Food Chemistry 174 (2015) 97–103

Contents lists available at ScienceDirect

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

Isolation of micro- and nano-crystalline cellulose particles and

fabrication of crystalline particles-loaded whey protein cold-set gel
Maede Ahmadi a, Ashkan Madadlou a,b,⇑, Ali Akbar Sabouri c
a
Department of Food Science & Engineering, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran
b
Center of Excellence for Application of Modern Technologies for Producing Functional Foods and Drinks, University College of Agriculture and Natural Resources,
University of Tehran, Karaj, Iran
c
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran

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

Article history: Micro- and nano-crystalline cellulose (MCC and NCC, respectively) particles isolated from cellulose filter
Received 12 August 2014 papers via acid digestion were characterised and loaded into a heat-denatured whey protein isolate (WPI)
Received in revised form 6 October 2014 solution which was subsequently cold-set-gelled. Both the MCC and NCC particles were rod-shaped and
Accepted 6 November 2014
had higher crystallinity degrees than had the cellulose source they were isolated from. The hydrodynamic
Available online 15 November 2014
diameter of NCC particles was 15 nm. Fourier transform infrared (FTIR) spectroscopy suggested more
surface hydroxyl groups on the NCC than the MCC particles and complete digestion of hemicellulose
Keywords:
on the cellulosic substrate by acid. MCC- and NCC-loaded WPI gel matrices were topographically less
Cellulose
Nanocrystalline cellulose
uniform and contained many more undulations in comparison to the crystal-free counterpart. It was
Microcrystalline cellulose found, using dynamic rheometry and penetration tests, that the crystal loading into WPI gels weakened
Whey protein the texture. Non-covalent interactions between the cellulose crystals and whey protein strands were
Gel proposed in the gel structure according to FTIR results.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction bonding of glucan residues (Khandelwal & Windle, 2014). The glu-
can sub-units in cellulose assemble and merge into nanofibrils,
Gelation is one the most important functional properties of with a highly ordered structure (Abdul Khalil, Bhat, & Ireana
whey proteins and is mainly attributed to b-lactoglobulin Yusra, 2012). Cellulose fibres, however, are not fully crystalline
(Gezimati, Creamer, & Singh, 1997). Amongst the several methods and have amorphous regions of a less ordered structure (Klemm,
known to induce whey protein gelation, the cold-set procedure is Heublein, Fink, & Bohn, 2005). Preferential hydrolysis of the amor-
regarded with great enthusiasm as it provides a route for the for- phous domains in cellulose fibres with mineral acids, enzymes and
mation of fine-stranded gels at ambient temperatures (Alting, microorganisms (and occasion accompaniment of mechanical
Hamer, de Kruif, & Visschers, 2003). Cold-set gelation encompasses disintegration), causes crystalline residues with various shapes,
two distinctly separate steps. Initially, whey protein solutions are crystallinities and dimensions (Beck-Candanedo, Roman, & Gray,
heated at pH values far from the proteins isoelectric point 2005). The most common method is acidic hydrolysis, as it is
(pI  5.1), and at low ionic strengths. This causes protein denatur- relatively inexpensive and provides highly crystalline products
ation and the formation of tiny soluble aggregates. This causes the (Hanna, Biby, & Miladinov, 2001). Sulphuric acid-hydrolysed
electrostatic repulsion amongst the proteins to be decreased cellulose suspensions are highly stable colloids, owing to the
(through pH manipulation and/or ionic strength elaboration), sulphate-bearing hydroxyl groups that establish considerable elec-
which results in protein aggregation. Finally a gel is formed (Ako, trostatic repulsion amongst cellulose-derived objects (Bondeson,
Nicolai, & Durand, 2010). Kvien, & Oksman, 2006). Nano-crystalline cellulose (NCC) particles
Cellulose, an abundant organic homopolymer, has a hierarchical are rod-like particles with a highly crystalline structure, high
architecture arising from the inter- and intra-molecular hydrogen aspect ratio, large surface area, unique tensile strength (0.8–
10 GPa), low density and high Young’s modulus (100–170 GPa)
⇑ Corresponding author at: Department of Food Science & Engineering, University (Habibi, Lucia, & Rojas, 2010). It can be used for the reinforcement
College of Agriculture and Natural Resources, University of Tehran, PO Box: 31587- of polymer composites and enhancement of their thermal stability
77871, Karaj, Iran. Tel.: +98 26 3224 8804; fax: +98 26 3224 9453. and mechanical properties. Another cellulose-derived product,
E-mail address: a.madadlou@ut.ac.ir (A. Madadlou). microcrystalline cellulose (MCC) is a white, tasteless, odourless

http://dx.doi.org/10.1016/j.foodchem.2014.11.038
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
98 M. Ahmadi et al. / Food Chemistry 174 (2015) 97–103

and crystalline powder. It is used as a suspension-stabilizer, thick- and stored at 4 °C for 16 h (Britten & Giroux, 2001). In preparation
ener, a filler or binder in tablets and a flow characteristics-modifier of MCC- and NCC-loaded gels, the heat-treated and cooled WPI
in various formulations (Adel, Abd El-Wahab, Ibrahim, & Al-Shemy, solution was enriched with either MCC or NCC particles
2011; Wang, Shang, Song, & Lee, 2010). (10 mg ml1) before gelation induction.
Incorporation of carbohydrate polymers into the composition of
cold-set whey protein gels results in mixed gels that exhibit differ-
ent structural, mechanical and sensorial characteristics compared 2.4. Hydrodynamic size measurement of NCC and MCC particles
with the gels made solely of proteins (Bertrand & Turgeon, 2007).
There is no report in the literature on the characteristics of The length and diameter of NCC and MCC particles in suspen-
cellulose crystalline particles-loaded whey protein gels. We antic- sion were measured with photon correlation spectroscopy method
ipated that loading of cellulose crystals into a heat-denatured using a dynamic light scattering particle size analyser (ZetaPALS,
whey protein solution would impact the textural and functional Brookhaven Instruments Co., NY, USA). The suspensions were
characteristics of the subsequently prepared cold-set gel. There- diluted with distilled water to 0.5 mg ml1, and subjected to
fore, microcrystalline and nanocrystalline cellulose particles were duplicate size measurements with 5 readings for each.
produced, characterised and incorporated into the gel matrix,
followed by examination of the gel characteristics.
2.5. Fourier transform infrared (FTIR) spectroscopy
2. Materials and methods
The cellulose filter paper, MCC particles, NCC particles, and gel
2.1. Materials powders were vacuum-dried at 50 °C and 180 mmHg. The dry
samples were mixed with potassium bromide, pressed into disks
Whey protein isolate (WPI), with at least an 88% protein and scanned with a Perkin Elmer FTIR spectrometer (Perkin Elmer
content, was a kind gift from Arla Food Ingredients (Vibyi, Co., MA, USA). The IR spectroscopic analysis was performed from
Denmark). WhatmanÒ filter paper number 1 (98% alpha cellulose) 450 to 4500 cm1 with resolution of 4 cm1.
was supplied by Whatman International Ltd. (Maidstone, England).
Sulphuric acid, sodium hydroxide, calcium chloride, glucono-d-
lactone and sodium azide were purchased from Merck (Darmstadt, 2.6. Morphological and topographical features of MCC particles, NCC
Germany). particles and gels

2.2. Preparation of MCC and NCC particles Atomic force microscopy (AFM) was used to characterise the
topography of the gels, as well as, that of NCC particles. The AFM
Micro- and nano-crystalline cellulose particles were prepared imaging was carried out by a Nanowizard II microscope (JPK, Ger-
many) in the intermittent contact mode. NCC samples were diluted
through the acid hydrolysis procedure. For preparation of NCC par-
ticles, filter papers cut into small pieces (2 g) were hydrolysed with with distilled water to 100 lg ml1 and then dripped onto a plastic
lamella and air-dried before microscopic imaging. Gel specimens
50 ml of sulphuric acid (64%) at 45 °C for 75 min under continuous
stirring at 200 rpm. The cellulose hydrolysate was centrifuged were cut from the interior of the samples, using a razor blade,
and then fixed within glutaraldehyde (2.5%) for 1 h. Subsequently,
(D-37520 osterode am Harz, Germany) at 9400g for 10 min,
and the precipitate was washed with 2 M NaOH. This was followed the specimens were dehydrated by immersing in a series of
aqueous ethanol solutions with increasing alcohol concentration
by repeated centrifugation and washing with distilled water,
several times, until the supernatant became turbid (Bondeson (40%, 60%, 70%, 90% and 100%) and finally were placed on the
lamella and air-dried before microscopic imaging.
et al., 2006). The supernatant, which contained NCC, was either
vacuum-dried at 60 °C and 180 mmHg or stored at 4 °C for the Scanning electron microscopy (SEM) (KYKY-EM3200, china),
with an accelerating voltage of 26 kV, was employed to capture
same-day analysis.
For preparation of MCC, filter paper pieces (2 g) were hydroly- the morphological features of MCC particles. MCC powders were
coated with gold to submit them electrically conductive.
sed with 50 ml of sulphuric acid (64%) at 45 °C for 5 min while
being stirred at 200 rpm. Subsequently, large amounts of water
were added into the reaction beaker to stop the hydrolysis. Then
the suspension was neutralized with 15 M NaOH and agitated 2.7. X-ray diffraction (XRD)
harshly (T 18 digital ULTRA-TURRAX, IKA, Staufen, Germany) at
24,000 rpm for 5 min. The ground suspension was centrifuged The crystallinity degree of cellulose filter papers, NCC and MCC
(D-78532, Hettich, Tuttlingen, Germany) at 1520g for 10 min, was measured through X-ray diffraction (XRD) analysis, carried out
and the precipitate was washed with distilled water. The centrifu- by using a PW3040/60 X’Pert diffractometer (Philips, Netherlands)
gation and washing steps were repeated several times to eliminate with Cu Ka radiation (k = 0.154056 nm). Operating voltage and
any residues of chemical substances (Ilindra & Dhake, 2008). The filament current were 40 kV and 30 mA, respectively. The samples
sediment, which contained MCC particles, was vacuum-dried at were scanned in the interval 5° 6 2h 6 30° using a step size of
60 °C and 180 mmHg. 0.02°.
The crystallinity index (CrI) of samples was calculated, using the
following equation (Eq. (1)) (Segal, Creely, Martin, & Conrad, 1959):
2.3. Gel preparation
 
WPI powder was dissolved in distilled water (80 mg ml1) con- I  I’
CrI ¼  100% ð1Þ
taining sodium azide (0.1 mg ml1) under continuous stirring at I
500 rpm for 60 min. The solution was kept at 4 °C for 12 h to allow
for complete hydration and then heated at 80 °C for 15 min, where I is the height of the peak at 2h = 22.6°, corresponding to the
followed by rapid cooling with tap water. The solution was then crystalline and amorphous fraction and I0 is the height measured at
injected with CaCl2 (4 mM) and glucono-d-lactone (6.7 mg ml1) 2h = 18°, corresponding to the amorphous fraction.
 M. Ahmadi et al. / Food Chemistry 174 (2015) 97–103 99

2.8. Textural analysis

2.8.1. Oscillatory rheological measurement

The small amplitude oscillatory shear test was employed to
measure the dynamic moduli of WPI and MCC-WPI and NCC-WPI
gels. A Bohlin Gemini 2 rheometer (Malvern Instruments Ltd.,
Worcestershire, UK) was used to perform the measurements. The
measuring geometry consisted of two parallel plates with a
diameter of 25 mm and a 1 mm gap size (specimen thickness). A
frequency sweep test at range of 0.01–1 Hz, with a strain of 1%,
was carried out to determine the storage (G0 ), loss (G00 ) and
complex (G⁄) moduli of the samples.

2.8.2. Penetration test

A texture analyser (Model M350-10CT, Rochdale, UK), equipped
with a 13 mm probe, was used for measuring the force required to
penetrate the WPI, MCC- and NCC-loaded WPI gels fabricated in
50 ml beakers as an index of gel hardness. Gels were penetrated
to a depth of 12 mm at speed of 50 mm min1.

2.8.3. Apparent viscosity of gel

Gel viscosity was measured by means of a Brookfield viscome-
ter (LVDV-II Pro, Brookfield Engineering Inc., USA) equipped with
the LV spindle No. 4 at shear rate of 1.22 s1. Viscosity was
determined in triplicate at 25 °C.

2.9. Statistical analysis

The data obtained were recorded as means ± standard devia-

tion. Significant differences between values at a p level of 0.05
(Duncan’s Test) were evaluated using SAS Software (version 9.1).

Fig. 1. Upper panel: AFM image of nanocrystalline cellulose (height image of

3. Results and discussion surface topography); lower panel: SEM image of microcrystalline cellulose.

3.1. Characterisation of MCC and NCC particles

rod-shaped MCC particles from kenaf bast and wood pulp. The
During the acid hydrolysis of cellulose, the acid protonates the almost identical morphology of MCC and NCC particles confirms
glucosidic oxygen or cyclic oxygen in the D-glucopyranose units the homologous structural hierarchy of cellulose. It is noteworthy
in the glucan chains of cellulose, and then (by water addition) that both the nano- and micro-crystalline cellulose rods did not
glucosidic bonds are slowly split (Lu & Hsieh, 2010). The amor- bend when dried for the microscopic imaging, which implies a firm
phous domains of cellulose are more sensitive to hydrolytic structure of crystalline particles, owing to their strong elastic
reactions and are rapidly destroyed (Hon & Shiraishi, 2000). The nature (Eichhorn et al., 2010).
longer the reaction time, the more intensive is the digestion of XRD exhibited a sharp high peak at 2h = 22.6° and two weak
the amorphous regions. Being negatively charged, by the sulphate peaks at 2h = 14.8° and 16.3° for all cellulose, NCC and MCC,
ester groups which had attached onto the glucose units during the samples (Fig. 2). These peaks are attributed to the crystalline cellu-
acid hydrolysis, the nanocrystalline cellulose suspension was lose I structure, which consisted of parallel glucan chains with
stable for longer. The microcrystalline cellulose suspension, how- repeating b-(1 ? 4)-D-glucopyranose units (Besbes, Alila, & Boufi,
ever, became increasingly viscous from day to day and almost 2011; Jiang, Esker, & Roman, 2010; Li & Renneckar, 2011). It is
gelled (data not shown). The hydrodynamic size measurement of suggested, based on the XRD results, that the crystalline cellulose
NCC particles suggested the presence of two populations of structure was preserved during acid hydrolysis. Li, Wang, and Liu
rod-like particles with different length distributions (112– (2011) reported that the crystal structure of cellulose was main-
243 nm and 453–844 nm) but identical diameter (15 ± 0.2 nm). It tained after acid hydrolysis.
is argued that acid hydrolysis of cellulose preferentially slacked/ The diffraction intensity at 2h = 18°, which is related to the
dissolved the amorphous and less ordered regions in the cellulose amorphous sections of cellulose (Segal et al., 1959), was weaker
fibres, leaving the crystalline regions intact. An atomic force for NCC particles (32 a.u.) than for cellulose and MCC particles
microscopy image of NCC is shown in Fig. 1 (upper panel). In agree- (55 a.u.). This reveals extensive destruction of the amorphous
ment with other reports (Kvien, Tanem, & Oksman, 2005; region of cellulose fibres during the longer reaction time in NCC
Petersson, Kvien, & Oksman, 2007), NCC particles were of rod-like isolation (Ghaderi, Mousavi, Yousefi, & Labbafi, 2014). The crystal-
shape with nanoscale diameters. linity indices (see Section 2.7) of the cellulose, MCC and NCC
The hydrodynamic size measurement of the microcrystalline particles were 84.8%, 86.9% and 89.1%, respectively. The harsher
cellulose particles suggested a diameter range of 165–243 nm the acid hydrolysis of cellulose, the higher was the crystallinity
and length range of 2183–4161 nm. A scanning electron micros- index of residual particles. Crystallinity indices of 72.5%, 74% and
copy image of the microcrystalline cellulose is shown in Fig. 1 75% have been reported for NCCs obtained from sweet potato
(lower panel). In similarity to the nanocrystalline counterpart, residue (Lu, Gui, Zheng, & Liu, 2013), bleached soft wood craft (Li
MCC particles were rod-shaped. Wang et al. (2010) also observed et al., 2011) and kenaf core woods (Chan, Chia, Zakaria, Ahmad, &
100 M. Ahmadi et al. / Food Chemistry 174 (2015) 97–103

2011) and was not present in the cellulose crystals due to acid
hydrolysis.

3.2. Characteristics of the gels

For the purpose of comparison, atomic force microscopy images

of WPI and MCC- and NCC-loaded WPI gels are illustrated in Fig. 4.
The height images of the gels (Fig. 4, right hand images) were
mapped by the rise and fall of the AFM cantilever, as the probe
tip scanned the surface of the gels. The height changes are shown
as bright and dark regions in the images, so that higher regions are
illustrated as brighter than others (Fishman, Cooke, & Coffin, 2004).
In the 3D gel images (Fig. 4, left hand images), it is observed that
the WPI gel was of much more uniform structure with fewer
undulations. In comparison with the WPI gel, the matrices of
both crystal-loaded gels were more heterogeneous. Arguably, the
integrity of protein gel matrix decreased due to incorporation of
NCC and MCC particles. The sulphate-bearing negatively charged
crystalline objects would likely repel the co-charged protein
strands (as long as pH was higher than the pI of proteins) and
interfere in protein network formation. Tavares and Lopes da
Silva (2003) pointed out that galactomannan presence negatively
affected the protein network development at pH of 5.0 via
hampering of the protein-to-protein interactions.
The frequency sweep test of gel samples was carried out to
investigate the influence of MCC and NCC loading on the dynamic
moduli of gel. The value of G0 was greater than that of G00 at any
Fig. 2. X-ray diffractograms of cellulose, nanocrystalline cellulose (NCC) and
frequency for all samples (Fig. 5). This reflects the major contribu-
microcrystalline cellulose (MCC).
tion of the elastic character in gel texture. Dynamic moduli were
frequency-dependent and increased with the increasing frequency.
This is attributed to the shorter time scale of the applied stress at
Dufresne, 2012), respectively. The considerably higher degree of higher frequencies resulting in less structural rearrangement and
crystallinity in NCC particles obtained in the present study is relaxation of fewer bonds (Sayadi, Madadlou, & Khosrowshahi,
attributed to the extremely high purity of the substrate used for 2013). Results show (Fig. 5) that crystal loading into the WPI gel
acid digestion, i.e. filter paper. It is noteworthy that non-processed decreased all three dynamic moduli because of the unpacking role
cellulose resources, including wood and bio-residues, encompass of crystals in the gel protein network. A supporting result has been
hemicellulose and lignin. In addition to cellulose sources, other reported for galactomannan-loaded WPI gel (Tavares & Lopes da
parameters, such as temperature and time of hydrolysis reaction Silva, 2003). The force required to penetrate the gels also decreased
affect the properties of the resulting cellulose crystals. These for crystal-loaded samples (Table 1). NCC decreased the penetra-
properties include crystallinity, moisture content, surface area, tion force more efficiently than did MCC, likely due to the higher
porosity and molecular weight (El-Sakhawy & Hassan, 2007). For number of negatively charged sulphate groups attached to the
example longer reaction times result in shorter nanoparticles with nanocrystal surface. This could provide a more intense repulsion
higher surface charges and smaller polydispersity index (Beck- between NCC particles and fusing protein strands and result in a
Candanedo et al., 2005). In addition, higher reaction temperatures less packed gel network. Notably, NCC was isolated through
(Habibi et al., 2010) and use of ultrasound (Pakzad, 2011) will a totally long duration acidic digestion but MCC was prepared via
reduce the size of nanoparticles. a short time pre-digestion with acid, followed by mechanical
The FTIR spectra of cellulose and its nano and microcrystalline disintegration. The apparent viscosity results (Table 1) followed a
offspring are demonstrated in Fig. 3a. The broad bands at 3000– similar trend to dynamic moduli and penetration force but the
3650 cm1 are attributed to the stretching vibration of free OH affect was not statistically significant (p > 0.05).
groups on the cellulose molecules. The absorption peak at Fig. 3b shows the FTIR spectra of WPI gel and cellulose crystal-
2900 cm1 corresponds to the stretching vibration of CH groups loaded WPI gels. The peak at 1537 cm1 corresponded to C@O
in cellulose. The peak at around 1640 cm1 corresponded to stretching of amide I in a-lactalbumin and b-lactoglobulin a-
absorbed water (Ashori, Babaee, Jonoobi, & Hamzeh, 2014; helical structure (Byler & Purcell, 1989). The peak observed at
Bajpai, Pathak, Chand, & Soni, 2013) and was of higher intensity 1649 cm1 is attributed to C–N–H in-plane bending and C–N
for NCC particles. This might reflect a greater number of surface stretching of the amide II band. Two peaks appeared at 1450 and
sulphate and/or hydroxyl groups in the NCC particles, and there- 3291 cm1 correlate to C–H bending and N–H stretching, respec-
fore they would be capable of forming ion–dipole and/or dipole– tively. The peak of O–H bending vibration of deionised carboxylic
dipole interactions with water molecules. It has been reported acid is observed at 1394 cm1 (Bagheri, Madadlou, Yarmand, &
(Pakzad, 2011) that the aspect ratio (length to diameter ratio) of Mousavi, 2013(. FTIR spectroscopy suggests that microcrystalline
cellulose increases towards the individualisation of sub-units. This and nanocrystalline cellulose particles were entrapped physically
would bring more hydroxyl groups into contact with the surround- (by non-covalent interactions) in the whey protein gel network
ing water molecules. Bending of the –C-6 CH2– group indicates an since no significant difference was observed in the spectra of gel
absorption peak at around 1430 cm1 (Lu & Hsieh, 2010). The peak samples. The potential interactions between the whey proteins
at 1155 cm1 in the FTIR spectrum of cellulose is reminiscent of and cellulose crystals could be, in essence, hydrogen bonding
C–O–C from hemicelluloses (Oksman, Etang, Mathew, & Jonoobi, and, partly, van der Waals interactions.
 M. Ahmadi et al. / Food Chemistry 174 (2015) 97–103 101

Fig. 3. FTIR spectra of (a) cellulose, nanocrystalline cellulose (NCC) and microcrystalline cellulose (MCC). (b) WPI gel, nanocrystalline cellulose-loaded WPI gel (NCC-WPI gel)
and microcrystalline cellulose-loaded WPI gel (MCC-WPI gel).

Fig. 4. AFM image of (a) WPI gel, (b) microcrystalline cellulose-loaded WPI gel and (c) nanocrystalline-loaded WPI gel.
102 M. Ahmadi et al. / Food Chemistry 174 (2015) 97–103

of cold-set whey protein gels and develop manipulated end-prod-

ucts. It might be of benefit to de-ionise the MCC and NCC particles
before injecting into whey protein solutions and therefore
minimise the repulsion between the crystals and protein strands.
However, this would decrease the water-dispersibility of the
cellulose crystals. A more comprehensive and detailed study is
required to highlight these aspects.

Acknowledgements

The authors are thankful to Iranian National Science Foundation

(INSF, Grant no. 90003665), Center of Excellence for Application of
Modern technologies for Producing Functional Foods and Drinks,
and the University of Tehran for the financial support of the
research.

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