The Influence of Natural Aging Exerting on the Stability of Some Proteinaceous Binding Media Commonly Used in Painted Artworks
Round 1
Reviewer 1 Report
I would like to congratulate the authors for their effort in constructing the manuscript. I believe there are several aspects that need to be improved, especially regarding the novelty of the work undertaken, the importance of the results in the context of a wealth of scientific information regarding the artificial ageing of materials. Another very important aspect is that, in the current form of the manuscript, one can understand that the samples were artificially aged using the SOL chamber for 900h, but were not analyzed after this time, but after another 10 years of storage in usual museum conditions. If this is true, we are talking about two separate things: on one hand, the artificial ageing in the SOL chamber (which could become irrelevant after (or is similar to) 10 years in the museum) and, on the other hand, natural ageing in museum conditions, which I believe is more relevant than the first.
Below are some punctual comments:
Page 1, line 2: "photothermal aging" implies accelerated ageing under the influence of light and temperature, but the paper talks about both artificial and natural ageing. Without knowing which one was longer, it is unclear and confusing to use this term.
Page 1, line 3: title mentions polychromy, but in the manuscript only one colour is discusses, ultramarine blue, so "monochrome", not "polychrome"
Page 2, line 67: an image of the samples could be inserted in a supplementary material
Page 2, lines 59-66: the paragraph only shows an enumeration of techniques, there is neither subject nor verb in the sentence. Please rephrase
Page 2, line 68: please explain the choice of the 3% concentration
Page 2, line 71: correct the name of the aging device
Page 2, line 72: the 900h exposure time corresponds to how many real years? The equivalent in real years should be estimated.
Page 2, line 76: if I understand correctly, the samples were artificially aged for 900 h then placed in museum conditions for an additional 10 years' time? What was the point of placing the samples in the Sol chamber? And if so, we are not talking about artificial, but natural ageing. If this is not correct, than the phrase must be modified for better clarity
Page 2, line 86: if the present study makes use of previously published working methodology, this should be expressed clearer
Page 3, line 100: please explain abbreviations were they appear for the first time in the manuscript
Page 3, Figure 1: figure needs to be redone; some of the images are partially overlapped by others. Images are not all the same size. I suggest keeping only some of the images in Figure 1, and put the rest in a supplementary material
Page 5, line 117: why are the values for Met, Lys and Tyr not listed in the table? What is the explanation behind the increase in Ala, Gly, Val, Leu, Pro concentrations, and the decrease of the other amino acids? Is this related to their aliphatic nature or their hydrophobicity?
Page 6, line 119: Table 2 has no relevance. If the authors are keen on keeping it, it should at best be inserted into a supplementary file, not the main text. Moreover, what is the point in presenting the average for each amino acid, when the difference varies so much for each element?
Page 6, lines 129-130: this is not entirely true: Hyp, for instance, an imino amino acid, registered a decrease. Ile and Phe, which are hydrophobic, aliphatic amino acids, registered a decrease. Other aliphatic amino acids, such as Ser, Asp, Glu also decreased. This sentence must be rephrased so as to accurately reflect the data shown in Table 1
Page 7, line 152: again, the authors speak of Tyr and Met as if they were included in the 11 amino acids analyzed, but they are not. These amino acids are discussed in the text but no data about them are presented
Page 8, line 183: this is in contradiction with the data shown in Table 1 and 2; Hyp, Glu, Ser, Ile registered a decrease, they did not increase
Page 10, line 253: it’s the first time that cinnabar appears in the discussion.
Page 11, line 263: the discussion should rather be changed to “natural” aging, as the ten years were in museum conditions, they did not represent accelerated aging
Author Response
Page 1, line 2: "photothermal aging" implies accelerated ageing under the influence of light and temperature, but the paper talks about both artificial and natural ageing. Without knowing which one was longer, it is unclear and confusing to use this term.
Since the artificial aging was much weaker than natural aging, so the title chose natural aging as key words
Page 1, line 3: title mentions polychromy, but in the manuscript only one colour is discusses, ultramarine blue, so "monochrome", not "polychrome"
Maybe painted artworks would be more proper.
Page 2, line 67: an image of the samples could be inserted in a supplementary material
It is a pity that the photo of the Vienna samples was missing (six-seven years ago). But here I can show you the picture of soil aging samples made before（almost the same appearance）.
Page 2, lines 59-66: the paragraph only shows an enumeration of techniques, there is neither subject nor verb in the sentence. Please rephrase
GC-MS analysis was performed with 7890A-5975C GC-MS (Agilent, USA), ABJ220-4NM balance (Kern, Germany), MS 3 DS25 vortex oscillator (IKS Klingelnberg GmbH), KQ-50E ultrasonic cleaner (Kunshan Ultrasonic Instrument, China), Anke LXJ-IIB centrifuge (Shanghai Anting Scientific Instrument Factory, China), QYN100-2 nitrogen purging apparatus (Shanghai Qiaoyue Electrical Company, China), MDS-8G microwave hydrolyzer (Shanghai Xinyi Microwave Chemical Technology, China), 10µL injector (Agilent, USA), LS120A balance (Switzerland Precisa). FTIR analysis was performed with Thermo Scientific Nicolet iN10 MX FTIR (Nicolet, USA), Tablet press (Specac, Britain) while SOL 2 Xenon artificial aging box (Dr K.Hönle, Germany) was the aging apparaturs.
Page 2, line 68: please explain the choice of the 3% concentration
Two sets of 3% (the proper concentration to mix pigments and draw paintings) Pig glue, whole egg, egg white, egg yolk and casein were prepared
Page 2, line 71: correct the name of the aging device
OL 2 Xenon artificial aging box had been corrected to SOL 2 Xenon artificial aging box
Page 2, line 72: the 900h exposure time corresponds to how many real years? The equivalent in real years should be estimated.
One hour in Xenon artificial aging box roughly equals to 18 hours in real time, so 900h exposure equals to 16200 hours (675 days, 1.85 years, approximately 2 years).
Page 2, line 76: if I understand correctly, the samples were artificially aged for 900 h then placed in museum conditions for an additional 10 years' time? What was the point of placing the samples in the Sol chamber? And if so, we are not talking about artificial, but natural ageing. If this is not correct, than the phrase must be modified for better clarity
Yes, this is true. To be honest, the Xenon aging samples were research contents of a Phd student, after finishing her Phd thesis concerning artificial aging, the samples were then placed in museum conditions. I was lucky to have the opportunity to test the samples after ten years’ natural aging.
Page 2, line 86: if the present study makes use of previously published working methodology, this should be expressed clearer
The pretreatments of the samples made use of previous published working methodology and the five main steps (ammonia extraction of the protein, clean-up of the upper extraction with the C4 sorbent pipette tip, hydrolysis, derivatization, injection) followed the paper[6].
Page 3, line 100: please explain abbreviations were they appear for the first time in the manuscript
All amino acids were explained in the abstract.
Page 3, Figure 1: figure needs to be redone; some of the images are partially overlapped by others. Images are not all the same size. I suggest keeping only some of the images in Figure 1, and put the rest in a supplementary material
The pictures were reformatted.
Page 5, line 117: why are the values for Met, Lys and Tyr not listed in the table? What is the explanation behind the increase in Ala, Gly, Val, Leu, Pro concentrations, and the decrease of the other amino acids? Is this related to their aliphatic nature or their hydrophobicity?
In PCA quantitative analysis, Met, Lys and Tyr were not included (Ref 1: Analytical Strategies for Characterizing Organic Paint Media Using Gas Chromatography/Mass Spectrometry. Ref 2: GC/MS Analytical Procedure for the Characterization of Glycerolipids, Natural Waxes, Terpenoid Resins, Proteinaceous and Polysaccharide Materials in the Same Paint Microsample Avoiding Interferences from Inorganic Media). In this paper, the disappearance of Met, Lys and Tyr were observed in chromatograms qualitively but not in a quantitative way.
As for the decline of hydrophilic and the climb of hydrophobic amino acids, it was speculated that the latter were surrounded by the former when the fresh proteins were in liquid states, with long-term dryness and aging, the hydrophilic amino acids outside were degraded so that the inner hydrophobic amino acids were exposed gradually.
Page 6, line 119: Table 2 has no relevance. If the authors are keen on keeping it, it should at best be inserted into a supplementary file, not the main text. Moreover, what is the point in presenting the average for each amino acid, when the difference varies so much for each element?
Table 2 had been deleted.
Page 6, lines 129-130: this is not entirely true: Hyp, for instance, an imino amino acid, registered a decrease. Ile and Phe, which are hydrophobic, aliphatic amino acids, registered a decrease. Other aliphatic amino acids, such as Ser, Asp, Glu also decreased. This sentence must be rephrased so as to accurately reflect the data shown in Table 1
The categories of amino acids were calculated in a combinative way despite there would be increase and decrease of individual amino acid. For instance, the aliphatic (the increase of Ala, Gly, Val, Leu> the decrease of Ile, Ser, Asp, Glu) and imino amino acids (the increase of Pro > the decrease of Hyp) both increased in a combinative view.
Page 7, line 152: again, the authors speak of Tyr and Met as if they were included in the 11 amino acids analyzed, but they are not. These amino acids are discussed in the text but no data about them are presented
In this paper, the disappearance of Met, Lys and Tyr were observed in chromatograms qualitively but not in a quantitative way.
Page 8, line 183: this is in contradiction with the data shown in Table 1 and 2; Hyp, Glu, Ser, Ile registered a decrease, they did not increase
The aliphatic (the increase of Ala, Gly, Val, Leu> the decrease of Ile, Ser, Asp, Glu) and imino amino acids (the increase of Pro > the decrease of Hyp) also increased in a combinative view.
Page 10, line 253: it’s the first time that cinnabar appears in the discussion.
HgS was changed to cinnabar (HgS)
Page 11, line 263: the discussion should rather be changed to “natural” aging, as the ten years were in museum conditions, they did not represent accelerated aging
Photothermal was changed to natural
Author Response File: 
Author Response.doc
Reviewer 2 Report
1. please specify the conditions under which the reference samples were kept.
2. Please justify the mock-ups preparation procedure. (e.g. why in that form and in that concentration?)
3. please justify the aging procedure used. Is any standard protocol used?
Author Response
- please specify the conditions under which the reference samples were kept.
In 2.3 Samples and aging
samples were aged under the SOL 2 Xenon artificial aging box which was similar with daylight energy in a spectrum range of 300~800 nm. Illuminance: 120 lux, irradiation intensity: 910 W.m-2, lamp temperature: 50 ℃, aged time: 900 hours (approximately 2 years natural aging). After the artificial aging, the aged samples were placed in the daily environment in Kunsthistorisches Museum. Illuminance: 30~50 lux, irradiation intensity: 230~380 W.m-2, temperature: 16~32 ℃, aging time: 10 years.
- Please justify the mock-ups preparation procedure. (e.g. why in that form and in that concentration?)
Two sets of 3% (the proper concentration to mix pigments and draw paintings) pig glue, whole egg, egg white, egg yolk and casein were prepared onto glass slides to form dry films. The third set of each binder with ultramarine was also made in the same way (simulating the painted layer composing of pigment and binders).
- please justify the aging procedure used. Is any standard protocol used?
In the first stage, the samples were aged in SOL 2 Xenon artificial aging box, which was research content of a Phd thesis concerning artificial aging. After that, the samples were placed in museum daily conditions.
Author Response File: Author Response.doc
Reviewer 3 Report
The research presents an interesting study about artificial ageing processes. However, the readability is severely affected by the incorrect usage of language, symbols and punctuation. With regard to English, the paper needs rigorous editing, possibly done by a mother tongue.
The paper can be published, but after some major revisions (see specific comments below).
1 – The title should be more pertinent to the paper, in addition to being grammatically correct. I suggest “The influence of photothermal aging exerting on the stability of some proteinaceous binding media commonly used in polychrome artworks”
34-47 – The introduction could be enriched. Then, please arrange spaces between brackets and words. Moreover, revise the language and terminology for clarity and correctness. The same suggestions apply to the whole manuscript.
70 - The sentence seems to contradict what the authors stated at line 88 and in the rest of the manuscript.
75 - Arrange the symbols so that the formula makes sense and it is correct.
100 - I would arrange chromatograms as three per line in order to make comparing the differences between each binder in the three forms (fresh, aged and agedB) easier.
102-107 - Please arrange spaces between words, parenthesis and other punctuation marks.
114 - Please rephrase the sentence. It doesn’t seem clear this way.
126 and following - For clearness, the whole paragraph should be revised. For instance, the contents' fluctuations were not calculated in Figure.2; perhaps they were calculated and then shown in Fig.2. Similarly, those calculations are “in accord with values reported in Table 1”. Moreover, the comment about electriferous and electrically neutral hydrophilic amino acids should be highlighted and perhaps compared to the previous ones: the 3.88% reduction in aromatic AA content seems not so different from the 3.93% related to electriferous ones.
131 - I wouldn't write "laws"; rather, "trends" would convey the desired meaning without being so sharp.
183-184 Please revise the sentence. It is not clear.
185-188 This statement should be supported by the literature.
200 - It isn't clear what the "reduction of intensity" the authors are talking about is.
233 - Figure 3 should be revised: in particular, the first spectrum should be named "A" and the band at 1533 cm-1 is reported twice. Then, spectra D and E are not in the correct order. Finally, in some cases the spectra are not readily readable. I suggest avoiding superimposing them, where possible.
211 - The sentence should be rewritten.
216-218 The sentence should be rewritten. It does not make sense this way.
224 - For consistency, the authors could indicate the corresponding wavenumbers. The enhancement they indicated does not seem so remarkable, though.
232 and following - Five secondary structures. Moreover, the authors should explain better the quantitative method they used to determine structures' contents reported in Table 5.
Author Response
1 – The title should be more pertinent to the paper, in addition to being grammatically correct. I suggest “The influence of photothermal aging exerting on the stability of some proteinaceous binding media commonly used in polychrome artworks”
Combining the suggestions of three reviewers, the title was changed to “The influence of natural aging exerting on the stability of some proteinaceous binding media commonly used in painted artworks”
34-47 – The introduction could be enriched. Then, please arrange spaces between brackets and words. Moreover, revise the language and terminology for clarity and correctness. The same suggestions apply to the whole manuscript.
The introduction and the whole manuscript had been corrected accordingly.
70 - The sentence seems to contradict what the authors stated at line 88 and in the rest of the manuscript.
Two sets of 3% (the proper concentration to mix pigments and draw paintings) pig glue, whole egg, egg white, egg yolk and casein were prepared onto glass slides to form dry films. The third set of each binder with ultramarine was also made in the same way (simulating the painted layer composing of pigment and binders). The first set of the five pure binders were used as fresh standards while the other two sets were aged under the SOL 2 Xenon artificial aging box which was similar with daylight energy in a spectrum range of 300~800 nm.
75 - Arrange the symbols so that the formula makes sense and it is correct.
W.m-2 had been changed to W.m-2.
100 - I would arrange chromatograms as three per line in order to make comparing the differences between each binder in the three forms (fresh, aged and agedB) easier.
The chromatograms were arranged as three per line.
102-107 - Please arrange spaces between words, parenthesis and other punctuation marks.
Chromatograms of the five fresh, natural aged binders (A: fresh pig glu-FPG, B: aged pig glue-APG, C: aged pig glue with ultramarine-APGU, D: fresh whole egg-FWE, E: aged whole egg-AWE, F: aged whole egg with ultramarine-AWEU, G: fresh egg white-FEW, H: aged egg white-AEW, I: aged egg white with ultramarine-AEWU, J: fresh egg yolk-FEY, K: aged egg yolk-AEY, L: aged egg yolk with ultramarine-AEYU, M: fresh casein-FC, N: aged casein-AC, O: aged casein with ultramarine-ACU).
114 - Please rephrase the sentence. It doesn’t seem clear this way.
What is more, the changes of amino acids contents in egg yolk were the most (-5.91%~4.62%), egg white (-4.49%~3.59%), whole egg (-4.41%~3.35%) and casein (-4.31%~2.93%) the medium while pig glue the least (-1.55%~1.85%).
126 and following - For clearness, the whole paragraph should be revised. For instance, the contents' fluctuations were not calculated in Figure.2; perhaps they were calculated and then shown in Fig.2. Similarly, those calculations are “in accord with values reported in Table 1”. Moreover, the comment about electriferous and electrically neutral hydrophilic amino acids should be highlighted and perhaps compared to the previous ones: the 3.88% reduction in aromatic AA content seems not so different from the 3.93% related to electriferous ones.
The paragraph had been revised accordingly.
131 - I wouldn't write "laws"; rather, "trends" would convey the desired meaning without being so sharp.
Laws had been corrected to trends.
183-184 Please revise the sentence. It is not clear.
Based on the above light and thermal aging discussions, the contents of Ile, Ser, Asp, Glu, Lys, Hyp, Met, especially aromatic Phe and Tyr showed decreasing trends while that of Ala, Gly, Val, Leu, Pro climbed. The aliphatic (the increase of Ala, Gly, Val, Leu> the decrease of Ile, Ser, Asp, Glu) and imino amino acids (the increase of Pro > the decrease of Hyp) also increased from a combinative view.
185-188 This statement should be supported by the literature.
As for the decline of hydrophilic and the climb of hydrophobic amino acids, it was speculated that the latter were surrounded by the former when the fresh proteins were in liquid states, with long-term dryness and aging, the hydrophilic amino acids outside were degraded so that the inner hydrophobic amino acids were exposed gradually[26].
200 - It isn't clear what the "reduction of intensity" the authors are talking about is.
Intensity was changed to strength.
233 - Figure 3 should be revised: in particular, the first spectrum should be named "A" and the band at 1533 cm-1 is reported twice. Then, spectra D and E are not in the correct order. Finally, in some cases the spectra are not readily readable. I suggest avoiding superimposing them, where possible.
Figure 3 had been revised.
211 - The sentence should be rewritten.
But the peaks in Amide II declined remarkably and that of Amide III disappeared in the mixture binders, suggesting that the mixture binders had lost the protein’s FTIR characteristics and ultramarine could speed up aging.
216-218 The sentence should be rewritten. It does not make sense this way.
The lipid typical peaks(CH2 asymmetric stretch, CH2 symmetric stretch, C=O stretch) in whole egg (2855~2925 cm-1, 1745~1712 cm-1) and egg yolk (2854~2925 cm-1, 1746~1712 cm-1) shifted and become weaker. Binder with ultramarine changed even intense, for instance, C=O in whole egg mixture binder had completely gone, stressing the accelerated aging function of ultramarine.
224 - For consistency, the authors could indicate the corresponding wavenumbers. The enhancement they indicated does not seem so remarkable, though.
CH3 symmetric bending vibration peaks (1377~1403 cm-1), which were important signs of aging
232 and following - Five secondary structures. Moreover, the authors should explain better the quantitative method they used to determine structures' contents reported in Table 5.
In 2.4.2, more detailed information was added. The atmospheric interference was firstly deducted with Ominic 8.2 for baseline correction, then deconvolution of the Amide I band was performed (band enhancement factor: 3, FWHM: 42.2cm-1). By using a seven point Savitzky-Golay function, the second order derivatives of the spectrogram could be obtained, after which the peaks of the five secondary structures (α-helix, parallel β-sheet, anti-parallel β-sheet, random coils, β-turns) were identified. Finally, the contents of each secondary structure were calculated with Gaussian curve fitting method.
Author Response File: Author Response.doc
Round 2
Reviewer 1 Report
Thank you for the effort of trying to respond to all of the comments in the first round of review. However, I feel that the paper still lacks a clear expression of what is the novelty of the work. It is very similar to that performed by the authors on the soil aged samples (reference 1), with the mentioning that in reference 1 the discussion of the results is much better.
There is still the need to properly revise some paragraphs, so the paper needs to be English-proofed, preferably by a native speaker.
Figures need better resolution, especially Figure 1.
Some specific remarks can be made, regarding:
- the fact that on page 1, line 19, authors mention that “Natural aging played a key role in degradation of the proteinaceous binders” – that is redundant. Of course, natural ageing affects proteins, if not we would be living much longer lives ?
- on page 1, line 23, authors say that samples were aged in natural condition. But they were not natural conditions, they were kept in museum conditions, right?
- page 2, line 58: egg yolk and egg white were not “acquired”, but they obtained
- page 4, line 134: “What is more, the changes of amino acids contents in egg yolk were the most (-5.91%~4.62%), egg white (-4.49%~3.59%), whole egg (-4.41%~3.35%) and casein (-4.31%~2.93%) the medium while pig glue was the least (-1.55%~1.85%).”
Most what? There is something missing there.
- page 6, line 144: the division into seven categories is confusing and does not explain the individual trends described in Table 1, especially given the fact that the seven categories partially overlap.
There could be more comments made, but overall, my reccomendation is to better highlight the novelty of the work undertaken, especially in the context of the existing literature, and to make sure that the speech is fluent.
Author Response
Thank you for the effort of trying to respond to all of the comments in the first round of review. However, I feel that the paper still lacks a clear expression of what is the novelty of the work. It is very similar to that performed by the authors on the soil aged samples (reference 1), with the mentioning that in reference 1 the discussion of the results is much better.
There is still the need to properly revise some paragraphs, so the paper needs to be English-proofed, preferably by a native speaker.
Figures need better resolution, especially Figure 1.
R: Figures were edited with better resolution.
Some specific remarks can be made, regarding:
- the fact that on page 1, line 19, authors mention that “Natural aging played a key role in degradation of the proteinaceous binders” – that is redundant. Of course, natural ageing affects proteins, if not we would be living much longer lives
R: “Natural aging played a key role in degradation of the proteinaceous binders” was deleted.
- on page 1, line 23, authors say that samples were aged in natural condition. But they were not natural conditions, they were kept in museum conditions, right?
R: Pig glue, whole egg, egg white, egg yolk, casein and mixtures with ultramarine were aged for 10 years in daily working environment in Kunsthistorisches Museum.
- page 2, line 58: egg yolk and egg white were not “acquired”, but they obtained
R: Pig glue (German Kremer, 63000), egg (egg white and egg yolk were obtained by separating whole egg) from the market
- page 4, line 134: “What is more, the changes of amino acids contents in egg yolk were the most (-5.91%~4.62%), egg white (-4.49%~3.59%), whole egg (-4.41%~3.35%) and casein (-4.31%~2.93%) the medium while pig glue was the least (-1.55%~1.85%).”
Most what? There is something missing there.
R: The changes of amino acids contents in egg yolk were the greatest
- page 6, line 144: the division into seven categories is confusing and does not explain the individual trends described in Table 1, especially given the fact that the seven categories partially overlap.
R：The contents of hydrophilic, electriferous hydrophilic, electrically neutral hydrophilic amino acids and aromatic amino acids got lowered with the average deductions of 6.70%, 3.93%, 2.77% and 3.88%. Whereas the contents of the hydrophobic, aliphatic (the increase of Ala, Gly, Val, Leu> the decrease of Ile, Ser, Asp, Glu) and imino amino acids (the increase of Pro > the decrease of Hyp) rose with average amounts of 6.70%, 1.17% and 2.71% respectively.
Amino acids could be divided into hydrophilic amino acids ( including electriferous hydrophilic, electrically neutral hydrophilic) and hydrophobic amino acids according to the polarity of the R side chain group. Meanwhile, Amino acids could be divided into aliphatic, aromatic and imino amino acids according to the side chain structure.
In this paper, the above two kinds of classification methods of amino acids were both used to discuss the changing characteristics due to aging.
There could be more comments made, but overall, my reccomendation is to better highlight the novelty of the work undertaken, especially in the context of the existing literature, and to make sure that the speech is fluent.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The term "natural ageeing" used in the title and all over the text, referring to the samples analyzed, does not correspond exactly to the experimental condition, since it has been used natural and accelarated ageeing. I suggest using both accelarated/artificial and natural ageing for material/methodoly description and results discussion.
Author Response
The term "natural ageeing" used in the title and all over the text, referring to the samples analyzed, does not correspond exactly to the experimental condition, since it has been used natural and accelarated ageeing. I suggest using both accelarated/artificial and natural ageing for material/methodoly description and results discussion.
R：Natural and artificial aging were used in the title and the whole text.
Author Response File: Author Response.pdf
Reviewer 3 Report
The paper has been appropriately revised.
Author Response
The title, introduction, Fig.1 and some expressions in the whole paper were improved.
Author Response File: Author Response.pdf
