Journal of Food Engineering 280 (2020) 109977

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Journal of Food Engineering

journal homepage: http://www.elsevier.com/locate/jfoodeng

Kinetic modelling of the heat stability of bovine lactoferrin in raw

whole milk
Haiyan Liu a, b, c, Irina Boggs d, Mike Weeks e, Qiming Li b, Huaxing Wu b, Paul Harris c, Ying Ma a,
Li Day a, e, *
a
School of Chemical Engineering and Technology, Harbin Institute of Technology, Harbin, Heilongjiang, 150090, China
b
R&D Center, New Hope Dairy Co, Ltd., Chengdu 610000, China
c
Dairy Nutrition and Function, Key Laboratory of Sichuan Province, Chengdu 610000, China
d
AgResearch Ltd, Ruakura Research Centre, 10 Bisley Road, Hamilton 3214, New Zealand
e
AgResearch Ltd, Grasslands Research Centre, Tennent Drive, Palmerston North 4442, New Zealand

A R T I C L E I N F O A B S T R A C T

Keywords: The thermal denaturation of the naturally occurring lactoferrin in raw bovine whole milk has been measured
Lactoferrin over a range of temperatures from 65 to 121 � C for holding times from 2 up to 300 s designed to mimic heating
Thermal denaturation processes used in the food manufacturing industry. Data were generated by heat treatments of raw whole milk
Industrial processing
from two different sources, New Zealand (NZ) and China, and with lactoferrin at two different levels of naturally
Iron saturation
occurring iron saturation. Denaturation rates for lactoferrin in the Chinese-sourced milk samples were at least
two-fold greater than lactoferrin in the NZ milk over the temperature range 65–80 � C. At 85 � C rates of dena­
turation were the same although more than 80-fold greater than at 65 � C whereas, at 95 � C, lactoferrin in the NZ
milk denatured at twice the rate of that in the Chinese milk. The differences in denaturation rates up to 80 � C
likely reflected the greater heat stability of the more iron-saturated NZ milk samples. From these data, a com­
bined kinetic and Arrhenius-based model has been developed and validated that allows calculation of native
lactoferrin retained after thermal processing at any temperature-time combination in the experimental range
(65–95 � C). This model should have applicability in the evaluation of existing processes and for the design of
processes to improve the retention of lactoferrin during food manufacture.

1. Introduction regarded as an important bioactive and functional protein (García-­

Lactoferrin is an iron-binding glycoprotein of about 80 kDa, a
member of the transferrin family, and present in the colostrum and milk
of mammals. The highest concentrations of lactoferrin are reported for
human colostrum (5–13 mg/mL) with mature human milk containing
1.5–4.0 mg/mL, which accounts for 15–20% of total human milk protein
(Elwakiel et al., 2019; Lo€nnerdal, 2004; Rai et al., 2014; Yang et al.,
2018). In contrast, lactoferrin concentrations of 0.5 mg/mL have been
reported for bovine colostrum, with mature milk containing 0.02 up to
0.2 mg/mL (Farrell et al., 2004; Masson and Heremans, 1971; Steijns
and Van Hooijdonk, 2000). Lactoferrin levels in bovine milk vary with
stage of lactation and somatic cell count (Cheng et al., 2008; Musayeva
et al., 2016).
Lactoferrin was first identified as a milk protein by Groves (1960)
and following extensive research over ensuing years came to be
Montoya et al., 2012; Giansanti et al., 2016; Iglesias-Figueroa et al.,
2019; Manzoni et al., 2018; Steijns and Van Hooijdonk, 2000). After its
approval as a food ingredient by the FDA in 2000 and the European
Commission in 2012, consumer demand for it has been continuously
increasing in recent years (Franco et al., 2018). Hence bovine lactoferrin
has been included in a variety of food and nutraceutical products either
as a purified extract or as a component of milk ingredients such as whole
or skim milk powders (WMP or SMP) or whey protein concentrates
(WPC) (Tomita et al., 2009; Wu et al., 2019). Purified bovine lactoferrin
has been manufactured from the mid-1980s by extraction from milk or
sweet whey (cheese or rennet whey) using large-scale cation-exchange
fractionation (Tomita et al., 2009), and is commonly added as an
ingredient to infant formula products (Lo €nnerdal, 2014; Satue-Gracia
et al., 2000), yoghurt and dietary supplements (Franco et al., 2010;
Franco et al., 2018; Steijns and Van Hooijdonk, 2000; Tomita et al.,

* Corresponding author. AgResearch Ltd, Grasslands Research Centre, Tennent Drive, Palmerston North, 4442, New Zealand.
E-mail address: li.day@agresearch.co.nz (L. Day).

https://doi.org/10.1016/j.jfoodeng.2020.109977
Received 10 November 2019; Received in revised form 31 January 2020; Accepted 11 February 2020
Available online 14 February 2020
0260-8774/© 2020 Elsevier Ltd. All rights reserved.
H. Liu et al. Journal of Food Engineering 280 (2020) 109977

depleted of endogenous lactoferrin (Sa �nchez et al., 1992), and where

apo- or holo-lactoferrin was added back to skim milk which was heated
to various temperatures prior to renneting and recovery of lactoferrin in
the whey (Brisson et al., 2007a).
It has been reported that the holo-lactoferrin is more resistant to
heat-induced changes than is the apo-lactoferrin, and both were more
heat sensitive when treated in milk than in buffer systems (Bokkhim
et al., 2013; Brisson, Britten and Pouliot, 2007b; Kussendrager, 1994;
Sa
�nchez et al., 1992; Sreedhara et al., 2010a). Also, both protein forms
were completely denatured by ultra-high-temperature (UHT) treatment
(Paulsson et al., 1993).
Although a considerable amount of research has been conducted on
the health benefits of lactoferrin, very little attention has been paid to
better understand the kinetic loss of native lactoferrin under various
thermal treatments, more especially with relevance to industrial
manufacturing processes, i.e. high temperature short times. Kussendr­
ager (1994) commented that kinetic approaches to lactoferrin heat
Fig. 1. Bovine lactoferrin structure showing the iron binding sites within the C- denaturation were important for predicting and controlling the effects of
lobe and the N-lobe. heating processes used industrially and he further noted that the kinetic
parameters of heat denaturation of lactoferrin would require determi­
nation in the process of interest as lactoferrin is strongly affected by its
Table 1 environment e.g. other proteins (caseins, whey proteins), pH or min­
Heat treatment temperatures and holding times applied to process the raw milk. erals. In this study, we have aimed to determine the heat stability of
Temperature (� C) Time (s) lactoferrin in its natural milk environment as a function of its heating
65 10 15 30 60 180 300
temperature and time and to develop a model based on the outcomes
70 5 10 15 30 60 120 that can be used to evaluate a variety of industrial processes and
75 5 10 15 30 45 60 implement process design improvements to retain more of this valued
80 5 10 15 30 45 60 protein in finished products.
85 5 10 15 30 45 60
95 2 5 10 15 30
121 2 4 6 10 2. Materials and methods

2.1. Bovine milk

2009).
The three-dimensional structure of bovine lactoferrin comprises two
lobes (C and N), linked by α-helix and β-sheet structures, with two do­
mains in each lobe (Fig. 1). Lactoferrin has two metal and anion-binding
sites so that it can bind two ferric ions with very high affinity thus
limiting the growth of various pathogenic microorganisms by denial of
essential iron. Naturally occurring native bovine lactoferrin typically
has an iron saturation of 12–22% (Bokkhim et al., 2013; Brisson, Britten
and Pouliot, 2007a; Kussendrager, 1994; Lo €nnerdal et al., 2011; Steijns
and Van Hooijdonk, 2000). Two additional conformational states of
lactoferrin include the open metal-free form termed apo-lactoferrin, and
an iron-saturated closed metal-bound form, known as holo-lactoferrin
(Majka et al., 2016). Some of the functional properties of lactoferrin is
strongly associated with its iron-binding properties. It has been shown
that oral delivery of lactoferrin prevented iron deficiency anaemia in
pregnant woman (Paesano et al., 2014; Rezk et al., 2016). Although
there is still no clear evidence on whether lactoferrin may promote ab­
sorption of iron in infants, lactoferrin is a common ingredient used to
fortify infant formula products, with primary purposes for promoting
bone growth, modulating immune functions and providing
anti-pathogenic effects (Lo €nnerdal, 2009, 2014).
For consumer safety, milk is required to be heat-treated under
carefully regulated conditions (typically at least a pasteurization of 72
�
C for a minimum of 15 s) whether it is to be used for extraction of
lactoferrin or to produce other milk products. For most applications, e.g.
infant formula, it is important for optimal performance of lactoferrin to
ensure it is delivered with maximal retention of quantity and quality
(Lo€nnerdal, 2014), hence the importance of having a good under­
standing of its stability to heat under a variety of industrial conditions.
The heat stability of bovine lactoferrin has been studied in water,
various buffers at different pH values (Kussendrager, 1994; Paulsson
et al., 1993; Sreedhara, Flengsrud, Langsrud, Kaul, Prakash and
Vegarud, 2010a; Sreedhara et al., 2010b) and in two studies in milk
where purified apo- or holo-lactoferrin was added back to skim milk
Two samples of Chinese bulk raw bovine milk, frozen and packed in
dry ice, were provided by New Hope Dairy Ltd (Hope Dairy Demon­
stration Farm, Meishan City, Sichuan Province, China). Two New Zea­
land (NZ) raw bovine milk samples were collected from a herd of 250
cows at the Tokanui Farm (Waikato, New Zealand) on two different days
during mid lactation (December). Samples were aliquoted and stored at
20 � C for lactoferrin analysis within one week.
2.2. Lactoferrin content determination
Lactoferrin content was determined by enzyme-linked Immunosor­
bent Assay (ELISA). Frozen milk samples were thawed at room tem­
perature, then diluted for analysis of lactoferrin level using a bovine
lactoferrin ELISA kit (Bethyl Laboratories, Montgomery, Texas, USA).
Samples were analysed in duplicate at dilutions of 1/100, 1/200, 1/400
for lower lactoferrin concentrations, 1/1,000, 1/2000 and 1/4000 for
higher concentrations. lactoferrin levels were determined by compari­
son with the reference standard provided with the kit which was diluted
2-fold from an initial starting concentration of 1000 ng/mL.
2.3. Heat treatment of raw milk
The NZ and Chinese raw milk samples were thawed, then heat
treated at eight different temperatures in the range from 65 � C to 121 � C
to cover industrial processes of high-temperature short-time (HTST)
pasteurization and UHT treatments at four to six different holding times.
The temperature-time combinations used are shown in Table 1. All heat-
treated samples were prepared in triplicate.
Heating to temperatures in the range 65–85 � C for various holding
times was performed by heating a 2 mL sample of milk using a heating
and cooling system designed by AgResearch. The sample heating
chamber consisted of two parallel 100 W Peltier thermoelectric plates
(RS Components Ltd, New Zealand) for the walls with a rubber gasket

2
H. Liu et al. Journal of Food Engineering 280 (2020) 109977

40 x 40 mm Type T thermocouple

Heat sink
with fan

SIDE VIEW
for control and
logging

Sample well
device and
Rubber +
- electronics box
+
gasket -

Fig. 2. Schematic (side view) of Peltier heating device.

Table 3
Table 2
First order model fit parameters for New Zealand raw milk (first set).
Lactoferrin concentration and iron saturation of New Zealand and Chinese raw
milk. Temperature C0 C0 SE k k SE
1
(� C) (μg/mL) (μg/mL) (s � 1000) (s 1 � 1000)
Lactoferrin (mg/mL) Iron saturation (%)
65 97.44 2.48 0.88 0.20
New Zealand milk 0.121 � 0.020 21.9 � 1.6 70 103.03 2.34 2.81 0.48
Chinese milk 0.097 � 0.009 11.6 � 0.5 75 86.04 2.07 5.02 0.80
80 93.78 3.76 10.02 1.50
85 93.60 6.00 31.64 3.96
between them across the base and two sides (see Fig. 2). The tempera­ 95 38.70 8.16 263.55 70.75
ture in the chamber was measured using a Type T thermocouple probe SE ¼ standard error.
and was controlled by means of a DAQ device (U6, Labjack Corporation,
USA) and DAQ Factory software (Azeotech Inc., USA). Accurate control
end-point heating temperature was typically reached in 30–45 s with a
of the heating rate and the target holding temperature was provided by
similar time taken for cooling to 10 � C.
means of a PID control loop feedback built into the software. Following
The high temperature heating in the range 95–121 � C was carried out
the heating/holding step, the sample was cooled in situ by switching the
using an oil bath. The milk sample was pipetted into a steel tube which
polarity of the electric feed to the Peltier plates and dissipating the heat
incorporated a temperature sensor. The sample was heated to the
through heat sinks and fans connected to the plates. This allowed the test
desired temperature in the oil bath, held for the appropriate time and
sample to rapidly cool before an aliquot was removed for analysis. The
then cooled rapidly by placing the tube in an ice-water bath until it
heating step was set at the desired end temperature and holding time.
reached room temperature.
The cooling steps were set at 20 � C hold for 1 s, 15 � C hold for 1 s and 10
The amount of native lactoferrin in the samples after heat treatment
�
C hold for 1 s, to allow the sample to cool down as fast as possible. The
was analysed using the ELISA method as described above. For high-
temperature treated samples, where final lactoferrin concentrations
were low, dilutions down to 1/20 were used.

2.4. Purification of lactoferrin from raw milk

Small scale purification of lactoferrin from the milk samples was

necessary to concentrate the protein to measure its iron saturation level
as well as to separate it from lactoperoxidase, which would interfere

Table 4
First order model fit parameters for Chinese raw milk (first set).
Temperature (� C) C0 C0 SE k k SE
1
(μg/mL) (μg/mL) (s � 1000) (s 1 � 1000)

65 68.9 1.2 0.65 0.13

70 68.4 1.3 1.57 0.38
75 68.7 2.8 5.08 1.36
Fig. 3. Effect of temperature on the lactoferrin denaturation rate constant, k. 80 55.7 4.9 23.08 4.47
Lines show the 1/k-weighted fit of the Arrhenius equation (eq. (5)), and dots 85 33.6 3.2 33.23 6.15
show the actual measured data points. 95 27.6 3.6 128.85 27.32

SE ¼ standard error.

3
H. Liu et al. Journal of Food Engineering 280 (2020) 109977

Table 5 2.5. Iron saturation determination of purified lactoferrin

Parameters for the Arrhenius dependence of k on temperature used to predict k
values at each experimental temperature. Bound iron was measured using a colorimetric assay at the New
NZ milk Chinese milk Zealand Veterinary Pathology testing laboratory (Hamilton, New Zea­
Parameter SE Parameter SE
land). The assay was done using a commercial quantitative iron deter­
mination test (Roche Diagnostics, Mannheim, Germany) where the
kref (s 1 x 1000) 11.45 1.29 13.83 2.19
lactoferrin-Fe complex was first acidified to pH < 2 using 200 mM cit­
Ea (kJ.mol 1) 225.56 8.80 162.94 12.90
ric acid to release bound iron. Ferric ion was then reduced to ferrous
SE ¼ standard error. state using ascorbate as the reducing agent. Finally, the ferrous ion and
FerroZine reagent was mixed to give a coloured complex. The saturation
Table 6 ratio was calculated from the moles of iron detected to the moles of
Model-predicted k values for lactoferrin denaturation at experimental lactoferrin in the sample using a molar ratio of 2:1 (Bokkhim et al.,
temperatures. 2013).
Temperature (� C) NZ milk Chinese milk

k k
2.6. Modelling lactoferrin heat stability
1 1
(s � 1000) (s � 1000)
The loss of lactoferrin on heating at each temperature was kinetically
65 0.38 1.98
70 1.22 4.18 modelled with the general rate law for a single-species reaction:
75 3.80 8.63
dC
80 11.45 17.47 ¼k​C​n ​ (1)
85 33.45 34.64 dt
95 261.83 128.88
where C is concentration, k is a rate constant (units: time 1.concen­
tration1 n) and n is the kinetic order (no units). Eq. (1) is integrated with
with iron detection due to its heme group. respect to time and concentration:
Skim milk was prepared by centrifugation of whole raw milk at 3000 Z Z
g for 20 min at 4 � C. The NZ and Chinese skim milk samples were sub­ C ​ n dC ¼ k dt (2)
jected to purification using the Akta chromatography system (GE
Healthcare). The samples were pumped through a HiTrap Sepharose For cases where n 6¼ 1 (nth order) the integrated form is:
cation-exchange column (GE Healthcare) with a flow rate of 1 mL/min, � �1 1 n
which was equilibrated with 20 mM phosphate buffer at pH 6.5. The C ¼ ðn 1Þkt þ ​ C10 n
(3)
bound proteins were eluted with a linear gradient 0–1 M NaCl. Fractions
were desalted using the Milicone filters with 3 kDa MWCO (Millipore) where C0 is the concentration at t ¼ 0.
and then freeze-dried. Lactoferrin in eluted fractions was detected by For n ¼ 1 (first order):
Western Blot, using an anti-bovine lactoferrin antibody (Bethyl). West­ C ¼ C0 expð ktÞ (4)
ern Blotting was performed as previously described (Broadhurst et al.,
2005). The purity of the preparation was estimated to be 90% by The derivation and application of these models are discussed else­
densitometry of the Coomassie blue stained gel (Shin et al., 2001). The where (Loveday, 2016).
protein content was determined by 280 nm absorbance using the A model was constructed using the following methodology.
ND-1000 Nanodrop spectrophotometer (Thermo Fisher Scientific).

Fig. 4. Denaturation curves for lactoferrin in New Zealand (left) and Chinese (right) milk derived from the model (solid lines) shown with the experimental data
points from the validation dataset ( 65, 70, 72, 75, 80, 85, 95 � C).

4
H. Liu et al. Journal of Food Engineering 280 (2020) 109977

Fig. 5. Predicted native lactoferrin (% remaining of original) in the New Zealand and Chinese milk samples (shown in red and grey lines respectively) when heating
in the Peltier system to 65–85 � C or oil bath (95 � C). The heating and cooling profiles are shown in blue. (For interpretation of the references to colour in this figure
legend, the reader is referred to the Web version of this article.)

Step 1. Data from a first set of temperature versus time treatments Step 3. The Arrhenius equation (5) was used to predict k values at each
were used to obtain C0 and k values using non-linear regression at each experimental temperature.
temperature in the range 65–95 � C for the NZ and Chinese milk samples.
Step 4. The model was validated for lactoferrin in the NZ and Chinese
Step 2. The temperature dependence of calculated k values from Step milk samples by comparing model predictions (Step 3) with data from a
1 was fitted to an Arrhenius equation. second set of temperature versus time treatments using the same tem­
� � �� perature time combinations.
Ea 1 1
k ¼ kref exp (5)
R Tref T Step 5. The kinetic (Eq. (4)) and Arrhenius (Eq. (5)) models were
combined to give a single model (Eq. (6)) that can be used to numeri­
where kref is the value of k at a reference temperature, Ea is the activation cally integrate time-temperature trajectories for lactoferrin denatur­
energy, R is the universal gas constant (8.314 J mol 1), T is absolute ation at any temperature in the range 65–95 � C:
temperature (Kelvin) and Tref is the reference temperature.

5
H. Liu et al.
� � � �� �
C Ea 1 1 rate constant of the loss of lactoferrin in the NZ and Chinese milk sam­
¼ exp kref exp t (6)
C0 R Tref T ples may reflect the differences in their iron saturation levels. The level
where C/C0 is the concentration of native lactoferrin at time, t, as a
proportion of the starting concentration.
Data were fitted using R 3.5.1 (R Core Team, 2019) with nls function
in the stats library.
3. Results and discussion
3.1. Lactoferrin content and iron saturation level in raw milk
Lactoferrin content and iron saturation level in the Chinese and NZ
raw milk samples are shown in Table 2. The average native lactoferrin
content in the NZ milk samples was 0.121 mg/mL, slightly higher than
the lactoferrin concentration of 0.097 mg/mL in the Chinese milk.
Lactoferrin concentration in raw milk has been reported to be between
0.02 and 0.1 mg/mL in skim milk (Farrell et al., 2004) and up to 0.2
mg/mL in whole cow milk (Steijns and Van Hooijdonk, 2000). A survey
of 198 Chinese Holstein cows showed that lactoferrin concentrations in
milk from individual cow varied between 0.031 and 0.486 mg/mL, and
was positively associated with stage of lactation and somatic cell count
score while being negatively associated with daily milk production
(Cheng et al., 2008). The lactoferrin concentrations in the raw bulk milk
from both NZ and China collected for this study fall within the reported
range for bovine milk.
Iron saturation levels of native lactoferrin have been variously re­
ported to be 19.2% in raw skim milk (Brisson et al., 2007a), 22% for a
commercial preparation (Kussendrager, 1994), 15–20% (Steijns and
Van Hooijdonk, 2000), 12% (Bokkhim et al., 2013) and 13.2 and 17.1%
for laboratory isolated and commercial preparations respectively
(Lo
€nnerdal et al., 2011). At 21.9%, the NZ milk samples is at the topmost
level reported and at 11.6%, the Chinese milk samples were similar to
the bottom-most reported level. It has been commented that lactoferrin
samples even with iron-saturation levels of 13.2% and 17.1% are still
largely present in the Fe-free (apo) form (Lo €nnerdal et al., 2011),
although others have stated that in practice, if the iron saturation level
of lactoferrin is between 0 and 6% it is in the apo form and between 76
and 100% it is in the holo form (Wu et al., 2019). So far there has not
been research to indicate that the level of iron saturation of lactoferrin
might vary with different farming systems.
3.2. Lactoferrin heat stability in milk
NZ and Chinese raw milk samples were isothermally heat treated at
temperatures from 65 to 95 � C for holding times from 2 s up to 300 s
(depending on temperature) to mimic HTST pasteurization and UHT
conditions. The amount of undenatured lactoferrin after heat treatment
was assessed by ELISA. The rate constants, k, for loss of native lactoferrin
in each of the NZ and Chinese milk samples as a function of temperature
are shown in Fig. 3. The results show that an increase in processing
temperature and holding time led to a rapid loss of native lactoferrin in
both NZ and Chinese milk samples, however the rate constant of the loss
as a function of temperature and heating times were different between
the milk samples sourced from NZ and China. When heating at 65–75 � C,
the rate of loss of native lactoferrin was higher in the Chinese milk than
the NZ raw milk samples. At the heating time of 60 s, about 10–33% of
lactoferrin was lost in the NZ milk between 65 and 75 � C, whereas the
levels of lactoferrin loss were about 35–43%, in the Chinese milk. For
temperatures above 80 � C, the loss of lactoferrin was much more rapid in
both samples. At 85 � C after 15 s, about 49% lactoferrin in the NZ milk
samples was lost and by 60 s 86% was lost. In comparison, ~76% was
lost in the Chinese milk after 15 s. Lactoferrin was almost completely lost
(~92%) in the Chinese milk after 60 s heating at 85 � C. At the ultra-high
temperature of 121 � C, all lactoferrin was lost just after 4 s.
The differences between the heat sensitivity of native lactoferrin and
6
Journal of Food Engineering 280 (2020) 109977
of iron saturation in the Chinese milk was about half of that in the NZ
milk (11.6% vs 21.9%). Although there has been no information on the
heat sensitive of native lactoferrin with different levels of iron satura­
tion, the work by Sa�nchez et al. (1992) showed that apo-form lactoferrin
was denatured more rapidly than iron-saturated lactoferrin (although
the levels of iron saturation were not reported). Similarly, the work by
Conesa et al. (2008) to determine the thermal stability of purified lac­
toferrins from different animal species by differential scanning calo­
rimetry also showed that the denaturation temperatures of
iron-saturated lactoferrins were higher than their respective native
lactoferrins, suggesting that the increased level of iron-binding in lac­
toferrin increases its structural stability. Although the difference in the
iron saturation levels of the milk samples used in this study was about
10%, our results confirm that such differences could still influence the
heat stability of native lactoferrin in bovine milk, particularly in the
temperature range that is commonly used for pasteurization of milk.
3.3. Kinetic modelling of the heat stability of native lactoferrin with
different iron saturation levels in raw whole milk
3.3.1. Determination of best fit lactoferrin denaturation model
The concentration of lactoferrin, as measured by ELISA, at each time
point after heating at each of the selected temperatures was used as a
measure of the native lactoferrin remaining after treatment. These
denaturation data were analysed with both Eq. (3) and Eq. (4), and the
first order model of Eq. (4) fitted the data from the first sample sets most
accurately, for both NZ and Chinese milk samples. The first order model
gave a statistically reliable fit, with highly significant parameters, and
residuals that were randomly distributed around zero. The fit parame­
ters of k, the rate constant for native lactoferrin loss and C0, the con­
centration of lactoferrin at time zero, are shown in Tables 3 and 4. Data
collected from the 72 � C heating runs were discarded because excess
noise in the data precluded accurate fitting. Kussendrager (1994) re­
ported that lactoferrin denaturation or unfolding in phosphate buffer,
cheese whey or cheese whey permeate followed first order kinetics in a
differential scanning calorimetry (DSC) study, while Sa �nchez et al.
(1992) also reported that lactoferrin denaturation followed first order
kinetics for lactoferrin in phosphate buffer and skim milk.
3.4. Temperature dependence of k values and rate constant predictions
Eq. (5) was fitted to the k values in Tables 3 and 4 using non-linear
regression with reciprocal k weighting to account for the fact that k
values varied over two orders of magnitude. Tref was chosen to be 80 � C.
The values for the parameters, Ea and kref, are given in Table 5 and the
dependence of k on temperature is shown graphically in Fig. 3. Eq. (5)
and the constants from Table 5, Ea and kref, were used to predict k values
at each experimental temperature (see Table 6).
3.5. Validation against independent data
Rate constants, k, for the second sample set were calculated for each
experimental temperature-time profile using Eq. (5) together with the
parameters, Ea and kref, derived from sample set one (see Table 5). These
generated k values were used in Eq. (4) to fit the second set of data for
both milk sources as a validation check on the model. As shown in Fig. 4,
the predicted denaturation curves generated by the model generally
fitted the experimental data well, especially at 75 � C and above.
3.6. Numerical integration of time-temperature trajectories
Denaturation profiles generated by the combined model (Eq. (6))
using the parameters in Table 5 are shown in Fig. 5 and include repre­
sentative Peltier heating and cooling profiles for heat treatments at 65,
H. Liu et al.
70, 75, 80, 85 and 95 � C for a holding time of 10 s at the target tem­
perature. The samples took 30–45 s to reach the end-point temperatures
of 65–85 � C in the Peltier system and about 80 s to reach 95 � C in the oil
bath. This demonstrates how the model can be used to estimate potential
losses of native lactoferrin over various heating steps in an existing
manufacturing process or for optimising a new plant design where
retention of native lactoferrin is of importance.
Assessment of the temperature of a heating step and its range of
control taken together with the time the lactoferrin is exposed to such
temperatures can be input into the model to estimate the losses of native
lactoferrin. Improved control of temperature and its in-process fluctu­
ations along with reduction of exposure time can effectively reduce
native lactoferrin losses. For example, heating at 72 � C for 120 s, the
model predicts retention of 72% of native lactoferrin, whereas if the
temperature rises to 75 � C or even 80 � C for the same period then native
lactoferrin retentions are reduced to 56% and 22% respectively.
Reducing the exposure time to 60 s increases retentions to 85%, 75% and
47% respectively.
From Table 6, the lactoferrin denaturation rate in the Chinese milk is
5-fold greater than in the NZ milk at 65 � C. This could be attributed to
the greater proportion of apo-lactoferrin in the milk (iron saturation
only 11.6% versus 21.9%). DSC studies have reported a denaturation
peak for apo-lactoferrin in the range 60.8–71 � C (Bokkhim et al., 2013;
Paulsson et al., 1993; Sa �nchez et al., 1992). At 70, 75 and 80 � C, lac­
toferrin in the Chinese milk was still denatured about 3.5-, 2.3- and
1.5-fold faster respectively than in the NZ milk although, by 80 � C in the
latter the rate of denaturation had increased 30-fold over that at 65 � C.
Holo-lactoferrin is reported to adopt a more compact conformation
because of the bound iron (Stǎnciuc et al., 2013) compared with the
more open conformation of apo-lactoferrin (Rastogi et al., 2016),
thereby contributing to its greater resistance to heat.
Increasing rates of denaturation around 75–80 � C likely reflect in­
teractions, both covalent and non-covalent between lactoferrin and
whey proteins, particularly β-lactoglobulin (Brisson et al., 2007b). At 85
�
C, lactoferrin in both NZ and Chinese milk samples denatured at the
same rate and at 95 � C, lactoferrin in the NZ milk denatured at twice the
rate of that in the Chinese milk. Loss of iron from holo-lactoferrin rea­
ches significant levels on heating to 90 � C (Sui et al., 2010) leading to an
open, more heat-labile conformation, perhaps accounting for the greater
reduction in heat stability of the more iron-saturated lactoferrin sample
in the NZ milk. At the ultra-high temperature of 121 � C, all lactoferrin
was lost just after 4 s (data not shown).
The values for Ea of 225.56 � 8.80 and 162.94 � 12.90 kJ mol 1 for
lactoferrin in the NZ and Chinese whole milk samples of 21.9% and
11.6% iron saturation respectively can be compared with the values
reported by Sa �nchez et al. (1992) of 183.97 and 157.51 kJ mol 1 for
holo-lactoferrin and apo-lactoferrin respectively when heated in skim
milk over the temperature range 72–85 � C. While lactoferrin in the
Chinese milk samples with an iron saturation of 11.6% yielded a similar
Ea to that for apo-lactoferrin in the study of Sa �nchez et al. (1992), the
lactoferrin in the NZ milk samples with an iron saturation of only 21.9%
yielded an Ea even higher than the value reported by S� anchez et al.
(1992) for the fully iron-saturated holo-lactoferrin. The constant, Ea, is
interpreted by some as an energy of activation with higher values
commensurate with greater heat stability (Sa �nchez et al., 1992), which
contrasts with the results for lactoferrin in the NZ milk.
4. Conclusions
A kinetic model has been developed and validated for the thermal
denaturation of native lactoferrin in raw whole bovine milk. The model
describes the loss of lactoferrin with increasing time and temperature
over the range 65–95 � C. The model’s predictions are better from 75 � C
and above and it has been validated for application in the temperature
range 65–95 � C.
Heat stabilities for lactoferrin in the Chinese-sourced milk were
7
Journal of Food Engineering 280 (2020) 109977
lower than those in the NZ milk up until heating at 85 � C where heat
stabilities were similar. At 95 � C denaturation rates for lactoferrin in the
NZ milk were twice those in the Chinese milk. The greater heat stability
of lactoferrin in the NZ milk can be attributed to the stabilising effect of
the higher iron saturation levels of lactoferrin in the NZ samples.
Heating at 121 � C denatured all lactoferrin in both milk samples within
4 s.
Declaration of competing interest
None.
Acknowledgements
The authors would like to acknowledge Simon Loveday, Di Lu and
Rex Humphrey for assistance with the preparation of the samples and
review of the manuscript.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jfoodeng.2020.109977.
Credit author statement
HYL conceptualised the project, provided study materials and
investigated the result findings.
IB performed experiments and collected data.
MW supervised experiments, data collection and analysis.
QML conceptualised the project and collected study materials.
HXW collected study materials.
PH designed the experiments and conducted data analysis.
YM conceptualised the project and supervised data analysis.
LD conceptualised and designed the research and wrote the paper.
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