.
Different letters superscripted on the results indicate significant difference
(P < 0.05) between each parameter tested.
Control LN, represents LN prepared with sandwiching layers made up of wheat
flour; 0.5MTG LN, 1.0MTG LN and 1.5MTG LN represent LN prepared with sand-
wiching dough layers prepared with SPI, wheat flour and 0.5, 1.0 and 1.5% of MTG
respectively.
Fig. 3. Capsaicin (%) release profile of LN after breakdown in MEC vessel during 1, 3, 5
and 7 cycles (38, 114, 190 and 266 s, respectively): Control LN (-), 0.5MTG LN (:),
1.0MTG LN (,) and 1.5MTG LN (6). Control LN, represents LN prepared with sand-
wiching layers made up of wheat flour. 0.5MTG LN, 1.0MTG LN and 1.5MTG LN
represent LN prepared with sandwiching dough layers prepared with SPI, wheat flour
and 0.5, 1.0 and 1.5% of MTG respectively. Each analysis was carried out on an indi-
vidual extract. Error bars indicate mean  standard deviations of 3 replicates.
in Control LN, 0.5MTG LN, 1.0MTG LN and 1.5MTG LN respectively
were released from the LN. Overall, the capsaicin release
percentage was in the order: Control LN > 0.5MTG LN > 1.0MTG
LN > 1.5MTG LN. This ranking implies that sandwiching the CED
with wheat flour/SPI layers with increasing amount of MTG did
have an effect on the capsaicin retention.
The capsaicin concentrations of the LN ranged between 12 and
17 mg/100 g before extrusion, which was already beyond the
tolerance of many people (1e10 mg). If all the capsaicin were
released at the same time, it would be too hot for most people to
eat. Yoshioka et al. (2004) investigated that the effect of capsaicin
on anti-obesity behavior occurs after passing through the mouth.
Thus, the less capsaicin released in the mouth, the more dose will
be released in the gastrointestinal tract to exert its function. It
might be hypothesized that with the eating styles of Asian who
tend to slurp the noodles with minimum chewing, MTG LN will
result in less capsaicin being released in the mouth. This idea needs
investigation. After the first extrusion, the release dose of capsaicin
in the mouth were 5.0, 3.2, 1.2 and 0.7 mg/100 g for Control LN,
0.5MTG LN, 1.0MTG LN and 1.5MTG LN, respectively. Even though,
consumers chose to chew the noodle extensively, there was only
3.2 mg/100 g of capsaicin released in the mouth for 1.5MTG LN after
7 cycles extrusion as compared to the 7.2 mg/100 g of that from
Control LN.
3.4.2. Simulated gastric and intestinal conditions
The capsaicin release percentages of LN under simulated gastric
and intestinal conditions are shown in Fig. 4. In all cases, a faster
release of capsaicin occurred in gastric condition as compared to
the intestinal condition. In the former, the capsaicin retention of the
LN was similar between the Control LN, 0.5MTG LN and 1.0MTG LN
samples during the first 90 min of incubation, which might be due
to the rapid breakdown of LN structure at low pH. However, after
90 min, the 1.0MTG LN started to exhibit a relatively slower release
percentage. In contrast to other LN, the 1.5MTG LN showed much
lower release of capsaicin (better capsaicin retention) during the
3 h period of gastric incubation. It could be that the cross-linked
structure of 1.5MTG LN was strong enough to inhibit the rapid
structural breakdown in gastric condition.
Fig. 4 also shows release percentage of LN under intestinal
conditions. Compared with Control LN, the MTG LN showed a lower
release percentage of capsaicin throughout incubation. The
percentage release of capsaicin from all MTG LN was quite similar in
 L.-Y. Li et al. / Food Hydrocolloids 30 (2013) 495e503 501

from 1.0MTG LN and w12% released from 1.5MTG LN. Even so, the
released capsaicin percentages of MTG LN were still lower than the
Control LN (44%).

3.5. Microstructure of LN

The SEM micrographs of the cross-section of Control LN before

Fig. 4. Capsaicin (%) release profile of LN during incubation at different pH conditions:
Control-Gas ( ), 0.5MTG-Gas ( ), 1.0MTG-Gas ( ), 1.5MTG-Gas
( ), Control-Int ( ), 0.5MTG-Int ( ), 1.0MTG-Int ( ), 1.5MTG-Int
( ). Control, represents LN prepared with sandwiching layers made up of
wheat flour. 0.5MTG, 1.0MTG and 1.5MTG represent LN prepared with sandwiching
dough layers prepared with SPI, wheat flour and 0.5, 1.0 and 1.5% of MTG respectively.
Gas and Int represent gastric and intestinal conditions, respectively. Each analysis was
carried out on an individual extract. Error bars indicate mean  standard deviations of
2 replicates.
the first 120 min of incubation. After 120 min, the 0.5MTG LN
started to show relatively faster release of capsaicin than 1.0MTG
LN and 1.5MTG LN. After 180 min of incubation, w32% of capsaicin
was released from the 0.5MTG LN compared to the w14% released
and after cooking are shown in Fig. 5A and B respectively. The three
layers of the noodle in which the core CED layer is sandwiched
between two sandwiching dough layers can be clearly seen. It is
possible to spot signs of the slight loss in denseness and the
occurrence of more hollow structure in the cooked LN that is due
probably to the impact of heat and the water absorption during
cooking. The microstructures of the sandwiching dough layers of
LN are also exhibited (Fig. 5CeF). MTG LN appears denser than the
Control LN. Increasing MTG level caused an increase in denseness of
the structure with more compact and less void structure make up.
The structures of uncooked (Fig. 5G) and cooked CED
(Fig. 5HeK) are also displayed. The oval shaped structures (Fig. 5G)
that stick to one another might be the starch granules, forming the
CED network. However, after cooking, the starch granules were
varied in number. Almost no starch granules could be observed in
the cooked CED from Control LN (Fig. 5H). This can be attributed to
the fact that starch swelled and the original granular form is
destroyed at a highly hydrated condition during cooking. A suffi-
cient amount of water could have penetrated through the surfaces
of the LN into the spaces containing the granules. After cooking

Fig. 5. Scanning electron micrographs of the cross-section of LN: (A) is uncooked control LN, 50. (B) is cooked Control LN, 50. (C), (D), (E) and (F) are the sandwiching layers of
cooked control LN, 0.5MTG LN, 1.0MTG LN and 1.5MTG LN respectively, 500. (G) is uncooked CED, 50. (H), (I), (J) and (K) are the CED of cooked control LN, 0.5MTG LN, 1.0MTG LN
and 1.5MTG LN respectively, 500. Control LN, represents LN prepared with sandwiching layers made up of wheat flour; 0.5MTG LN, 1.0MTG LN and 1.5MTG LN represent LN
prepared with sandwiching dough layers prepared with SPI, wheat flour and 0.5, 1.0 and 1.5% of MTG respectively.
502 L.-Y. Li et al. / Food Hydrocolloids 30 (2013) 495e503

Table 3 Acknowledgments
Sensory evaluation of cooked LN.

Control LN 0.5MTG LN 1.0MTG LN 1.5MTG LN The FRGS (PTEKIND/671199) and financial support of Graduate
Appearance 5.6  1.6a 5.5  1.7a 6.2  1.5a 5.5  2.0a Assistance-ship for Miss Li Ling Yun from University Sains Malaysia,
Smoothness 6.8  1.7a 5.8  1.7a 5.9  1.7a 5.9  1.2a Penang, are gratefully acknowledged.
Odor 6.3  1.2a 6.4  1.0a 6.3  1.2a 6.3  1.0a
Firmness 7.0  1.0b 6.2  1.6ab 6.2  1.6ab 5.7  1.5a
Springiness 6.7  1.4a 5.9  1.8a 6.0  1.6a 5.6  1.3a References
Chewiness 6.6  1.4b 6.0  1.5ab 5.7  1.5ab 5.4  1.4a
Flavor 5.9  1.9a 5.9  1.5a 5.7  2.0a 5.3  1.9a
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