LWT - Food Science and Technology 108 (2019) 261–267

Contents lists available at ScienceDirect

LWT - Food Science and Technology

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

Effect of anticaking agents on hardening and Maillard-induced protein T

aggregation in high-protein nutrition bars formulated with whey protein
concentrate
Xianghe Meng∗, Jian Ji, Xie Qi, Xiaohua Nie∗∗
Department of Food Science and Technology, Ocean College, Zhejiang University of Technology, Hangzhou, Zhejiang, 310014, PR China

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

Keywords: Hardening is a common problem of high-protein nutrition bars during storage, causing the products to be un-
High-protein nutrition bars acceptable for consumers. The object of this study was to prepare high-protein nutrition bars formulated with
Silicon dioxide whey protein concentrate (WPC), and evaluate the effect of anticaking agents (SiO2 and Ca3(PO4)2) on their
Calcium phosphate hardness, microstructure and Maillard-induced protein aggregation over storage at 37 °C. The increment in
Free amino groups
hardness of WPC bars was obviously slowed by SiO2 and Ca3(PO4)2, especially the former; along with an ap-
Protein structure
parent dispersive microstructure in CSLM image. The addition of SiO2 and Ca3(PO4)2 inhibited the progress of
Maillard reactions in WPC bars, evidenced by limited changes in L* value, free amino groups and SDS-PAGE
pattern. Fewer larger proteins were found in WPC bars with SiO2, which indicated that the used anticaking
agents diminished the formation of Maillard-induced protein aggregation. On the other hand, α-helix in whey
proteins was continuously transferred into β-sheet during storage. This conformation transformation was re-
strained by SiO2 and Ca3(PO4)2, further confirming less protein aggregation formed by Maillard reactions.
Therefore, the use of anticaking agents can inhibit Marllard reactions in high-protein nutrition bars, and is
effective for controlling the bars’ hardening.

1. Introduction
High-protein nutrition bars mainly contain protein (20–50%), car-
bohydrate, fat and some humectants with a water activity of 0.5–0.8.
They have received growing attention in sports nutrition, muscle
building, health supplement and weight reduction markets in recent
years. Whey protein, an abundant co-product of cheese industry, is
widely appreciated for its functional versatility, high nutritional value
and direct physiological effects when consumed in conjunction with
human performance activities (Kelly, 2019). It has been commonly used
in the manufacture of high-protein nutrition bars (Banach, Clark, &
Lamsal, 2014; Loveday, Hindmarsh, Creamer, & Singh, 2010; Lu et al.,
2016). However, high-protein nutrition bars often become hardened
over storage, that makes the products unacceptable to consumers. And
this problem intensifies when higher percentages of protein are used
(Rao, Kamdar, Guo, & Labuza, 2016). The bars' hardening is likely due
to protein aggregation induced by physico-chemical changes such as
water migration (Zhou, Liu, & Labuza, 2008), phase separation
(Mcmahon, Adams, & Mcmanus, 2009), Maillard reactions (Zhou et al.,
2008) and disulfide interaction (Zhu & Labuza, 2010). Protein ag-
gregation gives rise to strong and tight networks which leads to ‘hard’
or ‘tough’ texture. Previous researches primarily focused on the func-
tion of ingredients including protein type (Hogan, Chaurin, O'Kennedy,
& Kelly, 2012), protein hydrolysate (Rao et al., 2016), sugar type
(Mcmahon et al., 2009) on the hardening development of high-protein
nutrition bars.
High-protein nutrition bars are non-equilibrium thermodynamic
multicomponent food systems (Mezzenga, 2007). Limited amount of
water present makes the bar form a heterogeneous dispersion con-
taining fat droplets, non-fully hydrated protein powders and aqueous
phase of sugar syrup. Small molecules (water, glycerol and sugar) in
aqueous phase may migrate into dry protein powders (Lu et al., 2016),
driven by the difference of water activity between adjacent ingredients
(Purwanti, van der Goot, Boom, & Vereijken, 2010). This migration
increases the hydration of protein molecules and triggers some dele-
terious reactions (especially Maillard reactions), causing the formation
of protein aggregation (Potes, Kerry, & Roos, 2014; Rao, Roccasmith, &
Labuza, 2012). Therefore, minimising molecule migration represents

∗
Corresponding author.
∗∗
Corresponding author.
E-mail address: mengxh@zjut.edu.cn (X. Meng).

https://doi.org/10.1016/j.lwt.2019.03.077
Received 29 September 2018; Received in revised form 23 March 2019; Accepted 25 March 2019
Available online 27 March 2019
0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
X. Meng, et al.
another means by which the hardening of high-protein nutrition bars
should be controlled.
Anticaking agents are substances added to powder foods to prevent
caking, lumping or aggregation. They can compete with host powder
for water, and also create protective moisture barriers on the surface of
hygroscopic particles or physical barriers between ingredient particles
(Aguilera, Valle, & Karel, 1995; Lipasek, Ortiz, Taylor, & Mauer, 2012).
The effectiveness of anticaking agents in improving the flowability of
powder foods has been well documented (Lipasek et al., 2012;
Nishanthi, Chandrapala, & Vasiljevic, 2017). Yet there are few pub-
lications regarding the use of anticaking agents in high-protein nutri-
tion bars. Food-grade silicon dioxide (SiO2) and calcium phosphate
(Ca3(PO4)2) are generally recognized as safe (GRAS) (FDA, 2019; Yang
et al., 2016), and widely used as anticaking agents in food industry
(Chang, Karim, Abdulkarim, Yusof, & Ghazali, 2018; Lipasek, Taylor, &
Mauer, 2011). In this study, high-protein nutrition bars formulated with
whey protein concentrate (WPC) were prepared, and the effect of an-
ticaking agents (SiO2 and Ca3(PO4)2) on their hardness, microstructure
and Maillard-induced protein aggregation were investigated.
2. Material and methods
2.1. Materials
Whey protein concentrate (WPC, containing 75% protein), maltose
syrup (about 80% maltose), coconut oil, glycerin, soybean phospholi-
pids and food-grade anticaking agents (SiO2 and Ca3(PO4)2) were pro-
vided by Hangzhou Hengmei Co., Ltd (Zhejiang, China). Fluorescein
isothiocyanate (FITC), Nile red, ophthaladehyde (OPA), sodium do-
decyl sulfate (SDS), coomassie brilliant blue R-250, molecular weight
marker were purchased from Sigma-Adrich Co. (St. Louis, MO, USA).
Other chemicals and solvents were of analytical grade.
2.2. WPC bars preparation
WPC bars were prepared according to the method of Banach, Clark,
and Lamsal (2013). The composition was 45 g/100 g whey protein
concentrate, 48 g/100 g maltose syrup, 4 g/100 g coconut oil, 2 g/100 g
glycerol and 1 g/100 g soybean phospholipid. SiO2 and Ca3(PO4)2 were
used as anticaking agents at a level of 0.5 g/100 g bar. Briefly, maltose
syrup, coconut oil, glycerol and soybean phospholipid were mixed and
heated at 55 °C until melted, then blended with whey protein con-
centrate and anticaking agents for 1 min. The dough was put into a bar
frame (dimensions 160 × 140 × 20 mm), covered with plastic film and
rolled into shape to fit the frame. After balance at room temperature for
3 h, the set dough was cut into bars of approximately
70 × 20 × 20 mm. The bars were placed in foil bags, heat-sealed and
stored at 37 °C. The bars without anticaking agents were used as the
control. Three bars of each group were randomly taken out every seven
days and equilibrated for at least 2 h at room temperature prior to
analysis.
2.3. Hardness analysis
The hardness of WPC bars was determined by TAXT Plus Text
Analyzer (Stable Micro Systems, Ltd., Godalming, Surrey, England)
with a 250 N load cell and a flat-ended cylinder stainless steel probe
(4 mm diameter). The speed and trigger force were 1 mm/s and 0.1 N,
respectively. Three locations in each bar were tested. Three bars of each
group were used for hardness analysis.
2.4. Confocal scanning laser microscopy (CSLM) test
The microstructure of WPC bars was analysed using CSLM method,
as described by Mcmahon et al. (2009) with some modifications. The
square (about 8 × 8 × 2 mm) was sliced from the middle of the bar and
262
LWT - Food Science and Technology 108 (2019) 261–267
placed on a glass bottomed dish. After successively stained with
200 mg/L fluorescein isothiocyanate acetone solution for 1 min and
200 mg/L Nile red acetone solution for 1 min, the square was covered
with a coverslip and sealed with silicone. The sample was observed
using an IX71 FV300 confocal laser scanning microscope (Olympus
Optical Co., Tokyo, Japan) in fluorescence mode. Fluorescence was
captured sequentially using filters of wavelengths 512–532 nm for
fluorescein isothiocyanate and ≥585 nm for Nile red. The images were
analysed using FluoView version 5.0 software (Olympus Optical Co.,
Tokyo, Japan).
2.5. Colour measurement
The colour parameters (L*, a* and b*) of WPC bars were measured
by ColorQuest XE colorimeter (HunterLab, Reston, Virginia, USA). The
instrument was calibrated to standard white and black tiles before
measurement. Three different locations in each bar were tested. Three
bars of each group were used for colour measurement.
2.6. Free amino group content determination
Free amino group content was determined by the orthophthaldial-
dehyde (OPA) method of Rao et al. (2012) with some modifications.
WPC bars (1.0 g) were extracted with 50 mL of 20 g/L SDS solution for
1 h, followed by the centrifugation at 4,000×g for 30 min. The super-
natant (100 μL) was added to 4 mL of fleshly prepared OPA solution
(800 mg/L OPA, 0.05 mol/L sodium tetraborate buffer, 10 g/L SDS,
2 mg/L 2-mercaptoethanol, pH 9.0). The mixture was kept at room
temperature for 2 min. The absorbance at 340 nm was read against
distilled water using SP-75 UV–Vis spectrophotometer (Spectra instru-
ments Co., Ltd, Shanghai, China). The OPA solution was used as a
blank. Relative free amino group content (%) was calculated according
to the below equation.
Relative free amino group content (%) = (Asample on day x – Ablank) /
(Asample on day 0 – Ablank) × 100
2.7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE)
Protein pattern of WPC bars was analysed by reducing SDS-PAGE
with 5 g/100 g stacking gel and 12 g/100 g separating gel. WPC bars
(0.5 g) were extracted with 50 mL of 10 mmol/L phosphate buffer (pH
7.4) containing 20 g/L SDS for 1 h, followed by the centrifugation at
10,000×g for 30 min. The supernatant was adjusted to a protein con-
centration of 5 mg/mL, added with an equal volume of 5 × sample
buffer and heated at 100 °C for 10 min. After electrophoresis at 120 V,
the gels were stained with coomassie brilliant blue R-250 solution (1 g/
L) for 30 min and then destained using 0.1 g/mL methanol and 0.1 g/
mL acetic acid. Stained gels were scanned using the Gel Doc XR system
(Bio-Rad Laboratories, Inc., Shanghai, China).
2.8. Fourier transform infrared (FTIR) spectrum
FTIR spectrum of WPC bars was determined using a Nicolet 8210E
FTIR spectrometer (Nicolet, Madison, WI, USA) as previously reported
(Hogan et al., 2012). After being lyophilized for 24 h, WPC bars (10 mg)
were uniformly ground with KBr (200 mg). The spectra was recorded
from 400 to 4000 cm−1 using 16 scans with the resolution of 4 cm−1.
Three replicates were collected for each group. The amide I band (1600-
1700 cm−1) in each spectrum was analysed using Ominic software
(Version 5.2, Nicolet, Madison,WI, USA).
X. Meng, et al. LWT - Food Science and Technology 108 (2019) 261–267

2.9. Statistical analysis

All data were expressed as mean values ± standard deviations.

Statistical analyses were carried out using OriginPro 8.5 software
(OriginLab, Northampton, MA, USA) and SPSS 21.0 software (SPSS Inc.,
Chicago, IL, USA). One-way analysis of variance (ANOVA) followed by
Tukey's tests (p ≤ 0.05) was used to compare the control and treated
groups.

3. Result and discussion

3.1. Changes in hardness

Hardness is an important parameter of high-protein nutrition bars.

Fig. 1. Changes in hardness of WPC bars with and without anticaking agents
(SiO2 and Ca3(PO4)2) during storage at 37 °C. WPC bars were high-protein
nutrition bars formulated with whey protein concentrate. SiO2 and Ca3(PO4)2
were used as anticaking agents at a level of 0.5 g/100 g bar. Data are mean
values of 9 measurements on three samples, with standard deviation indicated
by vertical error bars. the control; WPC bars with SiO2; WPC bars with
Ca3(PO4)2.
It is often increased over storage, mainly due to protein aggregation via
physico-chemical changes. The changes in hardness of WPC bars with
and without anticaking agents during storage are shown in Fig. 1. The
hardness of all WPC bars was obviously increased, in accordance with
previous reports in high-protein nutrition bars (Banach, Clark, &
Lamsal, 2018; Hogan et al., 2012; Lu et al., 2016). WPC bars with SiO2
and Ca3(PO4)2 had slightly lower hardness than the control. That is, the
increment in WPC bars' hardness was significantly slowed by anticaking
agents, especially SiO2. And there were significant differences between
WPC bars with SiO2 and Ca3(PO4)2 in the late stage of storage. The
hardness of WPC bars with SiO2 and Ca3(PO4)2 on day 35 were 54.07%

Fig. 2. CSLM images of WPC bars with and without anticaking agents (SiO2 and Ca3(PO4)2) after storage at 37 °C for 0, 21 and 35 days. WPC bars were high-protein
nutrition bars formulated with whey protein concentrate. SiO2 and Ca3(PO4)2 were used as anticaking agents at a level of 0.5 g/100 g bar. Red indicates Nile red
staining (lipid-rich phase); green indicates fluorescein isothiocyanate staining (protein-rich phase); black indicates unstained phase. All the scale bars are 100.0 μm in
length. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

263
X. Meng, et al.
and 63.13% of control value, respectively. It has been suggested that
anticaking agents create moisture barriers on the surface of hygroscopic
particles or physical barriers between ingredient particles through
steric hindrance (Lipasek et al., 2012; Nurhadi & Roos, 2017). Espe-
cially, anticaking agents can compete for available water (Lipasek et al.,
2012) and thus reduce water activity difference between adjacent in-
gredients. Discontinuities in chemical potentials of water between in-
gredients or micro-regions are generally the major driving force for
bar's hardening (Purwanti et al., 2010). Hogan et al. (2012) and Li et al.
(2017) considered minimising water activity difference between dry
powder and liquid phases to reduce molecules migration as another
means for controlling bar's hardening. The addition of SiO2 and
Ca3(PO4)2 might restrain the migration of water into whey protein
powders and simultaneously limit maltose redistribution, thus pre-
venting protein aggregation. On the other hand, SiO2 had more im-
provement in WPC bars' texture than Ca3(PO4)2. It was likely attributed
to the variation in water absorbability of the used anticaking agents.
3.2. Change in microstructure
High-protein nutrition bar is regarded as a non-equilibrium multi-
component food system of dry protein powder with syrup, glycerol,
phospholipid and oil. Small molecules such as water, glycerol and sugar
in the matrix may migrate and redistribute, causing deterioration in
microstructure and texture (Lu et al., 2016; Mcmahon et al., 2009). The
changes in the microstructure of WPC bars with and without anticaking
agents (SiO2 and Ca3(PO4)2) during storage are shown in Fig. 2. Three
interspersed phases were observed in CLSM images, including protein-
rich phase (green), lipid-rich phase (red) and unstained phase (black).
The unstained phase was some unhydrated protein powders or aqueous
solution containing water, glycerol and maltose. Initial difference in
WPC bars microstructures was not apparent.
No evidence of constituent phase separation was observed in all
WPC bars, as reported by Hogan et al. (2012) and Mcmahon et al.
(2009). It indicated that the direction of water migration was from
aqueous solution into protein powders, instead of the direction from
whey protein powders into aqueous solution. With the prolongation of
storage, protein-rich phase in the control was obviously increased along
with the decrease in unstained phase, and rearranged in a more com-
pact network-like matrix. But limited microstructure changes were
observed in WPC bars with SiO2 and Ca3(PO4)2 over storage. Protein-
rich phase in WPC bars with anticaking agents appeared to be still a
dispersive structure with the distribution of unstained phase. Hogan
et al. (2012) reported that accelerated storage led to fusion of protein
particles and an almost complete loss of protein particles definition,
even the development of a more homogeneous matrix, which resulted
in a firmer texture. There is not enough water in high-protein nutrition
bar. Water migration is expected to cause protein powder hydrating,
swelling and subsequent crosslinking. Limited change in bar micro-
structure further confirmed that SiO2 and Ca3(PO4)2 markedly hindered
the migration of small molecules (especially water and maltose) into
whey protein powders, thus inhibiting the formation of network-like
matrice and hardened texture.
3.3. Changes in colour and free amino group content
Maillard reactions have been implicated in the hardening develop-
ment of high-protein nutrition bars (Banach et al., 2014; Zhou, Guo,
Liu, Liu, & Labuza, 2013). Colour and free amino group content are the
common indicators to reflect the progress of Maillard reactions. The
changes in colour and free amine group content of WPC bars with and
without anticaking agents (SiO2 or Ca3(PO4)2) are shown in Table 1 and
Fig. 3, respectively.
L* values of all the WPC bars were significantly decreased along
with marked increases in a* and b* values, as reported by Mcmahon
et al. (2009). The colour of WPC bars turned yellow even brown. It
264
LWT - Food Science and Technology 108 (2019) 261–267
suggested the onset of late stage of Maillard reactions. The variations in
L, a* and b* values of WPC bars were retarded to some extent by SiO2
and Ca3(PO4)2, especially after 21-day storage. On the other hand, the
content of free amino groups in WPC bars was sharply reduced with
storage time, in accordance with the finding of Warmbier, Schnickels,
and Labuza (2010) and Banach et al. (2018). The loss of free amino
groups was obviously delayed by anticaking agents, particularly SiO2.
But there were no significant differences between WPC bars with SiO2
and Ca3(PO4)2. On day 35, free amino group content was reduced by
40.91% in WPC bars with SiO2 and 45.62% in WPC bars with
Ca3(PO4)2, but 69.21% for the control. All of these results demonstrated
that SiO2 and Ca3(PO4)2 could block the progress of Maillard reactions
in WPC bars. In low and intermediate moisture foods, free amino
groups located within the interior of proteins or in sterically inhibited
positions are unavailable to participate in Maillard reactions (Loveday,
Hindmarsh, Creamer, & Singh, 2009). Anticaking agents inhibited the
hydration and consequent flexibility of whey proteins, thus causing less
free amino groups to react with maltose. Furthermore, there were less
maltose migrated into protein powders due to the decreased water ac-
tivity difference by anticaking agents. It should be noted that the loss of
free amino groups was well correlated to the increment of hardness
(correlation coefficient r = 0.86–0.98). That is, Maillard reactions lo-
gically take part in bar hardening; although some researchers con-
sidered Maillard reactions were not a significant factor for hard texture
(Loveday et al., 2010; Mcmahon et al., 2009).
3.4. Changes in protein patterns
Protein aggregation gives rise to strong and tight networks which
leads to hard or tough textures. To further illustrate the formation of
Maillard-induced aggregates, protein patterns of WPC bars with and
without anticaking agents (SiO2 or Ca3(PO4)2) were analysed by SDS-
PAGE under reducing condition, and are depicted in Fig. 4. Three
prominent bands near 66, 20 and 14.2 kDa were clearly observed on
day 0, indicating the presence of bovine serum albumin (BSA), β-lac-
toglobulin (β-LG) and α-lactalbumin (α-LA) respectively. The bands of
β-LG and α-LA progressively diffused and moved upward with storage
time. And there were several larger protein bands in the 97–116 kDa
range, along with the weakening of BSA and casein's bands. These
changes were similar to those of whey proteins conjugated with low
molecular sugar via Maillard reactions in a dry state (Li, Luo, & Feng,
2011; Liu, Kong, Han, Sun, & Li, 2014). Zhou et al. (2013) also found
protein aggregation via Maillard reactions in protein bar model system.
Maillard reactions can lead to the appearance of high molecular mass
polymers in protein patterns. In comparison with the control, there
were lower intensities of larger protein bands in WPC bars with SiO2
and Ca3(PO4)2, especially the former. And the upward-diffusion of β-LG
and α-LA bands were slowed by the addition of anticaking agents. It
indicated that SiO2 and Ca3(PO4)2 could limit the Maillard-induced
protein aggregation in the bars. On the other hand, the intensities of
larger protein bands in the range of 97–116 kDa were not changed in
WPC bars with SiO2 during 35-day storage. The modification of whey
proteins was relatively slow, and it might take weeks or months to
develop the extensive glycation and aggregation of protein molecules.
3.5. Changes in FTIR spectra
FTIR spectra of WPC bars with and without anticaking agents (SiO2
or Ca3(PO4)2) are shown in Fig. 5, and the secondary structure content
of whey protein is given in Table 2. Similar spectra were found in WPC
bars on day 35, regardless of the presence of anticaking agents. The
main absorbance peaks were found at 3200–3400 cm−1 (amide A, the
stretching of OeH groups and NeH groups), 2850–2920 cm−1 (amide
B, the stretching of CeH groups), 1746 cm−1 (the stretching of C]O in
ester bonds), 1664 cm−1 (amide I, the stretching of CeN and C]O in
CONH groups) and 1542 cm−1 (amide II, the stretching of CeN groups
X. Meng, et al. LWT - Food Science and Technology 108 (2019) 261–267

Table 1
Changes in colour of WPC bars with and without anticaking agents (SiO2 and Ca3(PO4)2) during storage at 37 °C.
Storage time (day) L* a* b*

Control SiO2 Ca3(PO4)2 Control SiO2 Ca3(PO4)2 Control SiO2 Ca3(PO4)2

aA aA aA dA cA cA dA dA
0 83.5 ± 0.5 83.9 ± 0.5 84.8 ± 1.1 4.3 ± 0.1 4.2 ± 0.1 4.1 ± 0.1 25.5 ± 0.3 24.3 ± 0.8 24.5 ± 0.3dA
7 79.9 ± 0.4bA 81.8 ± 1.3aA 80.9 ± 0.4aA 5.6 ± 0.1cA 4.8 ± 0.1cA 4.7 ± 0.1cA 29.2 ± 0.7cA 26.4 ± 0.6dC 28.7 ± 0.2cB
14 77.8 ± 1.0bA 78.2 ± 0.4bA 78.1 ± 0.2bA 6.8 ± 0.1bA 6.4 ± 0.1bB 7.0 ± 0.1bA 31.9 ± 0.5cA 28.8 ± 0.1cC 30.5 ± 0.3cB
21 73.5 ± 0.5cB 76.1 ± 0.9bA 76.7 ± 0.6bA 8.6 ± 0.3aA 7.7 ± 0.1bB 7.9 ± 0.1bB 34.8 ± 0.7bA 31.0 ± 0.6cB 31.1 ± 0.4bB
28 70.7 ± 0.8cB 75.9 ± 1.2bA 74.2 ± 0.6cA 9.8 ± 0.3aA 8.8 ± 0.1aB 9.0 ± 0.1aB 37.3 ± 0.9aA 32.1 ± 0.5bB 32.9 ± 0.6bB
35 68.3 ± 0.5dB 75.5 ± 0.6bA 74.0 ± 0.3cA 10.6 ± 0.2aA 9.1 ± 0.1aC 9.8 ± 0.1aB 37.2 ± 0.7aA 33.1 ± 0.1aC 34.6 ± 0.8aB

WPC bars were high-protein nutrition bars formulated with whey protein concentrate. SiO2 and Ca3(PO4)2 were used as anticaking agents at a level of 0.5 g/100 g bar.
The bars were stored at 37 °C for 35 days.
Data are mean values ± standard deviation of 9 measurements on three samples.
a–d different letters in the same column indicate significant difference (p ≤ 0.05) among the same WPC bars during storage.
A–C different letters in the same row indicate significant difference (p ≤ 0.05) among WPC bars with and without anticaking agents on the same day.

crosslinking via hydrogen bonds (Ramos et al., 2013). It confirmed that

Fig. 3. Changes of relative free amino group content in WPC bars with and
without anticaking agents (SiO2 and Ca3(PO4)2) during storage at 37 °C. WPC
bars were high-protein nutrition bars formulated with whey protein con-
centrate. SiO2 and Ca3(PO4)2 were used as anticaking agents at a level of 0.5 g/
100 g bar. Data are mean values of three triplicate, with standard deviation
indicated by vertical error bars. the control; WPC bars with SiO2; WPC bars
with Ca3(PO4)2.
and bending of NeH in CONH groups) and 1036 cm−1 (the stretching
of CeO and CeN groups) (Hu, Li, Zhang, Kou, & Zhou, 2018).
Amide I region (1600-1700 cm−1) is directly associated to protein
backbone conformation (Carbonaro & Nucara, 2010). The peaks at
1628 (parallel β-sheet), 1646 (random coil), 1667 (β-turn) and 1682
(antiparallel β-sheet) cm−1 in the control were shifted to 1630, 1650,
1666 and 1691 cm−1 in WPC bars with anticaking agents, respectively.
The peak at 1658 cm−1 (α-helix) in the control was shifted to
1664 cm−1 in WPC bars with Ca3(PO4)2, but not changed in WPC bars
with SiO2. A shift to a higher wavenumber suggested weaker
there was fewer water migrating into whey protein particles when
added with anticaking agents. For the control, the contents of α-helix
and β-turn were obviously decreased over storage, with concomitant
increases in β-sheet and random coil's contents. Considering the in-
creased α-helix and decreased β-sheet in whey protein isolate/glycerol
mixture (Hogan et al., 2012), Maillard reactions occurred in WPC bars
might be responsible for the conversion of α-helix to β-sheet and
random coil. Wang, Bao, and Chen (2013) also noted that the glycation
of whey protein isolated enhanced β-sheet proportion at the expense of
α-helical structure. The formation of β-sheet structures is commonly
found in aggregated proteins, and relate to the bars' hardening
(Nishanthi et al., 2017). On day 35, the highest α-helix content and the
lowest β-sheet content were observed in WPC bars with SiO2, followed
by WPC bars with Ca3(PO4)2 and the control. The phenomena coincided
with the loss of free amino groups (Fig. 3). The addition of SiO2 and
Ca3(PO4)2 could minimum the secondary structure transformation
through inhibiting the hydration and consequent Maillard reactions of
whey proteins, then stabilize the bars' texture during storage.
4. Conclusion
Hardening is one of the major problems for high-protein nutrition
bars, which greatly limits the shelf life and thus hinder the development
of such products. The present study clearly showed that anticaking
agents (SiO2 and Ca3(PO4)2) had an obvious effect to prevent the
hardening of high-protein nutrition bars formulated with whey protein
concentrate (WPC bars). It was presumed that anticaking agents could
limit the hydrating, swelling and Maillard-induced aggregation of whey
proteins due to their strong hydrophilic ability, thus slowing the dete-
rioration of bar's texture. Given low dosage and high efficiency, food-
grade anticaking agents would have potential applications in the
quality control of high-protein nutrition bars.
Acknowledgements
The contribution of funding from the key project of Zhejiang

Fig. 4. SDS-PAGE images of WPC bars with and

without anticaking agents (SiO2 and Ca3(PO4)2) after
storage at 37 °C for 0, 7, 14, 21, 28 and 35 days. WPC
bars were high-protein nutrition bars formulated
with whey protein concentrate. SiO2 and Ca3(PO4)2
were used as anticaking agents at a level of 0.5 g/
100 g bar. Electrophoresis was performed under re-
ducing conditions. M: molecular weight marker;
BSA: bovine serum albumin; CN: casein; β-LG: β-
lactoglobulin; α-LA: α-lactalbumin.

265
X. Meng, et al. LWT - Food Science and Technology 108 (2019) 261–267

Fig. 5. FTIR spectra of WPC bars in the 400-4000 cm−1 region (A) and the 1600-1700 cm−1 region (B) after storage at 37 °C for 35 days. WPC bars were high-protein
nutrition bars formulated with whey protein concentrate. SiO2 and Ca3(PO4)2 were used as anticaking agents at a level of 0.5 g/100 g bar.

Table 2
Changes in secondary structure content (%) of whey protein in WPC bars with and without anticaking agents (SiO2 and Ca3(PO4)2) during storage at 37 °C.
Storage time (day) α-helix β-sheet β-turn Random coil

Control 0 4.60 ± 0.16a 72.41 ± 0.43d 21.83 ± 0.2a 1.12 ± 0.24f

7 1.60 ± 0.09e 77.78 ± 0.21a 13.88 ± 0.2e 6.70 ± 0.14b
35 0.92 ± 0.03f 78.39 ± 0.27a 10.83 ± 0.2f 9.89 ± 0.21a
SiO2 0 4.76 ± 0.52a 71.32 ± 0.22e 22.11 ± 0.2a 1.81 ± 0.10e
7 3.52 ± 0.11b 73.62 ± 0.14c 19.31 ± 0.2b 3.58 ± 0.43d
35 2.47 ± 0.08d 74.44 ± 0.17b 16.50 ± 0.2d 6.59 ± 0.15b
Ca3(PO4)2 0 4.81 ± 0.16a 71.15 ± 0.40e 22.56 ± 0.2a 1.48 ± 0.12e
7 3.19 ± 0.32c 73.27 ± 0.21c 20.53 ± 0.1b 3.01 ± 0.33d
35 1.46 ± 0.02e 75.40 ± 0.24b 18.50 ± 0.16c 4.64 ± 0.06c

WPC bars were high-protein nutrition bars formulated with whey protein concentrate. SiO2 and Ca3(PO4)2 were used as anticaking agents at a level of 0.5 g/100 g bar.
The bars were stored at 37 °C for 35 days.
Data are mean values ± standard deviation of three scanning acquisitions.
a–f different letters in the same column indicate significant difference (p ≤ 0.05) among the same WPC bars during storage.

Province in China (NO. 2019C02069) is gratefully acknowledged.
References
Aguilera, J. M., Valle, J. M. D., & Karel, M. (1995). Caking phenomena in amorphous food
powders. Trends in Food Science & Technology, 6(5), 149–155.
Banach, J. C., Clark, S., & Lamsal, B. P. (2013). Characterization of extruded and toasted
milk protein concentrates. Journal of Food Science, 78(6), 861–867.
Banach, J. C., Clark, S., & Lamsal, B. P. (2014). Texture and other changes during storage
in model high-protein nutrition bars formulated with modified milk protein con-
centrates. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology,
56(1), 77–86.
Banach, J. C., Clark, S., & Lamsal, B. P. (2018). Extrusion modifies some physicochemical
properties of milk protein concentrate for improved performance in high-protein
nutrition bars. Journal of the Science of Food and Agriculture, 98(1), 391–399.
Carbonaro, M., & Nucara, A. (2010). Secondary structure of food proteins by Fourier
transform spectroscopy in the mid-infrared region. Amino Acids, 38(3), 679–690.
Chang, L. S., Karim, R., Abdulkarim, S. M., Yusof, Y. A., & Ghazali, H. M. (2018). Storage
stability, color kinetics and morphology of spray-dried soursop (Annona muricata L.)
powder: Effect of anticaking agents. International Journal of Food Properties, 21(1),
1937–1954.
FDA (2019). Electronic code of federal regulations title 21. http://www.ecfr.gov/Title_21/
Chapter_1/Subchapter_B/part_182/Subpart_B/182.1217/, Accessed date: 1 March
2019.
Hogan, S. A., Chaurin, V., O'Kennedy, B. T., & Kelly, P. M. (2012). Influence of dairy
proteins on textural changes in high-protein bars. International Dairy Journal, 26(1),
58–65.
Hu, Y., Li, Y., Zhang, W., Kou, G., & Zhou, Z. (2018). Physical stability and antioxidant
activity of citrus flavonoids in Arabic gum-stabilized microcapsules: Modulation of
whey protein concentrate. Food Hydrocolloids, 77(4), 588–597.
Kelly, P. (2019). Chapter 9 - whey protein ingredient applications. In H. C. Deeth, & N.
Bansal (Eds.). Whey proteins from milk to medicine (pp. 335–375). Cambridge,
Massachusetts: Academic Press.
Li, Z., Luo, Y., & Feng, L. (2011). Effects of Maillard reaction conditions on the anti-
genicity of α-lactalbumin and β-lactoglobulin in whey protein conjugated with
maltose. European Food Research and Technology, 233(3), 387–394.
Lipasek, R. A., Ortiz, J. C., Taylor, L. S., & Mauer, L. J. (2012). Effects of anticaking agents
and storage conditions on the moisture sorption, caking, and flowability of deli-
quescent ingredients. Food Research International, 45(1), 369–380.
Lipasek, R. A., Taylor, L. S., & Mauer, L. J. (2011). Effects of anticaking agents and re-
lative humidity on the physical and chemical stability of powdered vitamin C. Journal
of Food Science, 76(7), 1062–1074.
Liu, Q., Kong, B., Han, J., Sun, C., & Li, P. (2014). Structure and antioxidant activity of
whey protein isolate conjugated with glucose via the Maillard reaction under dry-
heating conditions. Food Structure, 1(2), 145–154.
Li, J., Wu, Y., Ma, Y., Lu, N., Regenstein, J. M., & Zhou, P. (2017). Effects of addition of
hydrocolloids on the textural and structural properties of high-protein intermediate
moisture food model systems containing sodium caseinate. Food and Function, 8(8),
2897–2904.

266
X. Meng, et al.
Loveday, S. M., Hindmarsh, J. P., Creamer, L. K., & Singh, H. (2009). Physicochemical
changes in a model protein bar during storage. Food Research International, 42(7),
798–806.
Loveday, S. M., Hindmarsh, J. P., Creamer, L. K., & Singh, H. (2010). Physicochemical
changes in intermediate-moisture protein bars made with whey protein or calcium
caseinate. Food Research International, 43(5), 1321–1328.
Lu, N., Zhang, L., Zhang, X., Li, J., Labuza, T. P., & Zhou, P. (2016). Molecular migration
in high-protein intermediate-moisture foods during the early stage of storage:
Variations between dairy and soy proteins and effects on texture. Food Research
International, 82(4), 34–43.
Mcmahon, D. J., Adams, S. L., & Mcmanus, W. R. (2009). Hardening of high-protein
nutrition bars and sugar/polyol-protein phase separation. Journal of Food Science,
74(6), 312–321.
Mezzenga, R. (2007). Equilibrium and non-equilibrium structures in complex food sys-
tems. Food Hydrocolloids, 21(5), 674–682.
Nishanthi, M., Chandrapala, J., & Vasiljevic, T. (2017). Compositional and structural
properties of whey proteins of sweet, acid and salty whey concentrates and their
respective spray dried powders. International Dairy Journal, 74(11), 49–56.
Nurhadi, B., & Roos, Y. H. (2017). Influence of anti-caking agent on the water sorption
isotherm and flow-ability properties of vacuum dried honey powder. Journal of Food
Engineering, 210(1), 76–82.
Potes, N., Kerry, J. P., & Roos, Y. H. (2014). Protein modifications in high protein-oil and
protein-oil-sugar systems at low water activity. Food Biophysics, 9(1), 49–60.
Purwanti, N., van der Goot, A. J., Boom, R., & Vereijken, J. (2010). New directions to-
wards structure formation and stability of protein-rich foods from globular proteins.
Trends in Food Science & Technology, 21(2), 85–94.
Ramos, Ó. L., Reinas, I., Silva, S. I., Fernandes, J. C., Cerqueira, M. A., Pereira, R. N., ...
Malcata, F. X. (2013). Effect of whey protein purity and glycerol content upon
267
LWT - Food Science and Technology 108 (2019) 261–267
physical properties of edible films manufactured therefrom. Food Hydrocolloids,
30(1), 110–122.
Rao, Q., Kamdar, A. K., Guo, M., & Labuza, T. P. (2016). Effect of bovine casein and its
hydrolysates on hardening in protein dough model systems during storage. Food
Control, 60(2), 621–628.
Rao, Q., Roccasmith, J. R., & Labuza, T. P. (2012). Moisture-induced quality changes of
hen egg white proteins in a protein/water model system. Journal of Agricultural and
Food Chemistry, 60(42), 10625–10633.
Wang, W. Q., Bao, Y. H., & Chen, Y. (2013). Characteristics and antioxidant activity of
water-soluble Maillard reaction products from interactions in a whey protein isolate
and sugars system. Food Chemistry, 139(1–4), 355–361.
Warmbier, H. C., Schnickels, R. A., & Labuza, T. P. (2010). Nonenzymatic browning ki-
netics in an intermediate moisture model system: Effect of glucose to lysine ratio.
Journal of Food Science, 41(5), 981–983.
Yang, Y., Faust, J. J., Schoepf, J., Hristovski, K., Capco, D. G., Herckes, P., et al. (2016).
Survey of food-grade silica dioxide nanomaterial occurrence, characterization,
human gut impacts and fate across its lifecycle. The Science of the Total Environment,
565(9), 902–912.
Zhou, P., Guo, M., Liu, D., Liu, X., & Labuza, T. P. (2013). Maillard-reaction-induced
modification and aggregation of proteins and hardening of texture in protein bar
model systems. Journal of Food Science, 78(3), 437–444.
Zhou, P., Liu, X., & Labuza, T. P. (2008). Effects of moisture-induced whey protein ag-
gregation on protein conformation, the state of water molecules, and the micro-
structure and texture of high-protein-containing matrix. Journal of Agricultural and
Food Chemistry, 56(12), 4534–4540.
Zhu, D., & Labuza, T. P. (2010). Effect of cysteine on lowering protein aggregation and
subsequent hardening of whey protein isolate (WPI) protein bars in WPI/buffer
model systems. Journal of Agricultural and Food Chemistry, 58(13), 7970–7979.