Food Chemistry 294 (2019) 152–159

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Physical and sensory characterisation of noodles with added native and T

denatured pea protein isolate
⁎
M.S.M. Weea, D.E. Loudb, V.W.K. Tana, C.G. Fordea,c,
a
Clinical Nutrition Research Centre (CNRC), Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research (A*STAR), Singapore
b
Food Science and Technology Programme, Department of Chemistry, National University of Singapore, Singapore
c
Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore

A R T I C LE I N FO A B S T R A C T

Keywords: Wheat noodles with added native or denatured pea-protein isolate were characterised for their starch-protein
Noodle interaction, degree of starch gelatinisation, starch digestibility, textural and sensory properties using light mi-
Pea protein croscopy, differential scanning calorimetry (DSC), in vitro digestion, textural profile analysis (TPA) and de-
In vitro starch digestibility scriptive analysis respectively. It was hypothesised that denatured proteins with an unfolded structure, would
Sensory
have greater interaction with starch, thereby reducing the extent of gelatinisation and subsequent glucose re-
TPA
DSC
lease compared to native proteins. Results showed that the addition of denatured pea protein to a wheat noodle
Light microscopy matrix produced a reduction in in vitro glucose release, which was supported by a lower degree of gelatinisation
and greater binding of protein to the starch matrix visually. Addition of native protein to the noodles had less
impact on degree of gelatinisation and glucose release. Addition of both proteins had a negligible effect on
product texture and sensory perceptual properties.

1. Introduction
Noodles are a widely consumed staple in Asia, and are usually
wheat- or rice flour based such as ban mian or bee hoon noodles.
However, prevalence of type-2 diabetes in Asia has been associated
with rice and noodle consumption (Zuniga et al., 2014) as Asians are
often more susceptible to higher postprandial glucose levels compared
to Caucasians (Dickinson, Colagiuri, Faramus, Petocz, & Brand-Miller,
2002; Rhee, 2015; Yoon et al., 2006). There is a shift towards con-
suming lower glycaemic alternatives such as wholegrain rice or noo-
dles, but it is still challenging to change eating habits embodied by
culture, or when a products sensory appeal is compromised through
reformulation. Therefore instead of interventions directed towards re-
ducing consumer intake of noodles, efforts through alteration of food
composition and structure may be more effective in reducing the me-
tabolic impact of carbohydrate consumption. Noodles, unlike rice, is a
secondary product where raw ingredients and processing methods can
be manipulated during production to influence its nutrient and meta-
bolic effects (Moss, Gore, & Murray, 1987).
Functional ingredients such as oat beta-glucan (Regand,
Chowdhury, Tosh, Wolever, & Wood, 2011), polyphenols (Koh, Wong,
Loo, Kasapis, & Huang, 2009) and hydrocolloids (Jang, Bae, & Lee,
2015; Koh, Kasapis, Lim, & Foo, 2009) have been incorporated into
carbohydrate foods to restrict starch digestibility via various mechan-
isms. For example, beta-glucan increases lumen viscosity which delays
intestinal nutrient absorption and postprandial glycaemia (Lazaridou &
Biliaderis, 2007). Alginate forms a physical barrier between the starch
and digestive enzymes via microencapsulation Koh et al., 2009), while
polyphenols from teas inhibit both α-amylase and α-glucosidase en-
zymes which digest the starch. Adding proteins to carbohydrates can
also be used to lower starch digestibility and postprandial glycaemic
index through starch-protein interaction either at the surface (López-
Barón, Gu, Vasanthan, & Hoover, 2017; Ryan & Brewer, 2007), or via
the formation of a continuous protein network entrapping swollen
starch granules (Laleg, Barron, Santé-Lhoutellier, Walrand, & Micard,
2016). Protein addition has the added benefit of increasing satiety
(Veldhorst et al., 2008), and may be a benefit among older populations
requiring higher protein intakes to maintain lean muscle mass.
The structure and composition of the added protein is an important
determinant of the extent to which it can interact with the starch
granules, and also the strength of the entrapping network formed
around the starch granules (Singh, Dartois, & Kaur, 2010). A protein’s
tertiary structure and whether it is in native or denatured state will
influence its binding capacity to starch, and is dependent on the

⁎
Corresponding author at: Clinical Nutrition Research Centre (CNRC), Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research
(A*STAR), Singapore.
E-mail address: ciaran_forde@sics.a-star.edu.sg (C.G. Forde).

https://doi.org/10.1016/j.foodchem.2019.05.042
Received 14 January 2019; Received in revised form 26 March 2019; Accepted 7 May 2019
Available online 08 May 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
M.S.M. Wee, et al.
extraction and post-extraction methods of the protein source. Dena-
tured proteins, with an unfolded tertiary structure, can interact to a
greater extent with the starch granules via exposed hydrophobic amino
acid groups or disulphide linkages, thereby limiting starch gelatinisa-
tion during cooking. Hydrolysed proteins also have more exposed
amino acid groups which in turn can lead to further interaction with
starch molecules (López-Barón et al., 2017, 2018), to limit gelatinisa-
tion. Subsequently, starch digestibility and glucose release is influenced
by the degree of starch gelatinisation, as starches which are partially
gelatinised have fewer leached amylose chains for rapid enzymatic di-
gestion compared to fully gelatinised starches (Jung et al., 2009; Parada
& Aguilera, 2009). Globular proteins including whey, pea and soy
proteins have a spherical folded tertiary structure which unfolds upon
denaturation. As such, there are distinctive functional differences be-
tween native and denatured states, making them suitable proteins for
testing whether denatured or native protein structures differ in their
binding capacity to starch.
Preliminary in-vitro results suggest that denatured or hydrolysed
protein may limit starch digestibility in a simple protein-starch system
(López-Barón et al., 2017, 2018). However, to date, the impact of
protein tertiary structure, in the form of native or denatured protein on
degree of starch gelatinisation and its subsequent breakdown and glu-
cose release in a real food system such as noodles have not been tested.
Additionally, incorporation of a functional ingredient may result in
incompatibility with the existing food matrix, and negatively affect the
products textural and hedonic qualities (Petitot, Boyer, Minier, &
Micard, 2010). The current study therefore aimed to test the impact of
added protein on starch gelatinisation and glucose release in a complex
carbohydrate food. We hypothesised that denatured (unfolded glob-
ular) proteins would bind more to starch than native versions of the
same protein, and through this would have a greater impact on the
degree of starch gelatinisation and starch digestibility. Added native
and denatured pea proteins were incorporated into noodles across a
range of different concentrations, and then compared for their degree of
starch-protein interactions, degree of gelatinisation, in-vitro starch di-
gestibility and instrumental and perceived texture properties.
2. Materials and methods
2.1. Materials
All-purpose plain flour (Redman, Singapore) was purchased from
the local supermarket with the nutritional composition of protein
(13.1%), fat (1.6%), carbohydrate (69.6%) and fibre (3.8%). Pea pro-
tein isolate (yellow pea; Pisum Sativum) was obtained from Roquette
(Nutralys S85F, Roquette, SA, Lestrem, France) with a minimum pro-
tein content of 84% and pH of 7.5. Denatured pea protein isolate was
achieved by heating 5% w/w native pea protein suspension at 85 °C for
30 min in a water bath. The solution was left to cool, frozen and then
freeze-dried for a minimum of 48 h (Virtis SP Scientific, Philadelphia,
USA).
2.2. Noodle samples
Noodle samples were prepared using a commercial Philips
(HR2365) noodle maker by mixing 200 g of dry ingredients (plain flour
and pea protein isolate) with 68 g of water (potable DI water), using the
dry to wet ingredient ratio of 3:1 as recommended by the manufacturer.
The samples were made with increasing pea protein isolate content on a
dry weight basis, as shown in Table 1. Dry ingredients were first dry-
blended in an airtight bag before being added to the automated mixing
compartment of the noodle maker where the dry mix and water were
kneaded into a noodle dough for a total of 8 min (setting 4), before raw
noodles were extruded (1 mm). Raw noodles (100 g) were weighed and
cooked in 1 L of boiling water for 5 min, and then placed immediately in
1 L of cold water for 2 min to prevent further cooking. Noodles were
153
Food Chemistry 294 (2019) 152–159
Table 1
Noodle formulations with 0 (control), 2.5, 5.0, 7.5 and 12.5% w/w pea protein
isolate.
Pea Protein Isolate (% w/ dry weight)
0 2.5 5.0 7.5 12.5
Flour (g) 200 195 190 185 175
PPI (g) 0 5 10 15 25
Water (g) 68.0 68.0 68.0 68.0 68.0
Total Protein (% w/w) 9.8 11.4 13.0 14.6 17.9
Total Carbohydrate (% w/w) 51.9 50.6 49.3 48.0 45.4
then rinsed under running water in a strainer for 1 min to remove ex-
cess starch, and tossed 30 times in the strainer to remove excess water.
All cooked samples were packed in airtight bags and refrigerated
overnight (24 h) to rest prior to analysis. All cooked noodle samples for
light microscopy, in-vitro digestion, textural profile analysis and sen-
sory evaluation were prepared in the same manner.
2.3. Light microscopy
The degree of protein-starch interaction in the cooked noodle was
characterised using light microscopy and the procedure was adapted
from Eelderink et al. (2015). Cooked noodles were sliced using a
cryostat (CM3050 S, Leica, Cryostat, Nussloch, Germany) to 50 μm
thickness at −20 °C and mounted on glass slides. The sliced noodle
sections were stained with 50% Lugol (KI/I2; Sigma-Aldrich Chemic, St
Louis, USA) solution for one minute for the starch (purple) and excess
Lugol solution was rinsed off with DI water. The sections were then
stained with 0.1% Ponceau 2R solution (Sigma-Aldrich Chemic, St
Louis, USA) in 50% glycerol (Sigma-Aldrich Chemic, St Louis, USA) in
water to stain the protein (red) for 10 min. Noodle cross-sections were
viewed at 5× magnification with a light microscope (SZ 61, Olympus,
Japan) and images were captured using the Image-Pro 6.2 program.
White balance was applied to the images using an image editing soft-
ware (Photoshop CS, Adobe, USA) to correct the background colour. A
minimum of three images were taken for each sample slice, and each
sample was sliced in triplicate (Table 2).
2.4. Differential scanning calorimetry (DSC)
Raw noodle samples were snap-frozen in liquid nitrogen and freeze-
dried (Virtis SP Scientific, Philadelphia, USA). The freeze-dried noodles
were weighed and pulverized with a mortar and pestle and subse-
quently mixed with a weighed amount of water in a 3:7 (powder-water)
ratio with water in excess. A small amount of the mixture (approxi-
mately 15 mg) was placed and weighed in 40 µl aluminium pan which
was then hermetically sealed. The analysis was carried out at a heating
rate of 10 °C/minute from 30 °C to 110 °C with a reference cell con-
taining approximately 15 mg of deionized water in another 40 µl alu-
minium pan (Mettler Toledo DSC822e, Griefensee, Switzerland). Each
sample was repeated in triplicates. The parameters onset temperature
(TO), peak temperature (Tp), and enthalpy of gelatinisation (peak area;
ΔHg) for each sample were retrieved or calculated using the Mettler
Toledo DSC822e software.
2.5. In vitro digestion
The in-vitro digestion protocol was adapted and modified from the
INFOGEST digestion method (Minekus et al., 2014). Briefly, cooked
noodles were subjected to oral (100 s), gastric (1 h) and intestinal (2 h)
phase digestions. The total glucose released at the end was quantified
using a colorimetric reducing sugar assay with dinitrosalicylic acid
(DNS). The cooked noodles (5 g) were finely chopped into 25 mm pieces
to standardise surface area and volume in duplicates. For the oral phase
M.S.M. Wee, et al.
Table 2
Thermal characteristics of noodles (raw) with added native and denatured protein at 0 (control), 2.5, 5.0, 7.5 and 12.5% w/w concentrations.
PPI (% w/w) Onset Temperature, To (°C) Peak Temperature, Tp (°C)
Native Denatured Native
0 54.2 ± 1.7 62.9
2.5 55.1 ± 0.3 57.0 ± 0.5* 62.9
5.0 55.9 ± 0.2 56.2 ± 1.8 63.0
7.5 56.7 ± 0.2 55.7 ± 1.2ǂ 63.8
12.5 56.5 ± 0.2 56.3 ± 1.5 63.7
ǂ
Significant difference compared to control noodles (p < 0.05).
* Significant difference compared to native protein noodles at the same concentration (p < 0.05).
of digestion, oral phase solution (5 ml) consisting of α-amylase
(12.5 mg/ml in simulated salivary fluid; 4 ml; Sigma-Aldrich, St. Louis,
MO, USA) and 7.5 mM CaCl2 (1 ml; Sigma-Aldrich, St. Louis, MO, USA)
was added to the noodles, vortexed, and digested at 37 °C for 1 min 40 s.
Gastric phase solution (10 ml; pH 3) consisting of pepsin (9.94 mg/ml in
simulated gastric fluid; 8 ml; Sigma-Aldrich, St. Louis, MO, USA), 0.3 M
CaCl2 (5 μl), DI water (1.445 ml) and 1 M HCl (0.55 ml) was added to
the noodles and vortexed after oral phase digestion for 1 h at 37 °C.
After gastric phase digestion, the intestinal phase solution (20 ml; pH 7)
consisting of pancreatin (2.585 mg/ml in simulated intestinal fluid;
16 ml; Sigma-Aldrich, St. Louis, MO, USA) and amyloglucosidase
(80 μl), 0.3 M CaCl2 (40 μl), DI water (3.48 ml) and 1 M NaOH (400 μl)
was added to the gastric chyme and digested for 2 h under constant
stirring at 350 rpm at 37 °C. Aliquots (0.5 ml) of the digesta from the
intestinal phase were taken out at 0, 5, 10, 15, 30, 45, 60 and 120 min
interval and added to absolute ethanol (1.5 ml) to inactivate the en-
zymes. Simulated intestinal fluid (0.5 ml) was added back to the digesta
to replace the withdrawn aliquot at each time point. The aliquots were
centrifuged at 10,000 rpm for 4 min at 23 °C and then kept overnight at
4 °C prior to reducing sugar analysis.
The ethanolic digesta (50 μl; duplicates) was added to 0.75 ml of
DNS reagent (Sigma-Aldrich, St. Louis, MO, USA), vortexed and placed
in a boiling water bath (100 °C) for 10 min and left to cool for another
10 min. The mixture was further diluted with DI water (2 ml) and the
absorbance read at 540 nm. The total amount of glucose released was
quantified using a standard D-glucose (Sigma-Aldrich, St. Louis, MO,
USA) curve (0 to 10 mg/ml).
2.6. Textural profile analysis (TPA)
Textural properties of the cooked noodles were measured by tex-
tural profile analysis (TPA) using a double compression cycle test on a
TA.XT plus Stable Micro Systems Texture Analyser (Stable
Microsystems Ltd., Surrey, England) with the Texture Expert program
(version 6.1). A 36 mm compression probe (P/36; Stable Micro Systems
Ltd, Surrey, England) was used and test settings were fixed at 30%
compression strain, pre- and post-test speed of 3 mm/s and test speed of
1 mm/s at 22 °C for all samples. Three strands of noodles were placed
on the texture analyser platform with a distance of 3 mm apart. The
TPA parameters hardness (g), adhesiveness (g.sec), springiness, cohe-
siveness, chewiness and resilience were obtained with a minimum of 10
measurements performed for each noodle sample. This procedure was
adapted from a previously published approach (Wee, Goh, Stieger, &
Forde, 2018). Briefly, hardness is the maximum force obtained during
the first compression, adhesiveness is the negative area after the first
compression, springiness is the ratio between distance compressed till
maximum force during second compression to the distance during first
compression, cohesiveness is the ratio of second compression area to
first compression area, chewiness is the product of hardness, cohe-
siveness and springiness, and resilience is the ratio between compres-
sion area on the withdrawal stroke to the compression stroke during the
first compression.
Food Chemistry 294 (2019) 152–159
Enthalpy of Gelatinisation, ΔHg (J/g starch)
Denatured Native Denatured
± 0.3 13.9 ± 2.7
± 0.1 63.1 ± 0.8 18.2 ± 2.2 10.7 ± 1.0*
± 0.0 63.4 ± 0.1* 16.0 ± 1.0 11.3 ± 0.4*
± 0.2 63.8 ± 0.1ǂ 15.7 ± 0.7 11.2 ± 0.7*
± 0.0 63.6 ± 0.4ǂ 13.5 ± 0.2 10.2 ± 0.5*
2.7. Sensory evaluation
A sensory panel (n = 20, 5 males, 24 ± 2 years old) was recruited
from a participant database at the National University of Singapore.
Participants were screened for eligibility and the recruitment criteria
included age (21–40 years old), normal weight range (BMI < 30 kg/
m2), non-smoker, no self-reported problems with taste or smell, no al-
lergies or intolerances, and not diabetic or pregnant. The eligible par-
ticipants provided informed consent and were compensated for their
time. This study (Ref: 2017/01160) was approved by the Domain
Specific Review Board of the National Healthcare Group, Singapore,
and complies with the Declaration of Helsinki for research involving
human subjects.
Participants underwent training to ensure familiarity with the taste
and texture attributes and scale anchors to evaluate the noodles using
the descriptive analysis (DA) method (Lawless & Heymann, 2010).
During the training session participants were briefed on the DA method
and were presented with 8 noodle samples including a control (0%
protein), 2.5, 7.5 and 12.5% native and denatured protein noodles.
Participants were provided with a provisional list of attributes and
definitions based on previous sensory trials that had profiled texture
differences among similar noodle samples (Choo & Aziz, 2010; Laleg
et al., 2017; Tang, Hsieh, Heymann, & Huff, 1999). Once familiarised
with all the noodle samples, participants were asked to refine and
clarify attribute definitions and scale anchors, to ensure the vocabulary
fully described all of the perceptual differences among the noodle
samples. The final list of attributes and definitions used to profile the
noodles were firmness (the force required to cut through the noodle
during first bite), chewiness (the length of time required to masticate
noodle to a state for swallowing), stickiness (the extent of which noo-
dles will stick to teeth and palate), springiness (the extent to which
noodles deform without breaking), surface roughness (the amount of
irregularities perceived on the surface of noodles), ‘floury’ texture (the
texture of pasty or mealiness in mouth during consumption), and
overall flavour (the noodle flavour associated with noodle products like
mee poh). All noodle samples were rated for each attribute on a 100-
point line-scale from ‘Not at all…’ (0) to ‘Extremely…’ (1 0 0) and
participants were free to use the full line scale to rate each attributes
intensity.
All 8 noodle samples were blind-evaluated in triplicates across three
separate 1-hour sessions, with a minimum of 24 h between sessions.
Sample presentation within each replicate was randomised and ba-
lanced using a Williams Latin squared design, to minimise response bias
due to presentation order. Each noodle sample (5 g) was presented in
individual bowls with 5 ml of diluted Soba sauce (0.2% w/v, Yamaki
Soba Tsuyu, Japan) that was added immediately prior to serving. Each
bowl was labelled with random three-digit numbers and served at room
temperature. Participants cleansed their palate thoroughly with filtered
water during a mandatory one minute inter-stimulus interval between
samples. All data was collected using computerized data acquisition
software (CompuSense Cloud, Guelph, Ontario, Canada), in sensory
booths under white lights.
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M.S.M. Wee, et al.
2.8. Statistical analysis
Statistical analysis was carried out using paired t-tests for com-
paring means of samples to control, and between samples with native or
denatured protein at a 5% significance level for DSC, glucose release
and TPA results. For sensory evaluation results, a linear mixed model
ANOVA was used to compare sensory differences among the native and
denatured protein noodles, across the different concentrations tested,
with significant main effects compared using a post-hoc Bonferroni test.
All statistical test in this study were set at a 5% significance level
(α = 0.05) (IBM SPSS Statistics for Windows, Version 25.0. Armonk,
NY: IBM Corp.).
3. Results
3.1. Starch-Protein interactions in noodles
Cooked noodle samples were stained with lugol and ponceau 2R
solutions to differentiate the starch (purple) and protein (red) fractions
respectively, and light micrographs of the stained regions were visually
compared across native and denatured protein noodles at different
concentrations. No visible differences in proteinaceous fraction were
detected between the control samples without added protein (Fig. 1a
and e). The addition of pea protein isolate to the noodle matrix was
recognised as red regions interspersed within the gelatinised starch.
Increasing protein concentrations corresponded with an increasing
density of the red proteinaceous fractions (Fig. 1). The protein fractions
were uniformly dispersed within the noodle matrix for both native and
denatured protein, although with some large aggregates ranging in size
from 50 to 100 μm, which increased in size at higher concentrations of
added protein (Fig. 1b–h). Compared to the native protein, there ap-
peared to be a greater assimilation of the denatured protein fraction
into the starch matrix at 7.5 and 12.5% w/w protein with an almost bi-
continuous distribution of protein and starch phases at the highest
concentration (Fig. 1h). The starch granules were smaller and more
discrete for the noodles with 12.5% w/w denatured protein (Fig. 1h)
compared to the native protein (Fig. 1d), suggesting a reduced extent of
gelatinisation (Huang & Moss, 1991; Laleg, Cassan, Barron,
Prabhasankar, & Micard, 2016).
3.2. Degree of starch gelatinisation in noodles
Thermal transitions observed on the endotherms at temperatures
between 60 and 65 °C corresponded to starch gelatinisation (Lu,
Donner, Yada, & Liu, 2016), with a single peak observed for all samples.
No peaks at pea protein denaturation temperature of approximately
85 °C (Mession, Sok, Assifaoui, & Saurel, 2013) were observed for the
noodles with native protein, due to the relatively low concentration of
protein in the noodles (Aguilera & Rojas, 1996) and fast heating rate.
The addition of both native and denatured proteins generally re-
sulted in higher To (not statistically significant). Denatured proteins had
a greater impact on Tp and ΔHg of the noodles compared to native
protein. A lower ΔHg indicated less endothermic energy absorbed to
complete the gelatinisation transition. The control sample is assumed to
have undergone 100% gelatinisation, therefore any sample with a ΔHg
lower than control indicates a smaller extent of gelatinisation (Edwards
et al., 2015). This was interpreted to mean that noodles with added
denatured protein had a reduced extent of gelatinisation compared to
noodles without added protein (control), although adding more dena-
tured protein did not further enhance this effect on ΔHg. However, the
noodles with added native protein had a higher ΔHg than the control
but decreasing ΔHg as protein concentration increased.
3.3. Starch digestibility of noodles
Starch digestibility was represented by the total amount of glucose
155
Food Chemistry 294 (2019) 152–159
released per gram starch (corrected for total starch content due to flour
substitution) after oral (100 s), gastric (1 h) and intestinal (2 h) phase in
vitro digestion. There was an overall trend for a decline (not statistically
significant) in total glucose released with the addition of protein
(Fig. 2a). Noodles with added denatured protein appeared to have the
greatest impact in attenuating glucose release at 7.5% w/w, although
differences were not significant amongst all samples with native or
denatured protein across all concentrations (p > 0.05). The amount of
glucose released with time was also qualitatively compared between
control noodles, and noodles with 12.5% native and denatured protein
(Fig. 2b). Starch was rapidly digested within the first 30 min as in-
dicated by the steep slope for glucose release, especially for the control
noodles, which had twice as much glucose release within the first 5 min
when compared to the native and denatured protein noodles. At
120 min, the control noodles had the highest total glucose released
(compared to baseline), followed by 12.5% native, with the lowest re-
lease from the 12.5% denatured protein noodles (Fig. 2b). Although
there was a trend for lower glucose release in the 12.5% denatured
noodle, these differences were not significant (p > 0.05).
3.4. Textural properties of noodles
The instrumental texture parameter of hardness increased sig-
nificantly with increasing protein concentration for both native and
denatured protein noodles, while other parameters such as springiness,
cohesiveness and chewiness decreased (Table 3). No distinct differences
were found between noodles with added native or denatured protein.
3.5. Sensory properties of noodles
Sensory evaluation (n = 20) results showed no significant differ-
ences (p > 0.05) between noodle samples with added native and de-
natured protein for the textural attributes firmness, chewiness, sticki-
ness and springiness (Fig. 3). The addition of up to 12.5% w/w of native
or denatured proteins to the noodles did not influence texture percep-
tion as well. However, protein addition at 12.5% w/v significantly af-
fected the visual characteristics of surface roughness and ‘floury’ tex-
ture for both native and denatured noodles. Increasing protein
concentration resulted in a decrease in springiness, although this was
not perceived as significant by the sensory panel.
4. Discussion and conclusion
The current study demonstrated that the addition of protein to a
noodle matrix produced a reduction in both starch gelatinisation and in
vitro glucose release, while having a negligible effect on product sensory
properties. In addition we demonstrate that this effect was slightly
higher through the addition of denatured compared to native pea
protein, and was likely due to the greater interactions of the unfolded
structure of the denatured protein with the starch in wheat flour.
Based on the light microscopy results, the denatured protein ap-
peared to bind more strongly to the starch as compared to the native
protein. This corresponded to the in-vitro glucose release results
whereby there was overall less glucose released for the noodles with
denatured protein, which was likely in part due to the differences in
binding observed. All of the networks observed were protein dispersed
in a continuous starch phase, with an almost bi-continuous distribution
of protein and starch phases at the highest protein concentrations (7.5
& 12.5% w/w). A continuous protein network with entrapped starch
granules was not observed in this study, due to the low pea protein
concentration (< 20% dry weight) which was likely below the con-
centration required for phase inversion (Laleg, Cassan, et al., 2016).
The mode of protein binding to starch was then likely through the in-
teraction on the starch surface rather than entrapping of starch granules
within a protein network. Specifically, pea protein could interact with
the starch granules or surface protein on the starch granules via
M.S.M. Wee, et al. Food Chemistry 294 (2019) 152–159

(caption on next page)

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M.S.M. Wee, et al. Food Chemistry 294 (2019) 152–159

Fig. 1. Light micrographs of starch (purple) and protein (red) distribution and interaction in noodles with added native (left) and denatured (right) PPI at 0 (A, E), 2.5
(B, F), 7.5 (C, G) and 12.5% w/w (D, H); scale bar represents 500 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)

Fig. 2. a) Total glucose released from in vitro digestion of noodles (120 min) with added native or denatured protein at 0 (control), 2.5, 5.0, 7.5 and 12.5% w/w
concentrations; b) Change in glucose released with time for noodles with no added protein (control), and 12.5% native and denatured protein.

hydrophobic, hydrogen bonding or disulphide bonding with the hy-

drophobic amino acid groups such as leucine, polar amino acid groups
such as glutamic acid, or sulphurous amino acids such as cysteine re-
spectively (Debet & Gidley, 2006; López-Barón et al., 2017; Pascal,
Thierry, & Evelyne, 1990; Ryan & Brewer, 2007). These interactions
may limit starch hydration or form protein-protein bonds which would
restrict starch gelatinisation during noodle cooking and subsequent
enzymatic access to starch during digestion. Interaction of starch with
protein likely via these mechanisms have been demonstrated in several
studies to reduce in-vitro starch digestibility (Fardet et al., 1998; Laleg,
Barron, et al., 2016; López-Barón et al., 2017; Lu et al., 2016), as well as
postprandial blood glucose in in-vivo studies (Jenkins et al., 1987).
The in-vitro glucose release results were further supported by our
findings from differential scanning calorimetry, which demonstrated
that denatured pea proteins, especially at higher concentrations (e.g.
7.5 or 12.5% w/w), were more effective at limiting starch gelatinisation
compared to native pea proteins. However, it should be noted that in Fig. 3. Spider plot of sensory attributes of noodles with added native or de-
vitro digestion included protein digestion using pepsin in the gastric natured protein at 0 (control), 2.5, 5.0, 7.5 and 12.5% w/w concentrations; *
phase, which would have altered enzymatic access to the starch gran- indicates a significant difference from control (p < 0.05).
ules not just dependent on extent of starch gelatinisation alone. In this
regard denatured proteins, while limiting starch gelatinisation to a digestion, denatured proteins would be hydrolysed to a greater extent
greater extent than the native proteins during cooking, would also be which would break down their network allowing greater enzymatic
more susceptible to pepsin digestion in the gastric phase due to their access to starch. These two opposing effects may explain why there
unfolded structure (Nielsen, Deshpande, Hermodson, & Scott, 1988). were no significant differences observed for in-vitro starch digestibility
While the native proteins would also undergo denaturation during the of noodles between native and denatured proteins despite the observed
cooking process, the cooking time of 5 min is unlikely to denature na- differences in gelatinisation enthalpies. Further, we observed that
tive proteins to the same extent. Therefore when subjected to pepsin

Table 3
Textural properties of noodles with added native and denatured protein at 0 (control), 2.5, 5.0, 7.5 and 12.5% w/w concentrations.
PPI (% w/w) Hardness (g) Adhesiveness (g⋅sec) Springiness (−) Cohesiveness (−) Chewiness (g)

0 Control 2124 ± 105 −102 ± 23 0.915 ± 0.011 0.586 ± 0.016 1139 ± 51

2.5 Native 2130 ± 128 −105 ± 14 0.878 ± 0.025 0.524 ± 0.017ǂ 980 ± 87
Denatured 2190 ± 67 −120 ± 13* 0.898 ± 0.031 0.565 ± 0.006*,ǂ 1111 ± 40*
5.0 Native 2269 ± 70 −114 ± 44 0.893 ± 0.033 0.521 ± 0.022ǂ 1068 ± 92
Denatured 2559 ± 116*,ǂ −153 ± 9ǂ 0.855 ± 0.030ǂ 0.534 ± 0.025ǂ 1168 ± 66
7.5 Native 2238 ± 72 −166 ± 31ǂ 0.856 ± 0.028ǂ 0.514 ± 0.026ǂ 988 ± 97ǂ
Denatured 2257 ± 91 −94 ± 5* 0.902 ± 0.024 0.516 ± 0.017ǂ 1050 ± 42ǂ
12.5 Native 2381 ± 111ǂ −188 ± 21* 0.842 ± 0.028ǂ 0.484 ± 0.013ǂ 970 ± 56ǂ
Denatured 2409 ± 102ǂ −117 ± 11ǂ 0.872 ± 0.029ǂ 0.519 ± 0.016*,ǂ 1090 ± 70*

ǂ
Significant difference compared to control noodles (p < 0.05).
* Significant difference compared to native protein noodles at the same concentration (p < 0.05).

157
M.S.M. Wee, et al.
noodles with added native protein had a decreasing ΔHg as protein
concentration increased. This may be due to the native protein being
folded, in which case increasing native protein concentration would
increase the interaction sites with the starch and serve as a physical
barrier to gelatinisation (Lu et al., 2016). Unfolded denatured proteins
would already have more exposed sites for interaction with the starch,
thus have limited concentration-dependent effects. Noodles with added
native protein also had generally higher ΔHg than both the control (not
statistically significant) and denatured protein noodles. One possible
explanation could be due to the dilution of existing gluten protein
network with the introduction of exogenous protein (Laleg, Barron,
et al., 2016). This would have weakened the protein network entrap-
ping the starch granules, leading to greater extent of gelatinisation. The
effect was likely exacerbated without additional protein-starch inter-
actions like in the case of denatured proteins.
Flour substitution with protein reduced the total gluten content,
resulting in increased hardness and a partial loss of elasticity (in terms of
springiness, cohesiveness and chewiness) as gluten is responsible for the
elastic texture of noodles. With gluten substituted, water-absorption
was also reduced which affected noodle softness (Laleg et al., 2017).
However, textural changes were only detected via the instrumental TPA
and not by the sensory panel. This is important as it highlights that
glucose release could be attenuated via food structural modification,
without compromising on noodle sensory properties. The overall fla-
vour intensity was also not significantly influenced by the addition of
pea protein, despite observations that adding legume flour or protein
imparts a ‘beany’ flavour (Laleg et al., 2017).
This study has some noteworthy limitations that merit considera-
tion. The degree of starch gelatinisation is strongly dependent on a fixed
cooking temperature and time, and longer cooking of the noodles
would further gelatinise the starch completely, and potentially remove
any differences between control and protein enhanced noodles. While
our findings demonstrate that addition of denatured pea-protein to a
real food system has the potential to reduce starch digestibility, these
findings may not apply for other plant proteins or carbohydrate foods
such as bread or pasta. Furthermore, the marginal reduction of glucose
release from in vitro digestion results may or may not translate to an
actual reduction of postprandial blood glucose when consumed by a
human participant, as in vivo physiological responses do not always
reflect differences identified by in-vitro experiments (Bohn, Carriere,
Day, Deglaire, Egger, Freitas, & Menard, 2017; Wee, Yusoff, Chiang, &
Xu, 2017). In addition, the small effect of denatured (and native) pro-
tein addition on starch digestibility may be due to the weak interaction
between starch and protein, which may not be strong enough to limit
gelatinisation sufficiently to significantly reduce glucose release. In-
teractions between added protein and starch could be further
strengthened via high-temperature drying (Petitot et al., 2009) or high
pressure cooking to induce protein aggregation (López-Barón et al.,
2017) and protein hydrolysis (López-Barón et al., 2018) to increase
number of amino acid groups available for interaction, or by addition of
other proteins such as wheat gluten or egg white to form stronger
protein networks (Laleg, Barron, et al., 2016). Drying the protein-en-
riched wheat noodles prior to cooking could also be a simple approach
to lowering starch digestibility, as dried noodles have a slower uptake
of water, and starch granules at the core of the noodle have been ob-
served to undergo reduced gelatinisation than the starch at the exterior
(Laleg, Barron, et al., 2016; Laleg, Cassan, et al., 2016). Future studies
should build on the current findings and focus on in-vivo glycaemic
responses to protein enhanced noodles in human subjects. To further
isolate the effect of protein denaturation on interaction with the starch,
other globular (e.g. soy and whey) and non-globular proteins should be
added and compared for their effect on glucose release from noodles.
Declaration of Competing Interest
C.G. Forde has received reimbursements for speaking at conferences
158
Food Chemistry 294 (2019) 152–159
sponsored by companies selling nutritional products, serves on the
scientific advisory council for Kerry Taste and Nutrition, and is part of
an academic consortium that has received research funding from
Abbott Nutrition, Nestec, and Danone. The other authors have no
conflicts of interest.
Acknowledgements
This research is supported by the Biomedical Science Institute
Industry Alignment Fund (IAF-PP); Food Structure Engineering for
Nutrition and Health (H17/01/a0/A11).
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