Food Hydrocolloids 129 (2022) 107613

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Elucidation on the destabilization mechanism of whipping creams during

static storage
Hua Xu a, 1, Lan Yang b, 1, Jun Jin a, *, Jing Zhang a, Pengkai Xie a, Yuhang Chen a, Longkai Shi a,
Wei Wei a, Qingzhe Jin a, Xingguo Wang a, **
a
State Key Laboratory of Food Science and Technology, Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, International Joint
Research Laboratory for Lipid Nutrition and Safety, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
b
Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Key Laboratory of Dairy Products Processing, Ministry of Agriculture, Inner Mongolia
Agricultural University, Huhhot, 010018, PR China

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

Keywords: The destabilization mechanism of whipping cream emulsions during static storage was elucidated systematically
Whipping cream in the present study. The results indicated that whipping creams mainly undergo three destabilization processes:
Flocculation initial steady state, watering-off, and agglomeration. These three processes sequentially occurred with breakages
Partial coalescence
of the dynamic equilibrium of hydrogen bonds, hydrophobic interactions, ionic bonds, and disulfide bonds.
Watering-off
Agglomeration
Flocculation, mainly caused by negative effects of non-adsorbed whey protein aggregates and free calcium ions,
played a key role in accelerating the destabilization process of watering-off. As the degree of flocculation
increased, fat droplets in a close contact were involved into partial coalescence, followed by forming a three-
dimensional matrix with whey protein/κ-casein complexes, which further facilitated the agglomeration pro­
cess. It is proposed that inhibition of the flocculation is the key to improve stability of whipping cream emulsions
during static storage.

1. Introduction during storage, which is named as “watering-off”. Also, viscosity of the

Whipping creams, generally consisting of 30–40% milk fat, have
wide applications in many foods such as desserts, pastries, cakes, and ice
creams (Sajedi, Nasirpour, Keramat, & Desobry, 2014). The most
important feature of whipping creams is their capacities to transform
into triphasic aerated system that stabilized by partially-coalesced fat
globules formed at air-water interface (Li, Li, Wang, et al., 2020). A
desired whipping cream not only needs a good whippability but also
requires a higher emulsion stability during storage and transport; that is,
it should achieve a subtle balance between stability and destabilization.
The development of sterilization technology, especially
ultra-high-temperature (UHT), has diminished microbial activities and
extended shelf-life of whipping creams from a couple days to more than
half a year. Such whipping creams become more popular for both dairy
manufacturers and consumers because no cold chains are required for
storage and transportation.
Whipping creams are prone to form an aqueous layer on the bottom
cream emulsions will increase over time, and a solid gel is eventually
formed on the top, which is called “agglomeration”. Both watering-off
and agglomeration resulted in shorter shelf-life and unappealing
appearance, which has puzzled dairy manufacturers for a long time. It is
necessary to understand the instability mechanism of whipping creams
during storage to put forward proper solutions. Creaming, flocculation,
Ostwald ripening and coalescence are the well-known and studied
instability mechanisms of oil-in-water emulsions (Petrut, Danthine, &
Blecker, 2016). Partial coalescence is a typical destabilization mecha­
nism of whipping creams during beating, which is also considered by
many researchers as a potential instability factor in emulsion storage
and transportation (Fredrick, Walstra, & Dewettinck, 2010; Nguyen,
Duong, & Vu, 2015; Wang, Hartel, & Zhang, 2021). Although such a
coalescence plays an essential role in changing fat globule dispersion,
there is still a controversy that needs to be addressed: whether partial
coalescence is the key factor for the watering-off or agglomeration of
whipping creams?

* Corresponding author.
** Corresponding author.
E-mail addresses: junjin@jiangnan.edu.cn (J. Jin), wangxg1002@gmail.com (X. Wang).
1
The two authors contributed equally to this work and share the first authorship.

https://doi.org/10.1016/j.foodhyd.2022.107613
Received 15 September 2021; Received in revised form 30 January 2022; Accepted 21 February 2022
Available online 23 February 2022
0268-005X/© 2022 Elsevier Ltd. All rights reserved.
H. Xu et al.
Over the past decades, a number of researches have been focused on
the effects of various fats (Liu et al., 2021; Moens, Tavernier, & Dew­
ettinck, 2018), proteins (Cao, Liu, Zhang, Wang, & Ren, 2020; Long,
Zhao, Sun-Waterhouse, Lin, & Zhao, 2016) and low-molecular-weight
emulsifiers (Cheng, Dudu, Li, & Yan, 2020; Zeng et al., 2021; Zhao
et al., 2013) on whipping properties of creams. For the cream emulsions,
ensuring an extended shelf-life is also equally important. However, the
emulsion stabilities were mostly evaluated by analyzing initial indices in
their fresh status, instead of storage period (Fu, He, Zeng, Qin, & Chen,
2020; Lazzaro et al., 2017; Li, Li, Wang, et al., 2020). Nowadays, the
destabilization mechanism of whipping cream emulsions during storage
is still unclear. In the present study, changes in creaming index (CI),
viscosities, particle size distributions, microstructures, physicochemical
characteristics, protein compositions, surface tensions, zeta potential,
and chemical bonds and interactions of the whipping cream emulsions
during storage were systematically examined, as well as their whipping
properties. The destabilization mechanism was further proposed and
discussed, which could provide a theoretical basis for manufacture of
whipping creams with improved emulsion stabilities and favorable
whipping properties.
2. Materials and methods
2.1. Materials
Whipping creams were donated by a local dairy supplier (Inner
Mongolia, China). Nile red was purchased from Sigma-Aldrich Chemical
Co. Ltd. (St. Louis, USA). All other reagents and chemicals were of
analytical grade and purchased from Sinopharm Chemical Reagent Co.,
Ltd. (Shanghai, China).
2.2. Storage tests of whipping creams
The whipping creams (350 g) were filled into a sealed bottle and
stored at 20 ± 1 ◦ C (surrounding humidity, 45–50%) for 0, 2 and 5
months. Sodium azide (0.02%) was added to inhibit bacterial growth.
2.3. Whipping properties of whipping creams
2.3.1. Whipping time
After storage at 8 ◦ C for 2 h, whipping cream emulsions were beaten
immediately using a household kitchen mixer (DDQ-B01K1, Bear,
Foshan, China) at a speed of 160 rpm, and the time frame for forming
strong foam and soft spike was recorded as whipping time.
2.3.2. Overrun
Overrun was determined by weighing a fixed volume of whipping
cream with the same volume of whipped cream using equation below:
m1 − m2
Overrun (%) = × 100
m2
where m1 and m2 represent the mass of original whipping cream (g) and
the mass of whipped cream (g), respectively.
2.3.3. Serum loss
The whipped cream (5 g) was placed on a 50-mesh sieve at ambient
temperature for 2 h. The serum loss was calculated as equation below:
m1
Serum ​ loss (%) =
m2
× 100
where m1 and m2 are the mass of serum loss (g) and the mass of whipped
creams (g), respectively.
2.3.4. Decorating performance
Fresh whipped creams were piled into a shaped topping using a
2
Food Hydrocolloids 129 (2022) 107613
nozzle and placed on petri dish at ambient temperature for 2 h.
Appearance of the sample was observed in a mini photo studio and
photographed.
2.4. Emulsion properties of whipping creams
2.4.1. Creaming index
Creaming stability was examined by a visual observation method as
described previously (Al-Maqtari et al., 2021). The extent of
watering-off was characterized by CI, which was calculated using the
following equation:
H1
CI (%) =
H2
× 100
where H1 is the height of the serum layer, and H2 is the total height of
whipping cream.
2.4.2. Viscosities
Viscosities were measured using a digital viscometer NDJ-5S (Li
Chen Inc., Shanghai, China) at a revolution rate of 12 rpm.
2.4.3. Particle size distributions
Particle size distributions of cream emulsions on the top, middle, and
bottom layers were determined using a Microtrac S3500 laser particle
size analyzer (Microtrac Inc., USA). The refractive index of dispersed
particles and distilled water were 1.59 and 1.33, respectively. Particle
size measurement was reported as the volume-average droplet size.
2.4.4. Confocal laser scanning microscopy (CLSM)
Microstructures of cream emulsions on the top, middle, and bottom
layers were observed by a Zeiss LSM880 confocal laser scanning mi­
croscope (Carl Zeiss Jena, Germany) equipped with a 40× objective.
Twenty microliters of Nile red (0.02%, w/v) were added to 100 μL of the
prepared whipping cream, followed by diluting for ten times using
distilled water. Approximately, 6 μL of the stained sample was placed
into a slide and covered with a coverslip in the absence of any air or
bubbles between the sample and coverslip. A 40× objective was used to
observe the sample. Nile red was excited at 488 nm with an argon laser,
and the emission were recorded at 500–755 nm.
2.4.5. Cryo-scanning electron microscopy (Cryo-SEM)
Detailed information on microstructures were further observed by a
scanning electron microscope (SU3500, Hitachi, Japan). Emulsion
sample was applied onto a gold planchette, which was then immersed in
liquid nitrogen; subsequently, the sample was fractured at − 140 ◦ C by a
cryogenically cooled knife, and sublimated at − 80 ◦ C for 10 min. The
sample was finally transferred into a SEM chamber and observed at
− 140 ◦ C under 5 kV.
2.4.6. Physicochemical characteristics
Samples on the top, middle, and bottom layers were subjected to
analyze the typical physicochemical indices. Moisture, fat, and protein
contents were determined according to GB 5009.3–2016, GB
5009.6–2016, and GB 5009.5–2016, respectively. Concentrations of free
calcium ion were measured by atomic absorption spectrometry refer­
enced to GB 5009.92–2016. Adsorbed protein fractions were determined
according to the method described by Guo et al. (2022). Oxidation of oils
and fats was evaluated by monitoring the peroxide values according to
the method of GB5009.227–2016.
2.4.7. Protein compositions
Protein compositions were determined by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970). The
concentrations of stacking and separating gels were 3% and 12.5%,
respectively. The sample was dissolved in the SDS-PAGE buffer in the
H. Xu et al.
Fig. 1. Visual appearance (a), creaming index (b), viscosity (c), whipping time (d), overrun (e), and decorating performance (f) of whipping creams during storage.
presence (reducing conditions) or absence (non-reducing conditions) of
DTT. After heating for 5 min in a boiling water bath and centrifuging at
10,000 g for 5 min, each sample (10 μL) was loaded to a cell. The gel was
stained with Coomassie Brilliant Blue (G-250) and scanned using a
computing densitometer (Molecular Imager Chemi DocXRS+, Bio-Rad,
USA). The intensities of bands were integrated using Image-Pro Plus
6.0 software.
2.4.8. Surface tensions
Surface tensions were measured according to the Wilhelmy plate
method using an automatic surface tensiometer (DCAT21, DataPhysics
Corporation, Germany) at 25 ◦ C.
2.4.9. Zeta potentials
Zeta potentials were measured using a Zetasizer Nano-ZS90 instru­
ment (Malvern Ltd., UK) at 25 ◦ C. Cream emulsions were diluted with
deionized water at a ratio of 1:200 (v/v) prior to the measurements.
2.4.10. Chemical bonds and interactions
Cream emulsions were solubilized in five chemical dissociation
agents to evaluate the contribution of different interaction forces (Yang
et al., 2021). The five selected solvents were: 0.05 mol/L NaCl (SA), 0.6
mol/L NaCl (SB), 0.6 mol/L NaCl + 1.5 mol/L urea (SC), 0.6 mol/L NaCl
+ 8 mol/L urea (SD), and 0.6 mol/L NaCl + 8 mol/L urea +0.5 mol/L
β-mercaptoethanol (SE). Each sample (2 g) was dissolved in 10 mL of the
different solvents, vortexed (2500 rmp, 10 min) and then centrifuged
(10000 g, 4 ◦ C, 15 min). Protein concentrations in the supernatant were
determined by the Bradford protein assay kit (KeyGEN BioTECH). The
solubilities of (SB-SA), (SC-SB), (SD-SC) and (SE - SD) represent the
contribution of ionic bonds, hydrogen bonds, hydrophobic interactions,
and disulfide bonds, respectively.
2.5. Statistical analyses
All the experiments were performed in triplicate, and the results
were presented as mean ± standard deviation. Statistical analyses were
conducted using SPSS version 18.0. The quantitative differences were
assessed by ANOVA with Duncan’s multiple range test, and a probability
3
Food Hydrocolloids 129 (2022) 107613
value of P < 0.05 was considered statistically significant.
3. Results and discussion
3.1. Destabilization phenomenon of whipping creams
There are scarcely any high fat-containing emulsions (e.g., 30–40%)
showed perfect stable during storage. As shown in Fig. 1a, freshly pre­
pared whipping creams were homogeneous without any watering-off
(CI = 0%; Fig. 1b); whereas a serum layer was easily observed after 2
months storage (CI = 2.51%; Fig. 1b), and the thickness gradually
increased on the bottom. Two layers, mainly an opaque cream layer and
a transparent serum layer with CI of 6.03% (Fig. 1b), were completely
formed after 5 months storage. Viscosities had a trend toward an in­
crease over time (from 250 to 3870 mPa⋅s) as presented in Fig. 1c,
indicating that agglomeration occurred during the storage. In contrast,
most cream products on the market undergo severe watering-off and
agglomeration within one month when stored at improved temperature.
Whipping properties reflect the capacity to form foam. Changes in
whipping properties of the whipping creams during storage are shown in
Fig. 1d–f. Fresh whipping creams exhibited a strong foam and soft spike
within 313.5 s, and the overrun was up to 158.62%. The whipped cream
had a smooth surface and kept a firm shape with no serum loss (data not
shown) after storage at ambient temperature for 2h. After 2 months-
storage, the cream emulsions showed a shorter whipping time (222.0
s) and a slightly decreased overrun (144.59%). Meanwhile, the whipped
sample still had a smooth surface and a clear outline without obvious
collapse. The whipping time and overrun significantly decreased to 49.5
s and 31.09%, respectively, as the storage time increased to 5 months.
Despite no serum loss and collapse, the surface of whipped cream
became very rough.
For commercially packed whipping creams, slight watering-off does
not affect the appearance and functionality in some cases as the thin
serum layer will be readily mixed again with the rest of emulsion by
gently shaking the package. However, subsequent agglomeration not
only causes heterogeneous appearance and thick texture but also
severely damages the whipping capacities, which is totally unacceptable
for consumers and dairy manufacturers.
H. Xu et al. Food Hydrocolloids 129 (2022) 107613

Fig. 2. Particle size distributions in each emulsion layer during storage.

Fig. 3. CLSM (a) and Cryo-SEM (b) images in each emulsion layer during storage.

3.2. Changes in particle size distributions during storage
Particle size is an important parameter to evaluate the cream emul­
sion stability during storage and processing (Dhungana, Truong, Bansal,
& Bhandari, 2019). Fig. 2 depicts the particle size distributions of
whipping creams in different layers coupled with mean particle sizes.
Fresh cream samples showed a narrow monomodal distribution ranging
from 1.38 to 7.78 μm with a mean particle size of 2.46 μm, indicating
that their fat droplets were homogeneously dispersed without aggre­
gation. After 2 months of storage, the distribution range of the top
layer-sample extended from 0.97 to 31.11 μm, and the mean particle size
was increased to 3.33 μm, suggesting that a small population of fat
droplets aggregates occurred. In contrast, the bottom layer-samples
exhibited a bimodal distribution with some particles in a range of
0.04–0.17 μm and others in a range of 0.82–13.08 μm, and the mean
particle size was decreased to 1.69 μm. The former was probably
attributed to whey protein aggregates or casein micelles, whose di­
ameters ranged from tens to hundreds of nanometers (Dauphas et al.,
2005; Fan, Peng, Pang, Wen, & Yi, 2021). After 5 months storage, par­
ticle sizes of creams on the top cream layer were distributed in the range
from 11.00 to 52.32 μm, and the mean particle size became extremely
large (up to 22.50 μm). Such a steep increase revealed that numerous

4
H. Xu et al.
Fig. 4. Moisture contents (a), fat contents (b), protein contents (c), adsorbed protein fractions (d), and concentrations of free calcium ions(e), peroxide values (f) in
each emulsion layer during storage. Note: *, the column represents the top, middle, and bottom layers of fresh samples.
large fat clusters were formed on the top layers; on the contrary, mean
particle sizes of the bottoms layer-samples further decreased to 0.83 μm.
As large fat droplets moved up, small fat droplets and non-adsorbed
whey protein aggregates were left in the bottom aqueous layer. No
substantial changes were observed in particle size distributions of the
middle layer-samples throughout the storage period.
3.3. Changes in microstructures during storage
To look for insight into the changes in microstructures of whipping
creams during storage, CLSM analysis was carried out on the samples
collected from different layers. As illustrated in Fig. 3a, fresh cream
emulsions showed a uniform and stable morphology. The fat droplets on
the top cream layers became flocculated after a storage of 2 months; in
contrast, both amounts and volume of fat droplets on the bottom
aqueous layer decreased sharply. As the storage time prolonged to 5
months, obvious large and irregularly shaped fat clusters were observed
on the top cream layer, indicating that cream emulsions underwent a
partial coalescence process. On the bottom aqueous layer, fat droplets
were hardly observed, also implying that there is a serious upward
movement of the fat globules. During the whole storage period, fat
droplets on the middle emulsion layer remained relatively uniform. The
changes of microstructures were further validated by observation of
complementary cryo-SEM images (Fig. 3b). It was observed that a
number of fat droplets were stacked closely together at 2 months stor­
age, confirming the unpleasant process of droplet flocculation. Partial
coalescence of the emulsions was then validated by large amounts of
closely packed fat droplets that were partially fused at 5 months storage.
These changes in microstructures corresponded well to the particle size
distributions in Section 3.2. It can be inferred that whipping creams
mainly underwent a two-fold process, by which fat droplets firstly
flocculated and then partially coalesced during storage.
5
Food Hydrocolloids 129 (2022) 107613
3.4. Changes in physicochemical characteristics during storage
Changes in physicochemical characteristics were examined by col­
lecting samples from top, middle, and bottom layers of the cream
emulsions, respectively, and the results are shown in Fig. 4. For the top
cream layer, fat contents significantly increased from 34.60% to 49.13%
(P < 0.05), while moisture contents dramatically decreased from
59.84% to 46.32% (P < 0.05) as the storage time prolonged from 0 to 5
months (Fig. 4a and b). The case is completely opposite on the bottom
aqueous layer. It is indicated that fat droplets were inclined to move
upwards owing to the density differential, which is explicable in terms of
the fact that fat droplets have a lower density than the surrounding
aqueous phase. Distributions of proteins and calcium ions were also
changed along with the water and fat droplets. As shown in Fig. 4c and
d, protein contents on the top cream layer were increased to 2.29%,
which were much higher than those on the bottom aqueous layer
(0.35%) after 5 months of storage; meanwhile, adsorbed protein frac­
tions of top layer-samples increased from 86.50% to 91.62%. The for­
mation of gel during storage was probably related to the accumulation of
proteins in the top layer. Fig. 4e shows that freshly prepared cream
sample contained 195.0 mg/L of free calcium ions. After 5 months of
storage, the concentrations of free calcium ions on the top, middle, and
bottom layers decreased to 37.4, 40.8 and 87.4 mg/L, respectively,
which indicated that large amounts of free calcium ions were bound
with proteins in cream systems during storage. It has been reported that
free calcium ions can decrease electrostatic repulsion between droplets
as the effects of electrostatic screening and ion binding, which was
associated with droplet flocculation (Keowmaneechai & McClements,
2002). Oxidation of fats and oils evokes strong off-flavors and deterio­
ration of health benefits, affecting shelf-life and quality of food products
(Rafałowski, Żegarska, Kuncewicz, & Borejszo, 2014). From Fig. 4f,
peroxide values remained at relatively low levels (0.45–0.58 mmol/kg)
over the entire storage period, suggesting a lower degree of lipid
H. Xu et al.
Fig. 5. Non-reducing (a) and reducing (b) SDS-PAGE patterns of proteins in each emulsion layer during storage.
oxidation.
3.5. Changes in protein compositions during storage
Milk proteins play an important role in emulsion stability due to their
amphiphilic features, which allows them to adsorb spontaneously at oil-
water interfaces during emulsification (Liang et al., 2017). Fig. 5 depicts
the SDS-PAGE profiles of proteins presented in each layer during stor­
age. The content of each component is listed in Table 1. Non-reducing
electrophoresis shows that protein bands of caseins (αs-casein and
β-casein) were clearly on both top and middle layer-samples, and the
contents showed little changes across storage period. In contrast, rela­
tive contents of αs-casein and β-casein in the bottom layer-samples were
decreased from 39.48% to 31.95% and from 48.02% to 36.01%,
respectively. Relative contents of high molecular weight (HMW) in the
bottom layer-samples were increased from 12.50% to 32.04%, sug­
gesting a higher degree of protein aggregates that failed to enter the gel
during testing. Whey proteins might be responsible for these aggregates
as hardly any bands of whey proteins were found in all samples in
Fig. 5a. Interestingly, the top and middle layer-samples contained higher
percentages of caseins (αs-casein and β-casein) and a lower percentage of
HMW compared with the bottom layer-samples.
Further protein analyses were conducted by reducing SDS-PAGE
patterns. Similar to the non-reducing conditions, relative contents of
αs-casein and β-casein in both top and middle layer-samples varied little
over storage time, while those in bottom layer-samples decreased from
6
Food Hydrocolloids 129 (2022) 107613
34.83% to 21.32% and from 31.40% to 18.37%, respectively. Due to the
breakage effects of added DTT on disulphide bonds, HMW smears of all
the samples disappeared, and whey proteins (mainly α-lactalbumin and
β-lactoglobulin) were clearly observed (Fig. 5b). Specifically, few
changes in relative contents of α-lactalbumin and β-lactoglobulin were
observed in top and middle layer-samples during the storage. In turn,
relative contents of α-lactalbumin and β-lactoglobulin in bottom layer-
samples increased from 14.27% to 24.69% and from 12.18% to
25.11%, respectively. After 5 months, relative contents of κ-caseins in
bottom layer-samples increased from 7.32% to 10.51%, and the increase
was much higher than that in top and middle layer-samples (8.17%,
8.30%). The changes in κ-caseins indicated that whey protein aggregates
may also interact with casein micelles, more specifically with κ-caseins,
forming whey proteins/κ-casein complexes (Livney, Corredig, & Dal­
gleish, 2003). Subsequent release of whey proteins/κ-casein complexes
could lead to the formation of gel (Chavan, Chavan, Khedkar, & Jana,
2011).
The SDS-PAGE results indicated that during the storage of whipping
creams, caseins tended to move upwards along with fat droplets, while
whey proteins mainly sank with aqueous phase in the forms of aggre­
gates. Caseins, comprising approximately 80% of the total milk protein
content, could adsorb preferentially at the oil-water interface over whey
proteins during the process of emulsification (Sliwinski, Lavrijsen, et al.,
2003; Wong, Camirand, Pavlath, Parris, & Friedman, 1996). Once ca­
seins adsorbed as a pre-adsorbed layer at interface, they would hinder
the sequential adsorption of whey proteins (Nylander & Wahlgren,
H. Xu et al. Food Hydrocolloids 129 (2022) 107613

Table 1 Zeta potential measurement is widely used to determine surface

Protein compositions of whipping creams in each emulsion layer during room charge density of food emulsions. Higher surface charge density gener­
temperature storage. ally provides an additional electrostatic repulsion between fat droplets
Non-reducing SDS-PAGE (Ziaeifar, Shahi, Salami, & Askari, 2018). As shown in Fig. 6b, droplet
Month 0 Month 2 Month 5
surface potential was most negative in the newly prepared cream
emulsions, being around − 24.7 mV, which could be explained by the
HMW (%) Top 12.50 ± 2.16 7.76 ± 0.50b 11.07 ±
fact that milk proteins are negatively charged at neutral pH (Swaisgood,
0.87b
Middle 8.03 ± 0.73b 11.77 ± 1996). However, the magnitudes of surface potentials became less
1.25b negative during storage. Zeta potential values of cream emulsions on the
Bottom 26.11 ± 0.50a 32.04 ± 0.44a top layers increased to − 14.80 mV after 5 months storage, suggesting
αs-casein (%) Top 39.48 ± 1.97 42.04 ± 0.35a 38.97 ± the decreases of electrostatic repulsion among the fat droplets (P <
0.52b
Middle 40.97 ± 41.57 ± 0.28a
0.05). As increasing fat droplets accumulated on the top cream layers,
0.70b steric stabilization, the inter-droplet repulsion associated with
Bottom 34.03 ± 0.23c 31.95 ± 1.35c compression and overlap of adsorption layers on droplets, would make a
β-casein (%) Top 48.02 ± 0.97 50.20 ± 0.64a 49.94 ± 0.37a major contribution for the emulsion stability (Dickinson, 2019).
Middle 51.01 ± 1.41a 46.66 ±
Chemical dissociation agents are frequently used to evaluated the
1.46b
Bottom 39.86 ± 36.01 ± 1.25c contribution of covalent and non-covalent bonding because they can
0.27b destroy the interaction forces among proteins or between proteins and
Reducing SDS-PAGE
water molecules (Hong, Xiao, Li, Li, & Xie, 2022). As shown in Fig. 6c,
Month 0 Month 2 Month 5 the intensities of the interactions in fresh whipping creams were in the
following order: hydrogen bonds (5.14 mg/mL) > hydrophobic in­
αs-casein (%) Top 34.83 ± 0.64 37.36 ± 0.45a 35.86 ± 0.90a
Middle 37.23 ± 0.99a 33.66 ± teractions (1.88 mg/mL) > ionic bonds (1.83 mg/mL) > disulfide bonds
0.52b (0.72 mg/mL). These interactions drive and maintain the steady state of
Bottom 24.97 ± 21.32 ± 1.15c fat droplets, and continually change over time. With the prolongation of
1.14b storage time, the contribution of ionic bonds and hydrogen bonds
β-casein (%) Top 31.40 ± 0.41 30.92 ± 1.05a 32.01 ± 0.85a
Middle 30.65 ± 0.33a 30.06 ± 1.05a
gradually reduced to 1.38 and 3.99 mg/mL, respectively; and the loss
Bottom 18.96 ± 18.37 ± means the disruption of hydrogen bonds and ionic bonds, leading to the
2.03b 1.39b changes in the molecular structure of milk proteins. After 2 months of
κ-casein (%) Top 7.32 ± 0.64 8.36 ± 0.63b 8.17 ± 0.83b storage, the contribution of hydrophobic interactions and disulfide
Middle 9.05 ± 0.38b 8.30 ± 0.79b
bonds increased to 3.51 and 0.84 mg/mL, respectively, indicating that
Bottom 12.11 ± 1.31a 10.51 ± 0.15a
α-lactalbumin (%) Top 14.27 ± 0.87 12.57 ± 13.91 ± hydrophobic interactions had the greatest contribution to watering-off
0.46b 0.76b process. After 5 months, the contribution of hydrophobic interactions
Middle 12.17 ± 14.53 ± decreased slightly to 2.52 mg/mL, but the contribution of disulfide
0.46b 0.61b bonds greatly increased to 2.39 mg/mL, suggesting that disulfide bonds
Bottom 23.68 ± 0.31a 24.69 ± 0.47a
β-lactoglobumin (%) Top 12.18 ± 0.88 10.79 ± 10.06 ± 0.85c
played an important role in agglomeration process. Combining the
0.48b SDS-PAGE analyses, it was inferred that the changes in these interactions
Middle 10.89 ± 13.45 ± are mainly associated with non-adsorbed whey protein aggregates that
1.29b 0.10b can form not only the larger aggregates with other whey proteins but
Bottom 20.28 ± 1.74a 25.11 ± 2.37a
also the whey protein/micelle complexes with caseins (Nicolai &
Values followed by different letters in the same column differed beyond a 5% Chassenieux, 2021).
significance.
3.7. Destabilization mechanism of whipping creams during storage
1994). Whey proteins were prone to undergo denaturation and aggre­
gation during high temperature-sterilization, and the denaturation de­ Whipping creams are thermodynamically unstable systems that tend
grees reached 60%–90% after UHT treatment (Akkerman et al., 2016; to undergo an destabilization phenomenon during storage. Taking all
Sliwinski, Roubos, Zoet, van Boekel, & Wouters, 2003). It has been the above results together, instability process of whipping creams during
pointed out that whey protein aggregates are poor at forming emulsions storage could be divided into following three stages: initial steady state,
due to their inflexibilities and slow diffusion rates at oil-water interface watering-off, and agglomeration (Fig. 7a). The steady state of droplets in
(Millqvist-Fureby, Elofsson, & Bergenståhl, 2001). Moreover, the pres­ freshly prepared creams depends on the balance of various intermolec­
ence of non-adsorbed whey protein aggregates in the aqueous phase ular forces (hydrogen bonds, hydrophobic interactions, ionic bonds, and
might induce “depletion flocculation” (Jenkins & Snowden, 1996; disulfide bonds). At this stage, cream emulsions are uniform with milky
Radford & Dickinson, 2004). Therefore, it can be inferred that the white appearance, and fat droplets show a homogeneous distribution
presence of non-adsorbed whey protein aggregates is an important risk without aggregation. However, the droplets have a tendency of upward
factors for emulsion destabilization of whipping creams. movement due to density difference between oil and aqueous phase,
which is also named as creaming (Loi, Eyres, & Birch, 2019).
3.6. Changes in intermolecular interactions during storage At the initial stage of storage, about 1–2 months, fat droplets in
emulsions frequently undergo collisions, which may result in an exten­
Surface characteristics, providing useful information about air-liquid sive flocculation and an accelerated creaming process, thereby further
interactions in cream emulsions, are the significant factors in determi­ causing the process of watering-off. In this case, non-adsorbed whey
nation of emulsion stability. Surface tensions are formed by the cohesion protein aggregates have a key role in droplet flocculation and aggrega­
and attributed to the attraction of atoms, ions and molecules (Geng tion. As the gap between two fat droplet surfaces becomes less than the
et al., 2021). Fig. 6a illustrates the changes in surface tensions of diameter of non-adsorbed whey protein aggregates, protein concentra­
whipping cream emulsions during storage. It was observed that the tions increase outside of the gap and creates an osmotic pressure
surface tensions were increased from 36.22 to 51.94 nN/m, indicating gradient inside and outside the interstitial space, thereby pushing the fat
that cream emulsions exhibited poor surface properties due to the droplets together (Fig. 7b); on the other hand, non-adsorbed whey
gelation and viscosity effects. protein aggregates interact with interfacial proteins and other non-

7
H. Xu et al.
Fig. 6. Surface tension (a), zeta potential values (b), and chemical bonds and interactions (c) on top the layer during storage.
adsorbed proteins via hydrophobic interactions, promoting the forma­
tion of intermolecular disulfide bonds mediated by thiol-disulfide
interchange reactions (Fig. 7c). In addition, it should be noted that
even without any addition of calcium ionic, also naturally present in the
raw milk (an important ingredient in the manufacture of whipping
creams). The presence of free calcium ions can reduce electrostatic
repulsion forces between droplets and contribute to the flocculation of
cream emulsions (Fig. 7d).
As the storage prolonged to over 4–5 months, the degree of watering-
off becomes more serious as increasing fat droplets are involved in the
flocculation and subsequent partial coalescence (Fig. 7e). Meanwhile,
whey proteins/κ-casein complexes released from casein micelles form a
three-dimensional matrix with partially coalesced fat droplets, further
making the whipping cream emulsions thicken and gelatinous. Coalesce
will be occurred in the upper layer in the next stage if storage temper­
ature is higher or storage time is long enough, eventually leading to
formation of a shiny oil layer, which is referred to as oiling-off (Pior­
kowski & McClements, 2014). This instability phenomenon rarely
happens during the shelf life of whipping cream products.
4. Conclusion
Destabilization mechanism of whipping creams during storage was
systematically elucidated in the present study. The emulsions mainly
undergo three destabilization stages, i.e., initial steady state, watering-
off, and agglomeration. At initial steady state, fat droplets in the
8
Food Hydrocolloids 129 (2022) 107613
emulsions had a homogeneous dispersibility; followed by gradually
moving upwards and flocculating, resulting in an increased viscosity and
an accelerated watering-off process. As storage time further prolonged,
increasing fat droplets were involved in flocculation, and partial coa­
lescence then occurred in the upper layers of the emulsions, thereby
leading to the agglomeration process. It is concluded that flocculation,
mainly induced by non-adsorbed whey protein aggregates and free
calcium ions, is the predominate factor to generate destabilization
process. Inhibition of the flocculation is therefore suggested to improve
the emulsion stability of whipping creams during storage. This study is
valuable for the manufacture of whipping creams with an extended
shelf-live and a good whippability.
CRediT authorship contribution statement
Hua Xu: Investigation, Writing – original draft, Formal analysis. Lan
Yang: Writing – original draft, Data curation, Validation. Jun Jin:
Conceptualization, Writing – review & editing. Jing Zhang: Data
curation, Visualization. Pengkai Xie: Data curation, Software. Yuhang
Chen: Data curation. Longkai Shi: Writing – review & editing. Wei
Wei: Validation. Qingzhe Jin: Methodology, Supervision. Xingguo
Wang: Funding acquisition, Project administration, Conceptualization.
Declaration of competing interest
The authors declared that they have no conflicts of interest to this
H. Xu et al.
Fig. 7. Schematic illustration of destabilization mechanism of room temperature-stored whipping creams during storage (a: instability process; b: depletion floc­
culation; c: hydrophobic interactions; d: bridging flocculation; e: partial coalescence).
work.
Acknowledgements
This work was supported by China Postdoctoral Science Foundation
(2021M691291), and the Fundamental Research Funds for the Central
Universities (JUSRP12004).
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