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8/9/2019 Inulins-Investigating the Influence of Inulin as a Fat
1/13
Investigating the influence of inulin as a fat
substitute in comminuted products using rheology,
calorimetric and microscopy techniques
Derek F. Keenan a,* , Mark A.E. Auty b,2, Linda Doran b,2, Joseph P. Kerry c ,3,Ruth M. Hamilla,1
aTeagasc, Food Research Centre Ashtown, Dublin 15, IrelandbTeagasc,
Food
Research
Centre
Moorepark,
Fermoy,
Co.
Cork,
IrelandcFood
Packaging
Group,
School
of
Food
and
Nutritional
Sciences,
University
College
Cork,
Co.
Cork,
Ireland
f o od s t ru c tu r e x xx ( 2 01 4 ) x xx –x x x
* Corresponding author. Tel.: +353 1 8059500; fax: +353 1 8059550.E-mail addresses: [email protected] (D.F. Keenan), [email protected] (Mark A.E. Auty), [email protected] (J.P. Kerry).
1 Tel.: +353 1 8059500; fax: +353 1 8059550.2 Tel.: +353 25 42222; fax: +353 25 42340.3 Tel.: +353 21 4903000; fax: +353 21 4903000.
a
r
t
i
c
l
e
i
n
f
o
Article history:
Received 16 January 2014
Received in revised form
17 April 2014
Accepted 16 June 2014
Available online xxx
Keywords:
SausageFat replacement
Inulin
Design of experiment (DOE)
Relaxation studies
Cryo-scanning electron microscopy
a
b
s
t
r
a
c
t
Thepresentmanuscript studied the effects of fat substitutionwith twocommercial inulins
on themagnetic resonance, rheological, calorimetric andmicroscopic properties of break-
fast sausages. Sausage formulations were evaluated using mixture design (D-optimal). A
total of 17 experimental treatments were employed, with each representing a different
substitution level for fat. Sausage batters were formulated to contain lean pork shoulder,
pork back fat/inulin, water, rusk andseasoning (44.3, 18.7, 27.5, 7 and2.5% w/w, respective-
ly). The resultant products’ water mobility, deformation and thermal behaviors were
analyzed for each treatment group using nuclear magnetic resonance (NMR), rheology,
differential scanning calorimetry (DSC), while their ultra-structural properties were ana-lyzed using light, confocal andscanning electronmicroscopy for selected extremes. Signifi-
cantmodelswere produced forwatermobilitywith inulin inclusions in sausages increasing
the relative protonpopulations of boundwater (T2b) values ( p < 0.0001)anddecreasing free
water (T22) population ( p < 0.0001). Inulin inclusions significantly altered the rheological
characteristicswith increases inboth thegel strength (G00 G00
0) andunit interactionstrength
( An) ( p < 0.0001, respectively). Complementary temperature-dependent behavior was ob-
servedusing rheology andDSCwhichshowed increasedelastic behavior (G0) circa 40 8C that
corresponded to the endothermic peaks for the onset of protein denaturation. Cryo-scan-
ning electron and confocal laser microscopy techniques permitted visualization of the
aggregation of inulin micro-crystals and distribution of fat within the cooked sausage
matrix. Overall, the work presented has improved our understanding of the fundamental
properties of sausage products and will enable a more scientific-based approach to future
product development.#
2014 Elsevier Ltd. All rights reserved.
FOOSTR-15; No. of Pages 13
Please cite this article in press as: Keenan, D. F., et al. Investigating the influence of inulin as a fat substitute in comminuted products using rheology, calorimetric and microscopy techniques. Food Structure (2014), http://dx.doi.org/10.1016/j.foostr.2014.06.001
Available online at www.sciencedirect.com
ScienceDirect
journal homepage: www.elsevier.com/locate/foostr
http://dx.doi.org/10.1016/j.foostr.2014.06.0012213-3291/# 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foostr.2014.06.001mailto:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.foostr.2014.06.001http://www.sciencedirect.com/science/journal/aip/22133291http://www.elsevier.com/locate/foostrhttp://dx.doi.org/10.1016/j.foostr.2014.06.001http://dx.doi.org/10.1016/j.foostr.2014.06.001http://www.elsevier.com/locate/foostrhttp://www.sciencedirect.com/science/journal/aip/22133291http://dx.doi.org/10.1016/j.foostr.2014.06.001mailto:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.foostr.2014.06.001
8/9/2019 Inulins-Investigating the Influence of Inulin as a Fat
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8/9/2019 Inulins-Investigating the Influence of Inulin as a Fat
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products represented two commercial forms of inulin)) were
developed using Design Expert software (v. 7.6.1, Stat-Ease
Inc., Minneapolis, MN, USA). Pork shoulder (95% lean) and pork
back fat (Granby Meats, Dublin, Ireland) were minced (model
PT-82/22 Mainca Barcelona, Spain) twice (5 mm plate size) and
bowl chopped with powdered inulin, ice water, seasoning and
rusk for 2 min. Sausage batter was piped into a cellulose casing
and blast frozen (air speed 3.75 m/s) and stored (20 8C) for allsubsequent analyses as outlined in a previous study (Keenan
et al., 2014). Sausages (five per treatment, vacuum-packed)
were cooked usingwaterbath immersion (85 8C) offive vacuum-
packed
sausages
per
treatment was
until they
had
achieved a
core temperature of 73 8C. Core temperature profiles were
recorded during the process using an Ellab E-Val TM TM9608
data module (Ellab [UK] Ltd., Norfolk, England) connected to a
laptop. A standard Ellab SSA-12080-G700-TS temperature probe
was inserted through an Ellab GKM-13009-C020 packing gland
(20
mm)
into
the
largest sample
in
the vacuum
bag.
2.2.
Time
domain
nuclear
magnetic
resonance
(TD-NMR)
Nuclear
magnetic
resonance
(NMR)
relaxation
measurements
were carried out as previously described (McDonnell et al.,
2013), on a Maran Ultra instrument (Oxford Instruments,
Abington, Oxfordshire, UK) with a resonance frequency for
protons of 23.2 MHz. Transverse relaxation (T2) times were
measured using Carr-Purcell-Meiboom-Gill (CPMG) pulse se-
quence
with
the
resultant relaxation
decays
analyzed
by
tri-
exponential unsupervised fitting in the RI Win-DXP software
(V. 1.2.3 Oxford Instruments, Abington, Oxfordshire, UK).
2.3.
Rheology
Rheological measurements were performed on a Physica MCR301 rheometer (Anton Paar GmbH, Graz, Austria) fitted with
parallel plate (50 mm; smooth) geometry running Rheoplus
software package (version 3.21, Anton Paar GmbH, Germany).
Sausage batterswere pressed fromtheir casingsand placed onto
the center of the base plate. The upper plate was moved into
position, i.e. the distancebetween the twoplates (gap)was set to
1 mm. Excess material was trimmed from the plate edges and
samples were allowed to rest for 5 min to achievea constant test
temperature
(25
8C
regulated
by the
rheometer’s
Pelltier
plate
and temperature hood), and for relaxation of residual stresses.
Viscoelastic properties were assessed by performing a prelimi-
nary amplitude sweep to identify the linear viscoelastic (LVE)
region of the samples and the strain (0.1%) that should be usedfor the resultant frequency sweep. A frequency sweep from 0.1
to 10 Hz was performedand the results for storagemodulus (G0),
loss modulus (G00), and complex modulus (G*) were recorded.
These data were modeled using the following power law
equations as suggested by Friedrich and Heymann (1998):
G0¼ G
0
0vn0 (1)
G00¼ G
00
0vn00 (2)
G¼
Anvn (3)
Thermal gelation properties were assessed as previously
described (Cofrades,Serrano,Ayo, Carballo,& Jiménez-Colmenero,
2008) with slight modifications. Before testing, samples were
rested (5 min) to achieve a constant test temperature (5 8C) and
relaxationof residual stresses. Thermal gelationwas inducedby
heating samples from5 8C to 85 8C at 1 8C min1 (Brunton, Lyng,
Zhang, & Jacquier, 2006). Temperature was controlled by the
aforementioned Pelltier plate and temperature hood. Samples
were sheared at a fixedfrequency of1.0 Hz with a strain of0.02%.
Sampleperimeterwas coated with a thin layerofpetroleumjellyto prevent dehydration during testing. Changes in the storage
modulus (G0) were monitored throughout the gelling process.
2.4. Differential scanning calorimetry (DSC)
Thermal transition properties were measured using a TA
Instruments DSC (Model No. DSC 2010, TA Instruments Inc.,
New Castle, DE, USA) equipped with nitrogen cooling. Indium
(melting point 156.6 8C) and baseline (empty pan) calibrations
were
carried
out
prior
to
testing.
Homogenized
(Robot
Coupé
Blixer 41 mono, Bourgogne, France) sausages samples
(15–20 mg) were weighed into aluminum pans and hermeti-
cally sealed. Samples were equilibrated at 10 8C and thenheated
from
10
to
90
8C
at
a
heating
rate
of
10
8C
min1
against a reference (empty) pan (McArdle, Kerry, Mullen, Allen,
& Hamill, 2011). Onset temperature, protein denaturation (T o),
the peak temperature (T p) and the denaturation enthalpy (DH)
were recorded. Samples were analyzed in triplicate.
Fig. 1 – Representative distribution of T2 relaxation times
for
breakfast
sausages:
(a)
comparison
between
raw
(
)
and
cooked
(–)
in
control
sausages
(run
10)
and
(b)
comparison
between
cooked
control
(–)
sausages
and
selected fat-substituted counterparts [- - - 100%
substitution
HP
(run
10)];
[
67%
substitution
HP:GR
1:1
(run 15)].
f o od s t ru c tu r e x xx ( 2 01 4 ) x xx – xx x 3
FOOSTR-15; No. of Pages 13
Please cite this article in press as: Keenan, D. F., et al. Investigating the influence of inulin as a fat substitute in comminuted products using rheology, calorimetric and microscopy techniques. Food Structure (2014), http://dx.doi.org/10.1016/j.foostr.2014.06.001
http://dx.doi.org/10.1016/j.foostr.2014.06.001http://dx.doi.org/10.1016/j.foostr.2014.06.001
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2.5.
Microscopy
Sausage
samples
(1
cm3)
were
flash
frozen
in
liquid
nitrogen
and stored at 80 8C. Sections were cut (20 mm) using a Leica
CM1950 cryostat (Leica Biosystems, Nussloch, Germany) after
equilibration to specimen chamber temperature (25 8C).
Light microscopy sections were stained with fast green and
iodine (ratio 10:1) stains and examined using a Leica DMLB
light
microscope
(Leica
Microsystems
AG,
Wetzlar,
Germany).
Confocal scanning laser microscopy (CSLM) was used in
conjunction with differential staining to visualize the distri-
bution of the fat component within the sausages. Sectionswere
stained
with
Fast
Green
(FCF)
and
Nile
Red
stains
and
examined under a Leica SP5 confocal microscope (Leica
Microsystems GmBH, Mannheim, Germany).
For Cryogenic Scanning Electron Microscopy (Cryo-SEM),
cooked samples were frozen in liquid nitrogen slush (210 8C)
and transferred to an Alto 2500 cryo preparation chamber
(Gatan
Ltd.,
Oxfordshire,
UK)
at
185
8C.
Samples
were
fractured used a cooled knife and then warmed to 95 8C
Table
2
–
Regression
coefficients
for
significant
quality
and
structural
parameters
of
sausages.
Dependent variables Independent variables R2 p model p lack of fit
X1 X2 X3 X1*X2 X1*X3 X2*X3 X1*X2*X3
DH p1 Y 1 1.05 0.22 0.17 0.85 0.0001 0.25
DH p2 Y 2 1.36 2.97 3.77 1.96 2.38 1.89 0.90 0.0001 0.78
G0
0 Y 3 8.73 10.66 11.67 0.89 0.0001 0.84
G0
0G
00
0 Y 4 8.47 10.40 11.42 0.89 0.0001 0.84 An Y 5 8.80 10.66 11.74 0.84 0.0001 0.86
n0 Y 6 0.18 0.37 0.32 0.40 0.0286 0.76
G0(30) Y 7 45.00 222.28 303.96 0.75 0.0001 0.87
G0(72) Y 8 47.10 114.40 128.10 0.35 0.0468 0.60
T2b (P) Y 9 15.40 27.71 30.03 6.86 11.39 30.63** 0.90 0.0001 0.79
T2b (T) Y 10 17.52 14.09 12.10 0.61 0.0013 0.98
T21 (P) Y 11 69.48 65.01 64.75 5.45 14.77 27.13** 0.70 0.0100 0.56
T21 (T) Y 12 40.30 26.87 21.94 0.96 0.0001 0.99
T22 (P) Y 13 15.51 7.08 5.12 0.96 0.0001 0.56
T22 (T) Y 14 171.68 140.96 136.22 0.85 0.0001 0.08
*,
**,
***Significant at p < 0.05, p < 0.01 and p < 0.001 respectively.
Fig.
2
–
Contour
plots
of
T2
relaxation
data
of
(a)
relative
proton
population
for
bound
water
(T2b
–
%);
(b)
time
constant
for
bound
water
population
(ms);
(c)
relative
proton
population
for
intra-cellular
water
(T21
–
%);
(d)
time
constant
for
intra-
cellular water population (ms); (e) relative proton population for extra-cellular water (T22 – %); (f) time constant for extra-
cellular
water
population
(ms);
for
fat
substituted
sausages
(A,
pork
fat;
B,
OraftiW GR;
C,
OraftiW HP;
where
A
+
B
+
C
=
18.7%
of total mixture).
f o od s t ru c tu r e x x x ( 2 01 4 ) x xx – xx x4
FOOSTR-15; No. of Pages 13
Please cite this article in press as: Keenan, D. F., et al. Investigating the influence of inulin as a fat substitute in comminuted products using rheology, calorimetric and microscopy techniques. Food Structure (2014), http://dx.doi.org/10.1016/j.foostr.2014.06.001
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for 5 min to remove surface ice. Fracture surfaces were sputter
coated with platinum for 120 s at 130 8C and the sample
transferred to the cold stage in the SEM instrument. Images
were acquired at 125 8C and 2 kV accelerating voltage in a
Carl Zeiss Supra 40VP field emission scanning electron
microscope (Carl Zeiss Ltd., Hertfortshire, UK).
2.6. Analysis of data
Mixture design experiments were designed and analyzed
using Design Expert (v. 7.6.1, Stat-Ease Inc., Minneapolis, MN,
USA)
as
previously
described
(Keenan
et
al.,
2014). All
parameters of NMR spectrometry, rheometry and calorimetry
were assessed and modeled using linear, quadratic, or
Scheffe’s special cubic models. Models were subjected to
analysis of variance (ANOVA) to determine the significance
( p
8/9/2019 Inulins-Investigating the Influence of Inulin as a Fat
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concentrations of 13–50% due to these limited surfactant
properties (Kim et al., 2001).
T21 was the second peak identified and occurred between
23–41 ms. T21 values represent water trapped by the dense
myofibrillar network, i.e. ‘intra-myofibrillar water’ (Bertram
et al., 2001).The T21 population represented the majority of the
water present in the sausage matrix for all samples and this
overall trend is consistent with the findings of other authors(Møller, Gunvig, & Bertram, 2010). T21 values were fitted to a
quadratic model which was found to be significant ( p < 0.01)
with a good fit to the experimental data (R2 = 0.70). The model
showed
that
linear
terms
were
significant
(
p
<
0.0140)
and
that
fat formulations resulted in higher T21 values than their inulin
substituted counterparts (Fig. 2c). Additional interactions for
the B and C components (GR and HP) were observed
( p
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implying a relatively weak structure, which is indicative of a
gel-like material consisting of a loosely ordered structure with
a viscoelastic consistency (Delgado-Pando, Cofrades, Ruiz-
Capillas, Triki, & Jiménez-Colmenero, 2012). Power law-fitted
parameters were derived from G0, G00 and G* moduli as
previously described (Campo & Tovar, 2008). G00 and G00
0
(Eqs. (1) and (2)) are the initial storage and loss moduli,
respectively, and are a measure of the resistance of a testmaterial to elastic (storage) and viscous (loss) deformation
(Zhou & Mulvaney, 1998) at an angular frequency of
0.5 rad s1. The parameters were fitted with a linear model,
which
was
transformed
using
a
natural
log
recommended
by
the Box–Cox method. The initial elastic behavior of sausages,
G0
0, increased in sausages which contained more inulin,
particularly in full substitutions with Orafti HP, compared to
intermediate formulations and fat-only controls (Fig. 4a). The
predicted model for G00 was found to be significant ( p
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and exhibited a lower frequency dependence. The reason for a
weakening of the protein conformation is unclear as inulin
appeared to add structural stability to the sausage matrix (as
evidenced by the other rheological parameters). NMR data
showed more bound water which could be attributed to the
inulin molecules. Typically, sausage emulsions are formed by
the salt-soluble myofibrillar proteins emulsifying the fat and
immobilization of water (Feiner, 2006, chap. 12). Less availablewater may have affected the emulsion structure, with less
protein being solubilized and hence reduced emulsification of
the fat component and consequently, increased its frequency
dependence.
3.2.2. Temperature-dependent behavior
Temperature sweeps showed that the thermal behavior of
samples in terms of their storage, loss moduli and phase angle
(G0, G00 and d) as a function of temperature (5–85 8C) (Fig. 5a–c).
Differences
in
thermal
rheological
properties
were
observed
between the different formulations due to their differing
compositions (presence or absence of fat/inulins) as expected
and are supported by the previous texture and rheologicalexperiments.
As
was
the
case
for
frequency
sweeps,
storage
modulus (elastic behavior) exceeded the loss modulus
(viscous behavior) over the temperature range, indicating
gel network (elastic) formation containing a substantial
emulsion structure (viscous). These observations were in line
with previous studies on the rheological thermal gelling
properties of low fat pork liver patés formulated with other
polysaccharides (Delgado-Pando et al., 2012). Fig. 5a and b
shows a slight decrease in both G0 and G00 moduli for samples
containing higher fat content, such as the full fat control (and
to a lesser extent the intermediate formulations) at 30–35 8C. If
we consider the absolute values of G0 at 30 8C in the mixturedesign, it shows that data was significantly ( p < 0.0001) fitted
to a linear model with a good fit to the experimental data
(R2 = 0.75) and showed that the linear components were the
most
significant
terms.
The
modeled
surface
shows
that
G0
values were lower in higher fat containing formulations than
their inulin containing counterparts (Fig. 4e). This observation
is consistent with the melting of pork back fat ( Jiménez-
Colmenero et al., 2012) and is in agreement with the DSC data
in the present study. This was followed by a dramatic increase
in
G0 and
G00 values
for
all
samples
between
40
and
80
8C
during
which the main rheological changes occurred (Fig. 5a and b).
This can be attributed to conformational changes in the meat
proteins that occur in this temperature range leading to thecharacteristic
stiff
elastic
matrix
of
meat
gels
The
absolute
values of G0 at 72 8C (finishing temperature of the sausages in
the present study) were successfully fitted ( p < 0.0468) to a
linear model (R2 = 0.35). Similarly, samples containing inulin
Fig.
6
–
(a)
Representative
thermal
behavior
of
control
( ~ )
sausages
and
selected
fat-substituted
counterparts
[&
–
100%
substitution
HP
(run
10)];
[
–
67%
substitution
HP:GR
1:1
(run
15)];
[+
–
50%
substitution
HP:GR
1:1
(run
16)];
[*
–
33%
substitution HP:GR 1:1 (run 5)]; and contour plots of thermal behavior: endothermic peaks (EP) (b) 1; and (c) 2; in
fat-substituted
sausages
by
differential
scanning
calorimetry
(DSC)
(A,
pork
fat;
B,
Orafti1 GR;
C,
Orafti1 HP;
where
A + B + C = 18.7% of total mixture).
f o od s t ru c tu r e x x x ( 2 01 4 ) x xx – xx x8
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had higher storage moduli than those containing fat (Fig. 4f).
The presence of inulin increased the storage and loss moduli
due to the formation of inulin’s own three-dimensional
network which differs to that of pork back fat. These data
corresponds to the thermal properties observed in the second
endothermic peak of DSC data (Fig. 6a). However, the effect
was not consistent for increasing levels of inulin, for example,
run 15 (containing 1/3 of each fat/inulin component) hadsubstantially higher G0 and G00 values than those containing
full substitution with inulin. Statistical analysis did not
support any possible interactive effect between the two inulin
types
to
account
for
this
phenomenon.
It
could
be
attributed
to
the heterogeneous nature of the sausage batter or due to the
mechanisms governing the formation of the inulin gel,
i.e. nucleation and crystallization, which are difficult to control
and rely on the mutual arrangement of the crystals that can
lead to gels with different rheological properties (Glibowski,
2010; Stasiak & Dolatowski, 2008). Furthermore, increases in G0
and G00 values were less pronounced in control formulations
compared to their inulin containing counterparts.
Further investigation of the absolute values of G0, G00 and d
and specific temperature points in the mixture designrevealed some interesting observations. Significant differ-
ences were observed between different formulations for G0
values from5 to 72 8C. However, this was only the case from 5
to
50
8C
for G00 values. This
could
indicate
that
conditions
that
conferred elasticity rather than viscosity were favored after
50 8C. Phase angle data showed no significant differences
Fig.
7
–
Light
micrographs
of
cryostat
sections
of
(a)
control
(full
fat)
sausages
at
4T; (b) 10T; and (c) 20T magnifications and
(d) fully fat-substituted sausages at 4T; (e) 10T; and (f) 20T magnifications.
f o od s t ru c tu r e x xx ( 2 01 4 ) x xx – xx x 9
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between formulations between 5 and 30 8C, implying a
similar underlying structure in all formulations. This
changed with increasing heat from 40 to 80 8C, with inulin-
formulated samples showing increases in viscoelastic prop-
erties (data not shown). These changes imply substantial
structural changes occurring during the application of heat
(as expected) and this changed significantly for different
levels of fat substitution (which is in agreement in with DSC,rheological and textural parameters shown in this study). A
decrease in G0 and G00 values was observed between 80 and
85 8C for sausage samples containing inulin, which were
more
notable in
samples
containing
the
inulin
form
Orafti
HP. This may be attributable to the greater degree of
polymerization (DP) of Orafti HP (>23) compared to Orafti
GR (>10). Inulin with high DP is thought to be thermally
unstable and temperatures above80 8C havebeen reported to
inhibit the formation of gels (Bot et al., 2004; Glibowski &
Wasko, 2008).
3.3.
Differential
scanning
calorimetry
(DSC)
Fig.
6a
shows
typical
DSC
heat
curves
for
fat
and
inulin-
enriched sausages, with two endothermic peaks obtained for
all samples. The first endothermic peak had an onset
temperature between 24 and 31 8C and corresponds to the
melting point of fat. This finding is in agreement with data
presented by other authors (Morin et al., 2004). A linear model
was
fitted
to
the
reaction
enthalpy
(data,
which
was
transformed using an inverse square-root power law recom-
mended by the Box–Cox method). The model was significant
( p
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from a system (Hinrichs, Prinsen, & Frijlink, 2001). This helps
to maintain the proteins native confirmation and prevents
denaturation (Barclay et al., 2010). Other reported findings of
increased DH values soy protein dispersions containing both
sucrose and inulin (using water as a solvent) compared to
controls18 were postulated as a thermal stabilization effect
on native soy proteins by inulin. Other contributory factors
which are responsible for the increased thermal propertiesobserved in the second thermal peak may have been due to
the water binding effect of inulin. This could limit the
available water to other matrix components, e.g. rusk,
thereby
limiting
hydration of
starch
and profoundly
modifying the thermal properties of starch gelatinization
( Juszczak et al., 2012).
3.4.
Microscopy
Lightmicroscopy conducted on
iodine
and
fast green stained
cryostat sections of control (full fat) and fully fat-substituted
sausages at different magnifications (4, 10 and 20) are
presented in Fig. 7a–f. Both treatments consisted of acontinuous
protein
phase (green)
containing fat
globules
and adipose cells (unstained). Some larger muscle fragments
were also visible. Large aggregates of partially gelatinized
starch granules (stained purple in iodine) were dispersed
within the protein matrix. These are most likely rusk
particles. No obvious structural differences were observed
between the
control
and
inulin-containing samples. The
presenceof fat and its distributionwithin the sausage matrix
was subsequently visualized using confocal scanning laser
microscopy (CSLM) and differential staining with fast green
and Nile red (Fig. 8a–c). Significant differences between the
sausage treatments were observed. Fig. 8a shows fat (stained
green) was abundantly present in full-fat control and was
coated by the protein/water continuous phase forming the
typical sausage emulsion-like structure. A large population
of adipose tissue is also visible in the bottom right of theimage. Fat substitution in different sausage formulations led
to a concomitant reduction of green stain, which can be
clearly seen in Fig. 8b and c representing 66% fat reduction
(run
15)
and
full fat
substitution, respectively.
The residual
green stain in the latter represents the native fat content of
the pork shoulder muscle. The presence of the inulin and its
distribution within the sausage matrix was visualized in a
fully fat-substituted formulation (Fig. 9c and d) using
cryogenic scanning electron microscopy (cryo-SEM) and
compared against
control (Fig. 9a and
b)
formulation (full
fat). Gelatinized starch granules from the added rusk could
be clearly identified in the sausage matrix (labeled SG –
Fig. 9a). Sausage treatment containing inulin had crystal-line
regions, often
in
the form of rounded spherulitic
structures. These structures were notpresent in the control
sample, which contained more conventional fat morphol-
ogy (labeled F – Fig. 9b), and it is strongly suggested that
they are inulin crystals (labeled I – Fig. 9d). Previous studies
(Cooper & Carter, 1986; Cooper & Steele, 1991)have reported
that inulin
particles normally
crystallize
from
water as
ovoids of 1–10 mm diameter, much like the observations of
Fig.
9
–
Cryo-scanning
electron
microscopy
(cryo-SEM)
of
(a
and
b)
control;
and
(c
and
d)
full
fat-substituted
sausages
(where
GS, gelatinized starch; F, fat; and I, inulin).
f o od s t ru c tu r e x xx ( 2 01 4 ) x xx – xx x 11
FOOSTR-15; No. of Pages 13
Please cite this article in press as: Keenan, D. F., et al. Investigating the influence of inulin as a fat substitute in comminuted products using rheology, calorimetric and microscopy techniques. Food Structure (2014), http://dx.doi.org/10.1016/j.foostr.2014.06.001
http://dx.doi.org/10.1016/j.foostr.2014.06.001http://dx.doi.org/10.1016/j.foostr.2014.06.001
8/9/2019 Inulins-Investigating the Influence of Inulin as a Fat
12/13
8/9/2019 Inulins-Investigating the Influence of Inulin as a Fat
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Cooper, P. D., & Carter,M. (1986). Anti-complementary action of polymorphic ‘‘solubility forms’’ of particulate inulin.Molecular Immunolology, 23, 895–901.
Cooper, P. D., & Steele, E. J. (1991). Algammulin, a new vaccineadjuvant comprising gamma inulin particles containing alum: Preparation and in vitro properties. Vaccine, 9, 351–357.
de Bruijne, D.W., & Bot, A. (1999). Fabricated fat-based foods. InA. J. Rosenthal (Ed.), Food texture: Measurement and perception
(pp. 185–227). Gaithersburg: Aspen Publishers Inc.Delgado-Pando, G., Cofrades, S., Ruiz-Capillas, C., Triki, M., &
Jiménez-Colmenero, F. (2012). Low-fat liver pâ tés enrichedwith n-3 PUFA/konjac gel: Dynamic rheological propertiesand technological behaviour during chill storage. MeatScience, 92, 44–52.
Feiner, G. (2006). Meat products handbook. Practical science andtechnology (1st ed.). Cambridge: Woodhead Publishing .
Friedrich, C., & Heymann, L. (1998). Extension of a model forcrosslinking polymer at the gel point. Journal of Rheology, 32,235–241.
Glibowski, P. (2010). Effect of thermal andmechanical factors onrheological properties of high performance inulin gels andspreads. Journal of Food Engineering, 99, 106–113.
Glibowski, P., & Wasko, A. (2008). Effect of thermochemical
treatment on the structure of inulin and its gelling properties. International Journal of Food Science and Technology,43, 2075–2082.
Hamm, R. (1975). Water-holding capacity ofmeat. InD. J. A. Cole& R. A. Lawrie (Eds.), Meat (pp. 321–338). London:Butterworth-Heinemann.
Hinrichs, W. L. J., Prinsen, M. G. H., & Frijlink, W. (2001). Inulinglasses for the stabilization of therapeutic proteins.International Journal of Pharmaceutics, 215, 163–174.
Honikel, K. O. (1983). Water binding and ‘‘fat emulsification’’during the processing of bru ¨ hwurst mixtures.Fleischwirtschaft, 62(11), 1179–1182.
Hsu, S. Y., & Sun, L.-Y. (2006). Comparisons of 10 non-meatprotein fat substitutes for low-fat Kung-wans. Journal of FoodEngineering, 74, 47–53.
Jiménez-Colmenero, F., Cofrades, S., Herrero, A. M., Fernández-Martı́n, F., Rodrı́guez-Salas, L., & Ruiz-Capillas, C. (2012).Konjac gel fat analogue for use in meat products:Comparison with pork fats. Food Hyrdocolloids, 26(1), 63–72.
Juszczak, L., Witczak, T., Ziobro, R., Korus, J., Cieślik, E., &Witczak, M. (2012). Effect of inulin on rheological andthermal properties of gluten-free dough. CarbohydratePolymers, 90, 353–360.
Keenan, D. F., Resconi, V. C., Kerry, J. P., & Hamill, R. A. (2014).Modelling the influence of inulin as a fat substitute incomminuted meat products on their physico-chemicalcharacteristics and eating quality using a mixture designapproach. Meat Science, 96, 1384–1394.
Kim, Y., Faqih, M. N., & Wang, S. S. (2001). Factors affecting gelformation of inulin. Carbohydrate Polymers, 46, 135–145.
Lee, C. M., Carroll, R. J., & Abdollahi, A. (1981a). A microscopicalstudy of the structure of meat emulsions and its relationshipto thermal stability. Journal of Food Science, 46(6), 1789–1793.
Lee, C. M., Hampson, J. W., & Abdollahi, A. (1981b). Effect of plastic fats on the thermal stability and mechanicalproperties of fat-protein gel products. Journal of the AmericanOil Chemists’ Society, 58(11), 983–987.
McArdle, R., Kerry, J. P., Mullen, A. M., Allen, P., & Hamill, R. M.(2011). Monitoring the effects of salt and temperature onmyofibrillar proteins in beef. In Proceedings of the 57thInternational Congress of Meat Science and Technology (Short
Paper 00958).McDonnell, C. K., Allen, P., Duggan, E., Arimi, J. M., Casey, E.,
Duane, G., et al. (2013). The effect of salt and fibre directionon water dynamics, distribution and mobility in pork
muscle: A low field NMR study.Meat Science, 95(1), 51–58.
Mendoza, E., Garcia, M. L., Casas, C., & Selgas, M. D. (2001).Inulin as a fat substitute in low fat, dry fermented sausages.Meat Science, 57, 387–393.
Møller, S.M.,Grossi,A., Christensen, M.,Orlien, V., Søltoft-Jensen, J., Straadt, I. K., et al. (2011). Water properties and structureof pork sausage as affected by high-pressure processing andaddition of carrot fibre.Meat Science, 87, 387–393.
Møller, S.M., Gunvig, A., & Bertram, H. C. (2010). Effect of starter
culture and fermentation on water mobility anddistribution in fermented sausages and correlation tomicrobial safety studied by nuclear magnetic resonancerelaxometry. Meat Science, 86, 462–467.
Morin, L. A., Temelli, F., & McMullen, L. (2004). Interactionsbetween meat proteins and barley (Hordeum spp.) b-glucanwithina reduced-fat breakfast sausage system.Meat Science,68, 419–430.
Pearson, A. M., & Gillett, T. A. (1996). Processed meats (3rd ed.).London: Chapman and Hall.
Peng, I. C., & Nielsen, S. S. (1986). Protein–protein interactionsbetween soybean beta-conglycinin (B1–B6) and myosin. Journal of Food Science, 51(3), 588–590.
Peressini, D., & Sensidoni, A. (2009). Effect of fibre addition onrheological and breadmaking properties of wheat doughs.
Journal of Cereal Science, 49(2), 190–201.Rodrı́guez, R., Jiménez, A., Fernandez-Bolan ˜ os, J., Guillén, R., &
Heredia, A. (2006). Dietary fibre from vegetable products assource of functional ingredients. Trends in Food Science andTechnology, 17, 3–15.
Schellman, J. A. (2003). Protein stability in mixed solvents:A balance of contact interaction and excluded volume.Biophysical Journal, 85, 108–125.
Shaarani, S. M., Nott, K. P., & Hall, L. D. (2006). Combination of NMR and MRI quantitation of moisture and structurechanges for convection cooking of fresh chicken meat.MeatScience, 72, 398–403.
Silva, R. F. (1996). Use of inulin as a natural texture modifier.Cereal Foods World, 41, 792–794.
Stasiak, D. M., & Dolatowski, Z. J. (2008). Efficiency of sucrose
crystallization from sugar beet magma after sonication.Polish Journal of Natural Science, 23(2), 521–530.
Swasdee, R. L., Terrell, R. N., Dutson, T. R., & Lewis, R. E. (1982).Ultrastructural changes during chopping and cooking of afrankfurter batter. Journal of Food Science, 47(3), 1011–1013.
Timasheff, S. N. (1998). Control of protein stability and reactionsby weakly interacting cosolvents: The simplicity of thecomplicated. Advances in Protein Chemistry, 51, 356–432.
Totosaus, A., & Pérez-Chabela, M. L. (2009). Textural propertiesandmicrostructure of low-fat and sodium-reduced meatbatters formulated with gellan gum and dicationic salts.LWT, 42, 563–569.
Tseng, Y.-C., Xiong, Y. L., & Boatright,W. L. (2008). Effects of inulin/oligofructose on the thermal stability and acid-inducedgelation of soy proteins. Journal of
Food
Science, 73(2), E44–E50.
Whiting, R. C. (1987). Functional properties of meat batters withdifferent sodium laurate additions. Journal of Food Science,52(5), 1126–1129.
Youseff, M. K., & Barbut, S. (2009). Effects of protein level andfat/oil on emulsions stability, texture microstructure andcolor of meat batters. Meat Science, 2, 228–233.
Zaidul, I. S. M., Absar, N., Kim, S.-J., Suzuki, T., Karim, A. A.,Yamauchi, H., et al. (2008). DSC study of mixtures of wheatflour and potato, sweet potato, cassava and yam starches. Journal of Food Engineering, 86, 68–73.
Zhou, N., & Mulvaney, S. J. (1998). The effect ofmilk fat, the ratioof casein to water and temperature on the viscoelasticproperties of rennet casein gels. Journal of Dairy Science,81(10), 2561–2571.
Ziegler, G. R., & Acton, J. C. (1984). Mechanisms of gel formation
by proteins of muscle tissue. Food Technology, 38(5), 77–82.
f o od s t ru c tu r e x xx ( 2 01 4 ) x xx – xx x 13
FOOSTR-15; No. of Pages 13
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