International Journal of Biological Macromolecules 178 (2021) 136–142

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

Impact of ice structuring protein on myofibrillar protein aggregation

behaviour and structural property of quick-frozen patty during
frozen storage
Fangfei Li a, Xin Du b, Yanming Ren c, Baohua Kong b, Bo Wang b, Xiufang Xia b,⁎, Yihong Bao a,⁎
a
College of Forestry, Northeast Forestry University, Harbin, Heilongjiang 150040, China
b
College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang 150030, China
c
Heilongjiang Province Agricultural Products and Veterinary Drug Feed Technical Identification Station, Harbin, Heilongjiang 150090, China

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

Article history: The goal of this study was to explore the cryoprotective effect of ice structuring protein (ISP) on the aggregation
Received 5 December 2020 behaviour and structural changes of myofibrillar protein (MP) from quick-frozen pork patties during frozen stor-
Received in revised form 24 January 2021 age. Frozen storage causes the formation of large protein aggregates and weakens MP structures. After adding ISP
Accepted 21 February 2021
into patties, MP had a more stable aggregation system, which was manifested by a uniform particle size distribu-
Available online 23 February 2021
tion and significantly higher absolute zeta potential (11.71 mV) than the control (9.56 mV) (P < 0.05). Atomic
Keywords:
force microscopy results showed that the surface roughness of MP aggregation decreased by 9.78% with ISP
Ice structuring protein after freezing for 180 d. Additionally, compared to patties without ISP, the MP carbonyl content from the ISP-
Quick-frozen pork patties treated patty decreased by 32%, and the free amino content increased by 14.99% during frozen storage. Results
Myofibrillar protein from circular dichroism spectroscopy and fluorescence spectroscopy showed that MP secondary and tertiary
Average particle size structure stability in patties improved with ISP. Overall, ISP has the potential to improve MP aggregation and
Structure unfolding structural stability during frozen storage.
© 2021 Elsevier B.V. All rights reserved.

1. Introduction strength and microstructure, during frozen storage [9]. Unfolding of

As a type of convenient food, quick-frozen pork patties should be
quickly frozen at −30 °C for 30 min and then placed at −18 °C for pro-
cessing, conservation and sale circulation [1]. These patties are popular
with consumers due to their convenient characteristics, satisfactory
quality and sensory properties [2]. Frozen storage is one of the most use-
ful methods for food preservation and can effectively prolong the shelf
life of meat and meat products by inhibiting microbial spoilage and re-
ducing enzyme activity and biochemical reaction rates [3,4]. However,
irreversible changes and quality deteriorations of meat products in-
duced by the formation of ice crystals, recrystallization, oxidative reac-
tions [5] and protein denaturation unavoidably occur during frozen
storage [6,7].
Myofibrillar protein (MP), which accounts for approximately
55–65% of meat muscle total protein, is responsible for many physico-
chemical properties of meat products. Improper MP aggregation and
structural degradation are critical factors that reduce protein functional-
ities, leading to the quality diversification of meat and meat products
[8]. For example, the formation of protein aggregates has been known
to yield decreasing gel qualities, such as water holding capacity, gel
⁎ Corresponding authors.
E-mail addresses: xiaxiufang@neau.edu.cn (X. Xia), baoyihong@163.com (Y. Bao).
the MP structure induced by a high denaturation temperature may
cause MP gel to soften and rot, which degrades the final juiciness and
organization status of meat products [10]. Also, the destruction of pro-
tein structural integrity results in the shrinkage of interfilaments [11],
which lead to water moisture and loss of patty after freezing and
thawing [4]. Zhang et al. [12] showed that MP oxidative denaturation
may lead to a reduction in shrimp texture properties and flavour.
Thus, MP properties are critical to improving meat product quality dur-
ing processing.
Emerging techniques, including high-intensity ultrasound [13], elec-
tric field-assisted freezing and high-pressure-assisted freezing [14],
have been used to inhibit meat quality deterioration by acting on the
MP structure. Cryoprotectants, such as alginate oligosaccharides [15],
xylooligosaccharides [16], ice structuring protein [17] and plant extract
[18], have also been reported to increase functional quality properties
when added to food. As a new type of antifreeze agent, ice structuring
protein (ISP) is a type of stress-tolerant protein [19] and has been dis-
covered in different organisms, including bony fish [20], insects [21],
plants [22], fungi [23] and diatoms [24]. The protective function of
ISPs are based on their unique properties, which include ice plane affin-
ity [25], thermal hysteresis [26], ice recrystallization inhibition ( [27]
and transient binding of an organism to ice [28]. ISP in frozen food can
control the growth morphology and aggregation of ice crystals [29,30],

https://doi.org/10.1016/j.ijbiomac.2021.02.158
0141-8130/© 2021 Elsevier B.V. All rights reserved.
F. Li, X. Du, Y. Ren et al.
which can enhance cell integrity and reduce tissue damage [31]. In fro-
zen meat systems, the inhibitory effect of ISP on meat quality deteriora-
tion [32] and peroxidation [30] during freezing processes has been
demonstrated. Previous studies have also shown that ISP can effectively
protect the quality of meat and meat products [33]. To date, information
concerning the impact of ISP on MP aggregation behaviour and struc-
tural properties in patties during frozen storage has not been reported.
The objective of this study was to identify the cryoprotective effect of
ISP on the aggregation behaviour and structural changes in MP and to
show the mechanism by which ISP maintains the MP characteristics of
quick-frozen pork patties during frozen storage.
2. Materials and methods
2.1. Chemical
The ice structuring proteins were procured by Nanjing Anfei Bio at a
purity above 95%. Piperazine-N,N′-bis-2-ethanesulfonic acid, sodium
chloride (NaCl), ortho-phthaldialdehyde, trichloroacetic acid,
hydrochloric acid, ethanol, ethyl acetate, guanidine, borax, 2,4-
dinitrophenylhydrazine, sodium dodecyl sulfate, guanidine hydrochlo-
ride, β-mercaptoethanol and L-leucine were purchased from Solabio
Corporation (Beijing, China). All chemicals were of analytical grade.
2.2. Patty preparation
Post-rigor pork shoulder and neck were purchased from Jiajiale
commercial abattoir (Harbin, Heilongjiang, China). Trimmed of visible
connectivity, the meat, ISPs (0% and 0.20%) and 12% ice water were
mixed for 5 min. The mixture was made into patties of approximately
100 g each (9 cm diameter and 2 cm thickness) and then kept at
−18 °C. A total of 30 patients were divided into 2 groups: the control
group (15 samples with 0% ISP) and the ISP group (15 samples with
0.20% ISP). Each group was randomly divided into 5 treatments and fro-
zen for 0, 30, 60, 90 and 180 d. Samples were thawed at 4 °C overnight
before analysis.
2.3. Myofibrillar protein preparation
MP was extracted via the following procedure and stored in a refrig-
erator (4 °C). Briefly, the minced patty was homogenized 3 times with 4
vol cold-extracted solution (pH 7.0) and then centrifuged at 6500 ×g for
15 min at 4 °C. The obtained pellet was washed 3 times with 4 vol
extracting buffer (0.1 M NaCl) and centrifuged at 6500 ×g for 15 min
at 4 °C again. The obtained MP extractions were kept at 4 °C.
2.4. Particle size distribution
MP particle size was measured with 1.5 mL of MP-dissolving liquid
(1 mg/mL) in a Mastersizer 2000 instrument (Malvern, UK). The aggre-
gate degree of the MP was determined by the following statistical pa-
rameters: d43-volume-mean diameter; d32-volume-surface-mean
diameter; and dV,0.1, dV,0.5, and dV,0.9 represented the size of the particle
for which 10%, 50% and 90% of the sample was below this size,
respectively.
2.5. Zeta potential
The MP zeta potential (0.1 mg/mL) was evaluated using a zeta po-
tential analyser (Zeta Plus, Brookharen, Holtsville, NY, USA), and certain
modifications were made based on the method described by Beliciu and
Moraru [34]. After the MP concentration was adjusted by double
steaming wate, 1 mL of MP was transferred to the measuring tank,
and the average measurement was 6.
137
International Journal of Biological Macromolecules 178 (2021) 136–142
2.6. Surface morphology
The micromorphology of MP aggregation was measured using
atomic force microscopy (AFM). MP (40 mg/mL) was deposited onto
glass slides heated at 75 °C for 20 min and then immediately blow-
dried. Results were acquired using an AFM instrument (Veeco Instru-
ments Inc., USA) in tapping mode.
2.7. MP primary structure
2.7.1. Carbonyl content measurement
The carbonyl group was measured using the method described by Li
et al. [4] and calculated using the absorption coefficient
(22,000 M−1 cm−1).
2.7.2. Free amino content determination
MP (0.25 mL, 1 mg/mL) was dissolved in a phosphate buffer
(pH 8.2), mixed with 2 mL of TNBS for 60 min, and kept away from
light at 50 °C. Absorbance was noted at 340 nm by a spectrophotometer,
and the contents of free amino acids were estimated using the L-leucine
standard curve.
2.8. MP secondary structure
The extracted MP (0.2 mg/mL) was characterized on a circular di-
chroism (CD) instrument (Jasco J-815, Tokyo, Japan) with a scan rate
of 100 nm/min and a spectral range from 200 nm to 260 nm.
2.9. MP tertiary structure
The MP tertiary structure was measured on a fluorescence spectro-
photometer (Hitachi Co., Tokyo, Japan) under an excitation wavelength
of 283 nm, a slit width of 5 nm, a scanning wavelength of 300–400 nm
and a scan speed of 240 nm/min.
2.10. Statistical analyses
Data were analysed using by analysis of variance (ANOVA) with
Statistix 8.1 (Analytical Software, St. Paul, MN). The means ± standard
deviations were considered significant when P < 0.05. All graphs were
generated using SigmaPlot 12.5.
3. Results and discussion
3.1. Protein aggregation behaviour
3.1.1. Particle size
The effects of ISP and frozen storage on myofibrillar protein aggrega-
tion was assessed with dynamic light scattering and are shown in
Fig. 1A and Table 1. At the start of frozen storage, a relatively uniform
and wide particle distribution was observed in the control and ISP
groups, which showed that the distribution of fresh samples was uni-
form and stable. During frozen storage, the first clear trend was an in-
crease in the average particle size of MP from all samples. For the
control, d43 and d32 increased by 41.82% and 41.55%, respectively, after
freezing for 180 d. The changes in the mean particle diameters were pri-
marily caused by myofibrillar protein unfolding and an increase in pro-
tein surface area [9]. Additionally, the disulphide bridges, hydrogen
bonds, and hydrophobic interactions between proteins were also
strengthened, which promoted the aggregation of MP and increased
the average particle size [35].
Compared to the control, the particle size gradually decreased with
the addition of ISP. After freezing for 180d, d43 and d32 of the samples
with ISP significantly decreased by 4.57% and 4.26% compared to those
of the control (P < 0.05). Similarly, d0.1, d0.5 and d0.9 of the samples
with ISP significantly decreased by 4.91%, 4.73% and 5.31%, respectively
F. Li, X. Du, Y. Ren et al. International Journal of Biological Macromolecules 178 (2021) 136–142

Frozen storage (d)

0 30 60 90 180
6 0

4 -5

Zeta potential
3

2 -10 Aa
Bb
1 Cb Aa
Ba
Db Ca
0 180 -15
90 Da
1 60 Eb Da Control
10 0
30
IS
P(d) ISP
Par 180
rage
t icl 60
90
ol sto
e si 100 30 ntr -20
ze (
μ 0 Co ozen
(A) m) 1000
F r (B)

Fig. 1. Effect of ISP on the particle size and Zeta potential of myofibrillar protein of pork patties during frozen storage. a–b indicate significant differences (P < 0.05) with the same frozen
storage of different treatments. A–E indicate significant differences (P < 0.05) with the same treatment after different frozen storage.

(P < 0.05). Results thus showed that ISP added to patties effectively de- (Fig. 2D) by AFM [41]. As shown in Fig. 2, the bright and dark regions
creased protein aggregation [36]. ISP inhibits the protein aggregation of (Fig. 2A and C) occur in convex and concave areas of the MP aggregation
pork patties during frozen storage, which can be associated with less surface, respectively. Additionally, the red arrows (Fig. 2B) indicate the
damage to muscle tissue. ISP can also inhibit recrystallization and pre- highest and the lowest locations on the cross-section of each image
vent irreversible mechanical damage to tissues from ice crystals. Addi- [9,42].
tionally, the protein denaturation induced by freezing can be delayed The surface morphology of MP aggregation was smooth and homo-
during frozen storage [27]. geneous before frozen storage. The MP aggregation surface had a
brighter and concave area, and a larger distance between the highest
3.1.2. Zeta potential and lowest points after freezing for 180 d. A significant increase from
The zeta potential is a critical stability indicator of myofibrillar pro- 23.8 nm to 64.4 nm in the average roughness after 180 d was also ob-
teins that can be used to evaluate the driving force for electrostatic in- served in the control group (P < 0.05). These observations indicated
teractions between charged biopolymers [37]. As shown in Fig. 1B, that the MP in patties underwent substantial molecular changes and
control MPs exhibited a relatively low net negative charge gradually formed protein aggregates during frozen storage [43]. The
(−15.23 mV) because weakly acidic proteins, such as aspartic acid consistent production of these aggregates was due to protein oxidation
and glutamic acid, form during frozen storage. As frozen storage length- and denaturation, which were caused by ice crystals growing after
ened, the zeta potential significantly increased to −9.56 mV (P < 0.05), freezing [44].
which could be due to partial protein denaturation and aggregate for- Compared to the control, samples from the ISP group had a
mation [38]. The results are similar to those in Fig. 1A, in which protein smoother and more homogeneous gel morphology and lower Rg after
aggregation occurred during frozen storage. the same storage time. Results showed that the behaviour of protein ag-
During storage, patties with ISP had significantly lower MP zeta po- gregation was inhibited by adding ISP. Kong et al. [31] found that ISP
tentials. Particularly after freezing for 180 d, the MP zeta potential with added to patties could decrease the degree of muscle cell damage by
ISP decreased by 22.48% compared to the control. Results showed that inhibiting recrystallization. Therefore, the adjunction of ISP could main-
the addition of ISP can inhibit MP aggregation after freezing because tain protein structure stability to some extent by preventing protein ox-
the formation of ice crystals was controlled by ISP, thus decreasing pro- idation during frozen storage [27].
tein freezing denaturation and aggregation [39]. The MP particle size
with ISP was more uniformly distributed and had a larger electrostatic 3.2. Protein structural property
repulsive force, which could prevent protein aggregation [40].
3.2.1. Carbonyl content
3.1.3. Surface morphology Carbonyl content is one of the most important characteristics to
The surface state of MP aggregation is shown in 2D images (Fig. 2A), evaluate changes in a protein's primary structure. The effect of ISP and
cross-sections of images (Fig. 2B), 3D images (Fig. 2C), and Rq values frozen storage on the carbonyl groups is shown in Fig. 3A. After being

Table 1
Effect of ISP on the particle size of myofibrillar protein of pork patties during frozen storage.

Frozen storage (d) Particle size (μm)

d43 d32 dV,0.1 dV,0.5 dV,0.9

Ea Ea Da Da
Control 0 86.41 ± 0.45 40.79 ± 0.89 20.21 ± 0.41 61.27 ± 3.51 179.61 ± 1.29Ea
30 90.47 ± 0.77Da 46.46 ± 0.44Da 22.73 ± 0.54Ca 68.07 ± 0.80Ca 186.96 ± 0.13Da
60 96.20 ± 0.27Ca 50.74 ± 1.32Ca 24.34 ± 0.57Ba 72.99 ± 1.00Ba 198.90 ± 0.96Ca
90 109.28 ± 0.73Ba 54.93 ± 0.77Ba 26.13 ± 0.71Aa 87.92 ± 1.16Aa 224.06 ± 4.16Ba
180 122.54 ± 0.85Aa 57.74 ± 0.19Aa 27.31 ± 0.05Aa 92.58 ± 0.84Aa 254.83 ± 3.43Aa
ISP 0 86.64 ± 1.16Da 41.48 ± 1.46Da 19.91 ± 0.82Ca 63.78 ± 1.47Da 181.22 ± 4.16Ca
30 89.08 ± 0.66CDa 40.14 ± 1.07Db 21.19 ± 0.89Ca 68.75 ± 0.94Ca 177.54 ± 2.04Cb
60 91.84 ± 1.92Cb 46.94 ± 0.04Cb 23.19 ± 0.60Ba 71.49 ± 1.12Ca 183.98 ± 4.26Cb
90 104.95 ± 1.42Bb 50.88 ± 1.48Bb 24.26 ± 0.22ABb 76.02 ± 0.26Bb 215.89 ± 1.17Bb
180 116.94 ± 2.03Ab 55.28 ± 1.04Ab 25.97 ± 0.76Ab 88.20 ± 1.28Ab 241.31 ± 3.50Ab
a–b
indicate significant differences (P < 0.05) with the same frozen storage of different treatments. A–E indicate significant differences (P < 0.05) with the same treatment after different
frozen storage.

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F. Li, X. Du, Y. Ren et al. International Journal of Biological Macromolecules 178 (2021) 136–142

Fig. 2. Effect of ISP on the surface roughness of myofibrillar protein (3.0 × 3.0 μm2 scan size) of pork patties during frozen storage. A: The scale displayed on the left ranges from 0 to 200 nm.
The straight line on the 2D image represent the incision location of cross-sections. B: The image cross-sections correspond to the section cut by the straight line on the 2D image. a–b indicate
significant differences (P < 0.05) with the same frozen storage of different treatments. A–C indicate significant differences (P < 0.05) with the same treatment after different frozen storage.

frozen for 180 d, there was a significant increase in carbonyl contents in
all samples (P < 0.05). Protein oxidation occurs frequently and leads to
the formation of carbonyl compounds [45]. During long-term frozen
storage, the formation of ice crystals caused damage to the muscle cell
ultrastructure, which released free radicals. This process aggravates ox-
idative changes in proteins and the formation of carbonyl groups [6,46].
Additionally, the NH and NH2 of protein side chains easily undergo oxi-
dative changes and form carbonyl compounds [16]. Cheng, Xu, Xiang,
Liu, and Zhu [47] also found that carbonyl accumulation in minced
pork slice proteins increased significantly during frozen storage.
Although no significant differences were detected at the beginning
of frozen storage in all samples (P > 0.05), the control sample showed
a sharp rise in carbonyl content compared to samples treated with ISP
after 30 d of frozen storage. After freezing for 180 d, patties with ISP
had lower carbonyl contents than patties without ISP, with a significant
difference of 32% (P < 0.05). The large ice crystals formed and destroyed
the muscle, which caused MP freezing and oxidative denaturation [4].
With ISP bound, the mechanical damage of muscle was controlled by
inhibiting the growth of ice crystals [48]. Therefore, the degree of pro-
tein degeneration decreased.
3.2.2. Free amino content
Changes in the free amino contents of proteins can be used to eval-
uate alterations in MP primary structure [49]. As shown in Fig. 3B, all
samples had significantly decreasing free amino contents after 180 d
(P < 0.05). The free amino acid contents of the control group and the

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F. Li, X. Du, Y. Ren et al. International Journal of Biological Macromolecules 178 (2021) 136–142

Carbonyl contents (nmol/mg protein)

Control 100 Control

Free amino (nmol/mg protein)

ISP Aa Aa ISP
3 Aa
Ba Bb Ba
Ca
Da
Cb Da
Ca 80 Db
Ab
2 Ab Eb
Bb
Da
Ea Ca Cb
1 60

0 40
0 30 60 90 180 0 30 60 90 180
(A) Frozen storage (d) (B) Frozen storage (d)

Fig. 3. Effect of ISP on the primary structure of myofibrillar protein of pork patties during frozen storage. a–b indicate significant differences (P < 0.05) with the same frozen storage of
different treatments. A–E indicate significant differences (P < 0.05) with the same treatment after different frozen storage.

ISP group decreased 23.09% and 12.99%, respectively. Proteins are sus- recrystallization and changed ice crystal morphology. Thus, cell-
ceptible to attack by reactive oxygen species while frozen [50]. The structure integrity is maintained, and the degree of protein oxidation
NH2 groups of amino acid residues could be transformed into carbonyl decreased [17]. Considering the result regarding carbonyl content, ISP
groups through a deamination process during frozen storage. Addition- can decrease oxidative damage to patties.
ally, derivatives then reacted with available NH2 groups, thus causing a
further decrease in free amino content [51,52]. Results thus responded 3.2.3. MP secondary structure
to changes in carbonyl contents. The stability of MP secondary structural characteristics can be de-
Compared to the control, the ISP group showed a marginally lower scribed by circular dichroism (CD) far-ultraviolet spectra, which com-
decrease in free amino contents during frozen storage. After the same prise α-helices, β-sheets, β-turns and random coils [57]. Fig. 4 shows
frozen-storage period, the samples with ISP had higher amino contents that the far-ultraviolet wavelengths of all samples exhibit typical nega-
than those without ISP. Particularly at 180 d, the difference in amino tive peaks at 190–250 nm. With longer frozen storage, the secondary
contents was as high as 14.99%. Mechanical damage to muscle caused structures of all samples showed a similar trend: the percentage of α-
by recrystallization leads to protein oxidative denaturation. The higher helixes and β-turns gradually decreased, while the fraction of β-sheets
amino contents in the ISP group indicated that ISP hindered and random coils significantly increased (P < 0.05). The protein

Fig. 4. Effect of ISP on the secondary structure of myofibrillar protein of pork patties during frozen storage. (Control: A and B; ISP: C and D). a–b indicate significant differences (P < 0.05)
with the same frozen storage of different treatments. A–C indicate significant differences (P < 0.05) with the same treatment after different frozen storage.

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F. Li, X. Du, Y. Ren et al. International Journal of Biological Macromolecules 178 (2021) 136–142

6000 0d 6000 0d
30 d
30 d
60 d
5000 60 d
90 d 5000 90 d

Fluorescence intensity

Fluorescence intensity
180 d 180 d
4000 4000

3000 3000

2000 2000

1000 1000

0 0
300 320 340 360 380 400 300 320 340 360 380 400 420
(A) Wavelength (nm) (B) Wavelength (nm)

Fig. 5. Effect of ISP on the tertiary structure of myofibrillar protein of pork patties during frozen storage. (Control: A; ISP: B).

oxidation aggravation induced by freezing causes a decrease in the per- protein aggregation, as shown by the MP particle size distribution,
centage of α-helical conformation [16]. Additionally, freezing destroys zeta potential and AFM. Additionally, the lower carbonyl contents and
hydrogen bonds and disorders the MP structure, which transforms the higher free amino contents of the ISP group showed that MP primary
helix structure to a random structure [40]. Jin et al. [53] showed that structure degradation was inhibited by ISP. Concurrently, the secondary
the protein structure stability of duck breast muscle decreased due to and tertiary structures of MP from pork patties with ISP tended to stabi-
the hydrophobic interaction of protein during frozen storage. lize after frozen storage. These results systematically showed the mech-
When the storage time was the same, patties with ISP exhibited anism by which ISP inhibits the frozen denaturation of patties based on
more stable secondary protein structures, as shown in the significantly the importance of the aggregation behaviour and structure of MP.
fewer changes in the contents of α-helices, β-sheets, β-turns and ran- Therefore, this study provides useful information for the future study
dom coils (P < 0.05). Results thus suggested that ISP decreased the de- of the cryoprotective effect of ISP on meat and meat products. Future re-
gree of hydrophobic residue exposure and inhibited interaction among search should focus on exploring the molecular interactions between
amino acid side chains. ISP can modify ice crystal morphology and in- the structure of ISP and MP spatial conformation at the molecular level.
hibit recrystallization, minimizing freezing and oxidation damage [31].
Nian et al. [27] also reported the same conclusion that antifreeze pro- Credit author statement
teins could maintain the secondary structural stability of large-mouth
bass MP during F-T cycles. Fangfei Li: Methodology, Software, Validation, Formal analysis, In-
vestigation, Writing-Original Draft, Funding acquisition.
3.2.4. MP tertiary structure Xin Du: Investigation, Software.
Fluorescence intensity (FI) describes the changes in MP tertiary Yanming Ren: Formal analysis.
structure via the reflection of tryptophan (Trp) residues towards the po- Baohua Kong: Conceptualization, Resources.
larity of the microenvironment [54]. Changes in the fluorescence inten- Bo Wang: Investigation, Formal analysis.
sity of MP are shown in Fig. 5. All samples exhibited a broad spectrum at Xiufang Xia: Conceptualization, Writing - Original Draft, Supervi-
336 nm, and as the frozen storage period increased up to 180 d, FI de- sion, Funding acquisition.
creased significantly (P < 0.05). The exposed and denatured indole Yihong Bao: Conceptualization, Resources.
side chains of Trp residues, which are located in the head and tail re-
gions of myosin, cause the formation of protein aggregation and the Acknowledgements
weakness of protein conformation affected by freezing [55]. Addition-
ally, MP unfolding and partial changes in protein conformation can This study was supported by Northeast Forestry University's re-
lead to the exposure of Trp in amino acids. The decrease in FI of MP search funding for talent introduced (grant no. 60201520109) and the
was also due to increased steric hindrance, which was caused by the ag- National Natural Science Foundation of China (grant no. 31571859
gregation of MP and the rise in hydrophobic interactions. This result is and 31771903).
consistent with a study that showed that the reductions in FI could be
due to the exposure and denaturation of Trp residue indole side chains References
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