Journal of Cultural Heritage 56 (2022) 56–64

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Journal of Cultural Heritage

journal homepage: www.elsevier.com/locate/culher

Original article

Optimization of GuHCl extraction protocol on collagen-based binders

in murals by response surface methodology
Zhanyun Zhu a,b,c,d, Jianghao Du e, Zhiyong Lu b, Xiuya Yao a, Xiaotong Jiang a,
Junchang Yang a,f, Liu Liu g,∗
a
Research and Practice Base of Conservation Science and Engineering, Department of History, College of Humanities, Xiamen University, Xiamen 361005,
China
b
Key Scientific Research Base of Conservation and Restoration for Murals as Collection and Materials Science in State Administration for Cultural Heritage,
Shaanxi History Museum, Xi‫׳‬an 710061, China
c
Department of Archaeology, Max Planck Institute for the Science of Human History, Jena D-07745, Germany
d
Joint International Research Laboratory of Environmental and Social Archaeology, Institute of Cultural Heritage, Shandong University, Qingdao 266237,
China
e
Center for Nano Energy Materials, State Key Laboratory of Solidification Processing, School of Materials Science and Engineering, Northwestern
Polytechnical University, Xi‫׳‬an 710072, China
f
Institute of Culture and Heritage, Northwestern Polytechnical University, Xi‫׳‬an 710072, China
g
Xiamen Academy of Arts and Design, Fuzhou University, Xiamen 361000, China

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

Article history: In this paper, response surface method (RSM) was used to study the effect of extraction parameters on
Received 27 October 2021 the extracting efficiency of guanidine hydrochloride (GuHCl) on residual protein from mural model sam-
Accepted 7 June 2022
ples. The central composite design (CCD) was applied to find the optimal combination of variables, includ-
Available online 18 June 2022
ing GuHCl concentration (x: 1.5-2.5 M), extraction time (y: 90-180 min), temperature (z: 50-70 °C) and
Keywords: ultrasound power (α : 120-280 W) during the extraction. Multiple regression analysis was employed to fit
Mural binders the experimental data into a second-order polynomial equation, and appropriate statistical methods were
Response surface method used for analysis. The optimal extraction conditions were determined as follows: GuHCl concentration of
Protein extraction 1.89 M, extraction time of 132 min, temperature of 57 °C, and ultrasonic power of 210 W. The extraction
GuHCl solution efficiency was 16.685 % under the optimal conditions, which was in agreement with the theoretically pre-
dictive value of 17.707 %. The extraction process under optimized parameters had no significant impact
on the collagenous structures, characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), ultraviolet (UV) absorption spectroscopy and circular dichroism (CD) spectroscopy.
© 2022 Elsevier Masson SAS. All rights reserved.

1. Introduction
Murals are important cultural heritage of the time-honored hu-
man civilization, reflecting valuable information on religion, cul-
ture, history, ethnicity, and society [1]. Research on the produc-
tion of murals through efficient technical means could obtain pre-
cious information and guide conservation, which is of great signif-
icance to heritage scientists and curators [2]. The choice of binding
materials is a key step during the ancient mural production. Un-
der the adhesive effect of binders on mineral particles, the paint
is formed by fully mixing inorganic minerals with binding media,
which have a great bearing on many physical and chemical proper-
ties of the paint matrices [3]. Therefore, scientific identification of
∗
Corresponding author.
E-mail address: liu.liu@fzu.edu.cn (L. Liu).
binding media could aid historical studies and restoration works
by revealing important technical and artistic information [4–7].
Protein identification techniques have been increasingly applied
in exploring cultural heritage, represented by widely-used pro-
teomic and immunological methods owing to their high accuracy
and strong specificity [8–14]. The extraction process of the resid-
ual protein from paint samples is always an essential part of these
methods, directly affecting the accuracy of the downstream identi-
fication procedures. Hence, it is necessary to conduct optimization
research on protein extraction.
Due to its rich sources and excellent thermal stability [15,16],
collagen is used as a routine material for mural binders [17].
Guanidine hydrochloride (GuHCl) serves as an extracting agent of
protein residues from mural samples due to its denaturation ef-
fects [18,19]. Previous studies [20] have revealed that 2 M GuHCl
has a better extraction efficiency on collagen-based mural binders
than other commonly used reagents for the residual proteins in

https://doi.org/10.1016/j.culher.2022.06.003
1296-2074/© 2022 Elsevier Masson SAS. All rights reserved.
Z. Zhu, J. Du, Z. Lu et al.
mural samples. Additionally, the protein extraction effect is cer-
tainly influenced by a number of variables, including the reagent
concentration, extraction time, temperature, and ultrasonic power
[21–23]. Therefore, it is essential to optimize the parameters of
the GuHCl extraction protocol on collagen-based binder, so as to
improve the overall protein extraction efficiency and upgrade the
technical process.
Response surface method (RSM) is a commonly used statistical
method for optimization of stochastic process in complex models
[24], as it is more efficient and less time-consuming than its coun-
terparts [25–28] by significantly reducing the number of experi-
ments on evaluating multiple variables and their interactions [29].
Furthermore, different from the orthogonal method to obtain mul-
tiple sets of discrete data, the predicted continuous model could
be built by RSM based on the regression equation. RSM has been
widely used in food science [30–32], medicine [33–35] and many
other fields. At present, no study has been found on the optimized
extraction of residual binders from mural samples using RSM.
In this paper, the parameters for protein extraction from model
mural samples using GuHCl were optimized, and possible modi-
fications of molecular structure caused by the optimized proce-
dure were assessed. The samples were made by subjecting the
mixture of Fe2 O3 pigment and rabbit skin glue to thermal ag-
ing. RSM was employed to simulate the influence of key vari-
ables (GuHCl concentration, extraction time, temperature, and ul-
trasonic power) on protein extraction efficiency. The bicinchoninic
acid (BCA) method was used for quantitative measurement of ex-
traction efficiency. Sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE), ultraviolet (UV) absorption spectroscopy,
and circular dichroism (CD) spectroscopy were adopted to charac-
terize the effects of optimum extracting variables on the protein
structure, so as to determine the most suitable extraction condi-
tions for collagen-based binders in murals.
2. Research aim
This work aims to determine the optimal parameters for the
GuHCl extraction protocol on collagen-based binders in murals, by
establishing a quantitative model showing the relation between
extraction efficiency and different experimental conditions using
RSM. Based on a recent finding that GuHCl is a suitable extraction
agent for collagen-based binders [20], this study investigates the
possible improvements of the above method, laying a solid foun-
dation for understanding the correlation between the extraction
parameters and the consequent effects. Moreover, it also provides
a reference for the extraction and analysis of binding media in
conservation practice. On this basis, incomplete characterization of
collagen-based binders derived from unsatisfactory extraction con-
ditions is expected to be avoided.
3. Materials and methods
3.1. Materials
Rabbit skin glue (NO. 63028) was provided by Kremer (Ger-
many). Fe2 O3 powder was supplied by Fuchen (China). Tetram-
ethylethylenediamine (TEMED), GuHCl, sodium tartrate, bicin-
choninic acid disodium salt, sodium dodecyl sulfate (SDS), and
ammonium persulfate were purchased from Sigma-Aldrich (USA).
Methanol, acetic acid, sodium hydroxide, sodium bicarbonate, an-
hydrous sodium carbonate, and copper sulfate pentahydrate were
purchased from Tianli (China). Coomassie brilliant blue G-250
Staining solution, bovine serum albumin (BSA), 1 × PBS, protein
marker (43-200 kDa), loading buffer, Tris-glycine running buffer, 30
% acrylamide solution, 1.5 M Tris-HCl (pH = 8.8) and 1.0 M Tris-HCl
(pH = 6.8) were purchased from Solarbio (China).
57
Journal of Cultural Heritage 56 (2022) 56–64
3.2. Sample preparation
Rabbit skin glue was sufficiently dissolved in deionized water at
40 °C by a vortex mixer (QL-901, Kylin-bell) to obtain a solution of
30 mg/mL. The solution was centrifuged after being cooled to at-
mospheric temperature by a Sigma 3K15 centrifuge (80 0 0 r/min,
10 min, 20 °C). Then the pipetted supernatant was completely
mixed with Fe2 O3 powder, which was commonly used as mural
pigment [36,37]. The ratio of solute to pigment was approximately
1: 2 (m/m). Aliquots of 100 μL mixture were transferred onto glass
slides and placed in darkness at atmospheric temperature for 6
months before the aging procedure. Afterward, the samples were
artificially aged in a forced convection oven (DHG-9030a, Huitai) at
100 °C for 2000 h. The thermal degradation process of the binder
under high temperature should follow Arrhenius equation [38–40],
where the aging effects on the samples for 20 0 0 h at 100 °C corre-
sponds to that for over 225 years at room temperature. The specific
calculation has been described in a previous study [20].
3.3. Protein extraction
In the present work, the GuHCl solution was used as the protein
extraction agent. The extraction steps were as follows: (1) samples
were separated from glass slides and fully blended with 300 μL of
x M GuHCl; (2) at z °C, the mixtures were subjected to the ultra-
sonic treatment of α W for y min; (3) after being cooled to room
temperature and centrifugation (15 min, 20 °C, 80 0 0 r/min), the
supernatants were obtained to determine protein concentration.
3.4. Quantitative determination of protein concentration
In this study, some modifications were performed based on the
BCA method by Smith et al. [41]. BSA was used as the standard
protein to plot the standard curves with different concentrations
of GuHCl. The supernatants were diluted with 1 × PBS, reaching
10 times the initial volume for the test. BCA disodium salt (0.10 g),
sodium tartrate (0.16 g), sodium bicarbonate (0.95 g), anhydrous
sodium carbonate (2.00 g), and sodium hydroxide (0.40 g) were
blended, and added deionized water to 100 mL (adjusting the pH
of the solution to 11.25) to prepare BCA reagent. Cu reagent was
obtained by dissolving CuSO4 ·5H2 O (0.40 g) in 10 mL deionized
water. BCA reagent and Cu reagent were sufficiently mixed at a ra-
tio of 50 : 1 (v/v) to obtain BCA working solution. Diluted solutions
for the test and BCA working solution were blended at a ratio of
10 : 1 (v/v) and heated at 37 °C for 30 min. After chilling to room
temperature, the protein concentration was accurately determined
using a micro-ultraviolet spectrophotometer (DS-11, Denovix). Each
experiment was repeated in triplicate.
3.5. Calculation of extraction efficiency
The following method was utilized to compare the extraction
efficiency of different values of parameters quantitatively. A total of
100 μL rabbit skin glue with an initial concentration of 30 μg/μL
was given to each sample. Thus, the total mass p of glue in each
sample was 30 0 0 μg (30 × 100). The extracting agent for a single
sample was 300 μL, the dilution ratio by 1 × PBS was 10, and
the final protein concentration was w μg/μL. As a result, the total
mass q of the extracted protein from each sample was 30 0 0w μg
(10 × 300 × w). Eq. (1) can be obtained as follows when η denotes
the extraction efficiency:
q 30 0 0w
η= × 10 0% = × 10 0% = w (1)
p 30 0 0
Z. Zhu, J. Du, Z. Lu et al.
3.6. Experimental optimization design
Design Expert 12 (Stat-Ease, USA) was used for RSM. In order to
further optimize the parameters of the extraction process, a cen-
tral composite design (CCD) was employed with four independent
variables, i.e. GuHCl concentration (M), extraction time (min), tem-
perature (°C), and ultrasonic power (W), at five levels. As shown in
Table 2, 30 groups of experiments should be conducted for analy-
sis, and the extraction efficiency η (%) corresponds to the response.
3.7. Microstructural analyses
In addition to recovering more proteinaceous residues, the ex-
traction process optimization also aims to cause less destruction
of the intrinsic structure of the extracted binder. To characterize
the possible impact of the optimized protocol on molecular struc-
ture of the extractive, comparative experiments were performed.
The optimized parameters were used to extract the protein from
model samples of the experimental group. In the control group,
the same volume of deionized water was utilized as the extraction
agent, with the extraction temperature of 60 °C, ultrasonic power
of 100 W, and time of 60 min before optimization [20].
3.7.1. Desalination
After extraction, the supernatants of experimental and control
groups were dialyzed using mini-dialysis devices (MWCO 3.5 kDa,
Thermo Fisher Scientific, No. 69550). Deionized water was replaced
every 2 h at room temperature. After dialysis for 48 h, the protein
solution was obtained for the following microstructural analyses.
3.7.2. SDS-PAGE
Based on Laemmli’s method [42], SDS-PAGE was utilized to
characterize the purity and molecular weight of the extracted pro-
teins. The extracted protein solution (40 μL) mixed with the load-
ing buffer (10 μL) was heated (100 °C, 10 min). After cooling to
atmospheric temperature and centrifugation (130 0 0 r/min, 3 min),
10 μL supernatant and 6 μL protein marker were used for elec-
trophoresis in 5 % stacking gel (under constant voltage of 120 V)
and 10 % resolving gel (under 150 V) in succession. The gels were
stained with Coomassie brilliant blue G-250 for 4 h and destained
with the destaining solution (methanol : glacial acetic acid : deion-
ized water = 3 : 1 : 6 [v/v]) for 6 h.
3.7.3. UV
An ultraviolet spectrophotometer (DS-11, Denovix) was em-
ployed to test the extracted supernatants of experimental and con-
trol groups after desalination with a spectral scan range of 200-
400 nm. The optical path length in the transmission measurements
was 10 mm. Deionized water was used as the baseline for tests.
3.7.4. CD
A circular dichrograph (ChirascanV100, Applied Photophysics,
UK) was utilized to perform CD tests on the supernatants of ex-
perimental and control groups after desalination. Three scans were
averaged in a wavelength range of 190-260 nm at 25 °C. Deionized
water was used as the baseline for tests.
4. Results and discussion
4.1. Single-factor results
The influence of GuHCl concentration on extraction efficiency
η is illustrated in Fig. 1a, with the extraction temperature of 60
°C, extraction time of 120 min, and ultrasonic power of 200 W. As
can be seen, with the rising GuHCl concentration, the extraction
58
Journal of Cultural Heritage 56 (2022) 56–64
Table 1
Independent variables and their levels used in the response surface design.
Independent Coded factor level
variables Symbol
-2 -1 0 1 2
GuHCl concentration (M) x 1.50 1.75 2.00 2.25 2.50
Extraction time (min) y 60 90 120 150 180
Extraction temperature (°C) z 50 55 60 65 70
Ultrasonic power (W) α 120 160 200 240 280
efficiency η firstly increased and then decreased, reaching maxi-
mum at the GuHCl concentration of around 2.00 M. This can be
explained that the electrostatic shielding effects, impairment of hy-
drophobic interactions, and breakage of hydrogen bonds by GuHCl
on proteinaceous molecules are enhanced with increasing GuHCl
concentration, which eliminates the aggregation and cross-linking
[43–45]. However, proteins can be denatured and structurally al-
tered by excessive concentration of GuHCl, thus affecting the ex-
traction effect [46].
The effect of extraction time on η is shown in Fig. 1b, with the
GuHCl concentration of 2.00 M, the temperature of 60 °C, and the
ultrasonic power of 200 W. The η increased with the extraction
time until 120 min and then decreased. The reagent-protein re-
action should certainly be promoted by appropriate extension of
extraction time. By contrast, the proteins for extraction can be de-
natured by chaotropic agents like GuHCl for unreasonably long ex-
tracting time, strengthening the hydrophobic interactions and de-
creasing extraction effect [22].
The effect of extraction temperature on η is depicted in Fig. 1c,
with the GuHCl concentration of 2.00 M, extraction time of 120
min, and ultrasonic power of 200 W. The η presents an increase
and then decreases with a rising extraction temperature, reaching
maximum at approximately 60 °C. This showed that partly increas-
ing the temperature of the extraction process upgrades the effect
of ultrasonic treatment and extraction agent, thereby improving
the reactivity. However, excessively high temperatures can further
denature the proteins and accelerate bond binding, thus decreasing
η [47].
The influence of ultrasonic power on η is shown in Fig. 1d,
with the GuHCl concentration of 2.00 M, extraction time of 120
min, and temperature of 60 °C. A proper increase in the ultrasonic
power can accelerate the reaction and target protein extraction by
the GuHCl solution. However, proteolysis will occur if the power is
over the normal level, finally reducing η.
Fig. 1 The effects of different parameters, (a) GuHCl concentra-
tion, (b) extraction time, (c) extraction temperature, and (d) ultra-
sonic power on the extraction efficiency η
4.2. Optimization of extraction parameters
4.2.1. Model fitting
As a routine experimental design of finding an improved or op-
timal process setting for over 3 factors [32,48], CCD was used in
this experiment. The range and central point values of 4 inde-
pendent variables were chosen in accordance with the results of
single-factor experiments. Independent variables and their levels
used in the response surface design are shown in Table 1.
Table 1 Independent variables and their levels used in the
response surface design.
Table 2 shows 30 runs for optimizing the 4 independent vari-
ables. The results revealed that η ranged from 12.5 % to 17.7 %,
reaching a maximum of 17.7 % with the GuHCl concentration of
2.00 M, extraction time of 120 min, extraction temperature of 60
°C and ultrasonic power of 200 W. By applying multiple regression
analysis on the experimental data, the predicted response variables
and the test variables were correlated according to the following
Z. Zhu, J. Du, Z. Lu et al.
Fig. 1. The effects of different parameters, (a) GuHCl concentration, (b) extraction time, (c) extraction temperature, and (d) ultrasonic power on the extraction efficiency η.
second-order polynomial equation:
η = 17.4 − 0.5125x + 0.47917y − 0.47083z + 0.2375α − 0.25625xy
− 0.08125xz − 0.19375xα − 0.26875yz − 0.33125yα − 0.25625zα
− 0.84479x2 − 0.74479y2 − 0.61979z2 − 0.94479
where η denotes the extraction efficiency; x, y, z and α are coded
factors of test variables, namely GuHCl concentration, extraction
time, extraction temperature and ultrasonic power, respectively.
Table 2 The central composite design and the response values
for extraction efficiency η
The fit statistics for extraction efficiency (η) for the calculated
quadratic predictive model are shown in Table 3. The determina-
tion coefficient (R2 = 0.9632) of the quadratic regression model
by analysis of variance (ANOVA) suggested that 96.32 % of the to-
tal variations were explained by the model, confirming the great
agreement between the predicted and experimental values [49].
The error analysis showed that the lack of fit value (0.1052) was
insignificant at the 95 % confidence interval level, indicating the
validity of model [50]. In addition, the coefficient of the varia-
tion (CV = 2.91 %) was much smaller than 10 %, indicating a high
degree of precision for the model and reliable experimental val-
ues [51,52]. Moreover, the value of adequate precision (15.69) was
evidently larger than 4, indicating adequate discrimination of the
model [53]. Furthermore, the p-value of the model was extremely
low (< 0.0 0 01), showing the significance of model terms. Taken to-
gether, all these statistical parameters confirmed the reliability of
the model.
Journal of Cultural Heritage 56 (2022) 56–64
Table 3 Analysis of variance for the response surface quadratic
model of protein extraction efficiency
The regression coefficient values of Eq. (2) are illustrated in
Table 4. For each term in the model, the small p-value and big F-
(2) value meant a significant influence on the extraction efficiency η
[54]. Table 4 showed that all linear coefficients (x, y, z, and α ), all
quadratic term coefficients (x2 , y2 , z2 , and α 2 ), and partially inter-
action coefficients (xy, yz, yα , and zα ) were significant (p < 0.05).
Thus, the GuHCl concentration, extraction time, extraction temper-
ature and ultrasonic power were all important factors affecting η
of the extraction process, especially the first 3 parameters owing
to their small p-values (< 0.0 0 01). Other interaction coefficients,
including xz and zα , exerted no significant effect on η (p > 0.05).
Table 4 Regression coefficients of the predicted quadratic poly-
nomial model
4.2.2. Optimization of extraction conditions
RSM was used to illustrate the effects of GuHCl concentration,
extraction time, extraction temperature, and ultrasonic power on
the response. The regression equation could predict the effects of
the four parameters on the extraction efficiency η. Through visual
methods, such as forming three-dimensional response surface plots
and two-dimensional contour plots, the interaction types among
the 4 measured variables and the relationship between the re-
sponse of each variable and the experimental level can be illus-
trated [55]. The effects of GuHCl concentration, extraction time,
temperature, and ultrasonic power on protein extraction efficiency
η are shown in Fig. 2. Each plot in Fig. 2 presented the result un-
59
Z. Zhu, J. Du, Z. Lu et al. Journal of Cultural Heritage 56 (2022) 56–64

Fig. 2. The 3D response surface and 2D contour plot illustrating the effects of GuHCl concentration, extraction time, temperature and ultrasonic power on protein extraction
efficiency η.

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Z. Zhu, J. Du, Z. Lu et al. Journal of Cultural Heritage 56 (2022) 56–64

Table 2
The central composite design and the response values for extraction efficiency η.

Independent variables Response Extraction

efficiency (%)
Run GuHCl concentration (M) Extraction time (min) Extraction temperature (°C) Ultrasonic power (W)
No. x y z α η
1 -1 -1 -1 -1 12.9
2 1 -1 -1 -1 13.5
3 -1 1 -1 -1 16.2
4 1 1 -1 -1 15.1
5 -1 -1 1 -1 13.6
6 1 -1 1 -1 12.9
7 -1 1 1 -1 15.0
8 1 1 1 -1 13.8
9 -1 -1 -1 1 15.2
10 1 -1 -1 1 14.1
11 -1 1 -1 1 16.4
12 1 1 -1 1 14.7
13 -1 -1 1 1 13.8
14 1 -1 1 1 13.1
15 -1 1 1 1 14.5
16 1 1 1 1 12.5
17 -2 0 0 0 15.2
18 2 0 0 0 13.0
19 0 -2 0 0 13.9
20 0 2 0 0 15.1
21 0 0 -2 0 15.6
22 0 0 2 0 14.4
23 0 0 0 -2 12.6
24 0 0 0 2 14.8
25 0 0 0 0 17.6
26 0 0 0 0 17.3
27 0 0 0 0 17.6
28 0 0 0 0 17.0
29 0 0 0 0 17.2
30 0 0 0 0 17.7

Table 3
Analysis of variance for the response surface quadratic model of protein extraction efficiency.

Sources Degrees of freedom Coefficient Sum of squares Mean square F-value p-value

Model 14 73.82 5.27 28.07 < 0.0001 significant

Residual 15 2.82 0.19
Lack of fit 10 2.44 0.24 3.21 0.1052 insignificant
Pure error 5 0.38 0.076
Cor. total 29 76.63
R2 0.9632
Adj-R2 0.9289
CV (%) 2.91
PRESS 14.59
Standard deviation 0.43
Adequate precision 15.69

Table 4 elliptical, while those of Fig. 2b and c were nearly circular. It can
Regression coefficients of the predicted quadratic polynomial model.
be concluded that a circular contour plot means the ignorable in-
Parameter Sum of squares Mean square F-value p-value teractions between corresponding variables, while an elliptical one
x 6.30 6.30 33.56 < 0.0001 indicates the opposite results [56]. The finding was in line with
y 5.51 5.51 29.34 < 0.0001 the result that interaction coefficients xy, yz, yα and zα were sig-
z 5.32 5.32 28.33 < 0.0001 nificant (p < 0.05), while xz and xα had no significant effect on η
α 1.35 1.35 7.21 0.0170 (p > 0.05). All the six 3D response surface figures showed obvious
xy 1.05 1.05 5.59 0.0319
bending, indicating that the change of each independent variable
xz 0.11 0.11 0.56 0.4649
xα 0.60 0.60 3.20 0.0940 significantly affects the η.
yz 1.16 1.16 6.15 0.0255 Fig. 2a shows the 3D graphic surface and contour plot of the
yα 1.76 1.76 9.35 0.0080 combined effects of GuHCl concentration and extraction time on
zα 1.05 1.05 5.59 0.0319
the η, which increased firstly and then decreased with the growth
x2 19.58 19.58 104.21 < 0.0001
y2 15.22 15.22 81.00 < 0.0001 in GuHCl concentration (1.75-2.25 M) and extraction time (90-150
z2 10.54 10.54 56.09 < 0.0001 min), reaching maximum at 1.75-2.00 M and 120-150 min. The ef-
α2 24.48 24.48 130.35 < 0.0001 fects of GuHCl concentration and extraction temperature on ex-
traction efficiency η are shown in Fig. 2b. It can be seen that
η achieved a maximum at 1.75-2.00 M and 55-60 °C. For both
der the influence of two factors, while keeping the other two at factors, excessively rising values could obviously cut down the η.
their middle level (the center point of the experiment ranges). As Fig. 2c shows the 3D plot and contour plot with varying GuHCl
can be seen, the 2D contour plots of Fig. 2a, d, e and f were all concentrations and ultrasonic powers. With the increasing GuHCl

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concentration, η rised until around 1.90 M and then went down.

With the ultrasonic power of about 210 W, η achieved the maxi-
mum. Fig. 2d presents the influence of extraction time and tem-
perature on η, which reached the maximum predicted value at
120-150 min and 55-60 °C, respectively. Fig. 2e shows the relation-
ship between extraction time and ultrasonic power on η. The area
of the smallest ellipse in the contour was large, corresponding to
105-150 min and 180-240 W. Furthermore, the change of the two
variables in this range had a limited effect on the improvement
of η. Fig. 2f shows the 3D response surface plot and the contour
plot at varying temperatures and ultrasonic powers. The maximum
predicted value guided by the surface was limited in the smallest
ellipse in the contour diagram that indicated a perfect interaction
between the two independent variables [57]. The η reached the
maximum at 56-59 °C and 200-220 W.
Fig. 2 The 3D response surface and 2D contour plot illustrat-
ing the effects of GuHCl concentration, extraction time, tempera-
ture and ultrasonic power on protein extraction efficiency η
In general, the optimum values of the test variables were GuHCl
concentration of 1.89 M, extraction time of 132 min, extraction
temperature of 57 °C, and ultrasonic power of 208 W. Under these
conditions, the maximum extraction efficiency η was predicted to
be 17.707 %. For precise control of the parameter, the optimum ul-
trasonic power was modified to be 210 W.

4.2.3. Validation of the model

In order to compare the predicted results with the experimen-
tal ones, verification was performed under the determined opti-
mal conditions. The result indicated that the experimental values
(16.685 %) were in good agreement with the predicted ones (17.707
%) (insignificant at the 5 % confidence level). This represented that
the model expressed in Eq. (2) was accurate and satisfactory. Fig. 3. The protein electropherogram obtained from experimental and control
groups, where lane a was the marker, lane b was the experimental group, lane c
4.3. Protein microstructural analysis under optimized conditions corresponds to the control group.

4.3.1. SDS-PAGE
The protein electropherogram obtained from experimental and
control groups is depicted in Fig. 3. Only a smeared band existed in
lanes b and c because of aging, different from the specifically dis-
tinct bands of α 1 , α 2 and β chains in collagen/gelatin [58–60]. The
distribution of bands in lane b was not evidently different from
that in lane c, indicating that the optimal parameters had no re-
markable effect on the purity and molecular weight of extracted
proteins.
Fig. 3 The protein electropherogram obtained from experimen-
tal and control groups, where lane a was the marker, lane b was
the experimental group, lane c corresponds to the control group.

4.3.2. UV
The UV absorption spectra of proteins from experimental and
control groups were illustrated in Fig. 4. A strong peak related
to the C=O, –COOH, and –CONH2 groups in gelatin [61,62] ap-
peared near 222 nm for both curves, while no peak representing
aromatic amino acids was shown at 280 nm. Little difference ex-
isted in the peak intensities at 220-230 nm between experimen-
tal and control groups, suggesting that optimized extraction condi- Fig. 4. UV spectra of extracted proteins for experimental and control groups.
tions caused little damage to the C=O, –COOH, and –CONH2 groups
in the polypeptide chain.
Fig. 4 UV spectra of extracted proteins for experimental and curves in Fig. 5 were consistent with the characteristics of gelatin,
control groups including the disappearance of the positive peak at 220 nm and
the red shift of the negative peak [64]. No significant difference ex-
4.3.3. CD isted between the two curves. It can be seen that compared with
The CD absorption spectra of the proteins from experimental the control group, the experimental group had no significant effect
and control groups are shown in Fig. 5. It is well-known that an on the protein conformation during the extraction.
obvious negative peak exists at 190-200 nm, and a slight posi- Fig. 5 CD spectra of proteins from experimental and control
tive peak occurs around 220 nm for natural collagen [63]. The two groups

62
Z. Zhu, J. Du, Z. Lu et al.
Fig. 5. CD spectra of proteins from experimental and control groups.
5. Conclusions
This study demonstrated that RSM could be used to optimize
the extraction of proteins in mural samples. The response and con-
tour plots in RSM were employed to predict the influence of 4 vari-
ables (i.e. GuHCl concentration, extraction time, temperature, and
ultrasonic power) on extraction efficiency. The experimental value
for extraction efficiency varied from 12.5 % to 17.7 %. A GuHCl con-
centration of 1.89 M, extraction time of 132 min, extraction tem-
perature of 57 °C, and ultrasonic power of 210 W were found to be
the optimum combination for protein extraction from model sam-
ples, forming an extraction efficiency of 17.707 %. Compared with
the control group, the experimental group under optimized extrac-
tion conditions had no significant impact on the purity, molecular
weight and protein structure.
The optimized parameters and statistics obtained in this arti-
cle can provide fundamental support for the extraction and identi-
fication of binding materials from murals. Specifically, the deter-
mined protocol here could serve as a reference for conservators
when handling murals containing collagen-based binders, with a
higher potential for extracting more target proteins, and improving
the success rate of the subsequent identification. Still, it should be
noted that the optimal extraction parameters for ancient samples
may vary on a case-by-case basis.
The ultimate objective of any innovation in conservation science
is to be executed on ancient samples. The present study focuses on
a fundamental step toward this objective, addressing the issue of
how the GuHCl experimental parameters influence the extraction
effects of collagen-based binders, providing a solid groundwork for
future case studies. In fact, ancient samples tend to be highly het-
erogeneous and unpredictable, as they may contain other types of
protein (ovalbumin, casein, etc.), organics (such as lipids, resins),
and environmental contaminants, in addition to collagens at vari-
ous degradation stages. In the next step of our research, a number
of ancient samples are expected to be selected, to discuss the re-
lation between different natures of samples and the possible vari-
ance in extraction effects.
Acknowledgement
This work was supported by National Natural Science Founda-
tion of China (Project No. 51908464), China Ministry of Education
Humanities and Social Sciences Project (Project No. 18XJCZH011),
63
Journal of Cultural Heritage 56 (2022) 56–64
Open Research Fund of Key Scientific Research Base of Conserva-
tion and Restoration for Murals as Collection and Materials Sci-
ence in State Administration for Cultural Heritage, The Alexan-
der von Humboldt Foundation, Joint International Research Labo-
ratory of Environmental and Social Archaeology (Project No. JoIn-
RLESA202202), The Fundamental Research Funds for the Central
Universities (Project No. 20720212015), and XMU Undergraduate
Innovation and Entrepreneurship Training Programs (Project No.
202110384177).
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