Physicochemical Stability of the Pigment Produced by Pseudofusicoccum adansoniae: Influence of pH, Temperature, Additives, and Light Exposure

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Physicochemical stability is sought after in natural pigments, as their color must remain unchanged throughout the food's shelf life. The aim of this study was to evaluate how different process variables affect the physicochemical stability of the pigment produced by Pseudofusicoccum adansoniae. The main question addressed by the research is that how does pH, temperature, ions, additives, storage temperature, and light exposure on color stability in Pseudofusicoccum adansoniae, which have not reported before, thus, the research topic is original. In general, this manuscript was well-written and results are significant, the conclusions consistent with the evidence and arguments presented in the data sheet and the results addressed the main question. I have the only the following minor comments:

 

Comments:

1. There were only three key word, should increase at least one more;

2. The first letter of each key word should be capital;

3. For the methodology part need to be modified, why did colorimetric measurements were taken at 0, 1, 3, 6, 9, 12 and 24h, any reference for it or explanation for it?.

4. For the result part, there are some key information missing, such as: Table 2, +-data stands for. It should mention in the Table;

5. In general, the references were appropriate, but the format of the references is not consistent, some have DOI link, some not, it should be the same.

Author Response

Reviewer 1

Physicochemical stability is sought after in natural pigments, as their color must remain unchanged throughout the food's shelf life. The aim of this study was to evaluate how different process variables affect the physicochemical stability of the pigment produced by Pseudofusicoccum adansoniae. The main question addressed by the research is that how does pH, temperature, ions, additives, storage temperature, and light exposure on color stability in Pseudofusicoccum adansoniae, which have not reported before, thus, the research topic is original. In general, this manuscript was well-written and results are significant, the conclusions consistent with the evidence and arguments presented in the data sheet and the results addressed the main question. I have the only the following minor comments:

Comments:

• Reviewer Comments: There were only three key word, should increase at least one more; The first letter of each key word should be capital;

Response: Thank you for pointing this out. We have added another keyword and put each word in capital letters

 

• Reviewer Comments: For the methodology part need to be modified, why did colorimetric measurements were taken at 0, 1, 3, 6, 9, 12 and 24h, any reference for it or explanation for it?.

Response: Thank you for pointing this out. The paragraph has been rewritten and explained “Spectrophotometric and colorimetric measurements were performed at 0, 1, 3, 6, 9, 12, and 24 hours, with the time intervals determined based on preliminary testing.”

 

Reviewer Comments:  For the result part, there are some key information missing, such as: Table 2, +-data stands for. It should mention in the Table;

Response: Errors in data presented in Table 2 are expressed as Mean Standard Deviation (MSD) as mentioned in the Table’s Caption.

- Reviewer Comments: In general, the references were appropriate, but the format of the references is not consistent, some have DOI link, some not, it should be the same.

Response: Thank you for your observation. The missing reference details have been verified.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript on the physicochemical stability of a new biopigment is potentially of interest for the broad readership of "Applied Science". However, information is missing to appreciate the context of the specific topic and the scope of the findings. Moreover, some figures are altered presumably because of an edition problem, so a proper reviewing is impossible. Therefore, I have to reject the present version of the manuscript but I encourage resubmitting a completed version, under consideration of the following points.

 

1. The general context developed in lines 26-63 in very well documented and presented. Between the lines 63 and 64, authors should comment their interest for "Pseudofusicoccum adansoniae". Why is this biopigment of potential interest for industrial applications? What are the specifications regarding physicochemical stability?

 

2. Lines 96-97: where does this particular temperature range come from: standard testing conditions or specific applications?

 

3. Lines 249-345. The section mainly overviews the literature on the stability of biopigments: perhaps some parts might be transferred in introduction? In any case, authors should comment more extensively their own results and how they rank with other pigments.

 

4. The graphs of figures 1, 3 and 4 are truncated..

Author Response

Reviewer 2

 

This manuscript on the physicochemical stability of a new biopigment is potentially of interest for the broad readership of "Applied Science". However, information is missing to appreciate the context of the specific topic and the scope of the findings. Moreover, some figures are altered presumably because of an edition problem, so a proper reviewing is impossible. Therefore, I have to reject the present version of the manuscript but I encourage resubmitting a completed version, under consideration of the following points.

 Response: We realized that the graphs format in the PDF version, created at the submission to the journal's website has been altered. This error was addressed in the revised version. We apologize for this.

• Reviewer Comments: The general context developed in lines 26-63 in very well documented and presented. Between the lines 63 and 64, authors should comment their interest for "Pseudofusicoccum adansoniae". Why is this biopigment of potential interest for industrial applications? What are the specifications regarding physicochemical stability?

 

Response: we have introduced a paragraph highlighting the importance of studying the pigment, in the revised manuscript, as suggested: “As it is an isolated and identified fungus, with no available data in the literature regarding the characteristics of the pigment it produces, studying these properties becomes essential for future applications”.

 

• Reviewer Comments: Lines 96-97: where does this particular temperature range come from: standard testing conditions or specific applications?

Response: Thank you for pointing this out. The paragraph has been rewritten and explained “Spectrophotometric and colorimetric measurements were performed at 0, 1, 3, 6, 9, 12, and 24 hours, with the time intervals determined based on preliminary testing.”

 

• Reviewer Comments: Lines 249-345. The section mainly overviews the literature on the stability of biopigments: perhaps some parts might be transferred in introduction? In any case, authors should comment more extensively their own results and how they rank with other pigments.

 

Response: We respectfully appreciate the reviewer’s comment on this issue, but the journal's layout adopts a separation between the Results and Discussion sections. The results obtained were initially presented in the appropriate section and subsequently discussed and compared, in the Discussion section, with scientific literature data on pigments produced by other fungi. The discussion covered all evaluated parameters whenever comparable data were available.

 

• Reviewer Comments: The graphs of figures 1, 3 and 4 are truncated.

 

Response: As explained previously, the problem with the Figure’s format has been addressed in the revised manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

In this publication, the authors study the physicochemical stability of a pigment produced from Pseudofusicoccum adansoniae

 

The authors focused on ONLY spectral changes. However, the reason of the spectral changes is a set of chemical transformations of one or more colored compounds.

The authors' results indicate that the key colored compound of the extract is thermally and photo-unstable; is a pH-sensitive compound, and is also capable of chelating ions. These properties are typical of many natural dyes and are not a new fact.

If complex irreversible processes can be responsible for thermal and photostability, then reversible processes are most likely responsible for pH sensitivity and binding of metal ions.

1) I recommend that the authors conduct similar experiments - changing the pH from 3 to 10 and then from 10 to 3. If the color returns, this will mean that nothing has happened to the pigment (it passes from one form to another without destroying the structure). Otherwise, it makes sense to talk about the destruction of the pigment.

2) Also, I do not recommend to testify to the interaction or its absence by the change in optical density at one wavelength. It is necessary to provide full absorption spectra in Supplementary materials, and not to limit oneself to optical density at one wavelength. This is especially true for interactions with metal ions. In some cases, a shift in the maximum of optical density can be observed, leading to a change in the color of the solution and indicating the interaction of the components. However, for one wavelength, the optical density can remain the same (the isosbestic point is the point where the optical density of the substance before and after the reactions coincide) and considering only one wavelength, one can make an incorrect conclusion about the stability/instability of the compound.

Therefore, I recommend that the authors rethink the experiments they obtained, taking into account the understanding of the chemistry of the key dye. There are also two technical comments:

 

1. Some text may have been lost – line 23: “keywords: … endophytic fungus-” (the line ends with a “-”)

Line 28 “he growing…” (the sentence does not start with a capital letter)

2. All figures (figure 1 – figure 4) should be redone. This may be a problem with the final document, but they are not displayed correctly in the final document. The entire range of values should be displayed on both the Y and X axis.
3. It is necessary to format the list of references in accordance with the rules for authors, including adding DOI for many references.

 

Taking into account the above, the material needs to be significantly reworked.

Author Response

Reviewer 3

 

In this publication, the authors study the physicochemical stability of a pigment produced from Pseudofusicoccum adansoniae

 

The authors focused on ONLY spectral changes. However, the reason of the spectral changes is a set of chemical transformations of one or more colored compounds.

- Reviewer Comments: The authors' results indicate that the key colored compound of the extract is thermally and photo-unstable; is a pH-sensitive compound, and is also capable of chelating ions. These properties are typical of many natural dyes and are not a new fact.

Response: We appreciate the reviewer comment on that issue and agree that fungal pigments use to share common properties; however, to date, there are no descriptions in the literature regarding the pigments produced by this specific, recently isolated and identified fungal species. Therefore, we consider this initial characterization to be a relevant contribution to the scientific community, providing important insights for directing future research.

 

- Reviewer Comments: If complex irreversible processes can be responsible for thermal and photostability, then reversible processes are most likely responsible for pH sensitivity and binding of metal ions.

I recommend that the authors conduct similar experiments - changing the pH from 3 to 10 and then from 10 to 3. If the color returns, this will mean that nothing has happened to the pigment (it passes from one form to another without destroying the structure). Otherwise, it makes sense to talk about the destruction of the pigment.

Response: We appreciate your valuable observation. We clarify that the experiments were conducted individually for each pH value, with specific buffer solutions prepared for each tested condition. A progressive pH adjustment was not performed throughout the assays. We acknowledge the importance of the suggestion regarding the reverse experiment; however, we regret to inform you that its execution is currently unfeasible.

2) Also, I do not recommend to testify to the interaction or its absence by the change in optical density at one wavelength. It is necessary to provide full absorption spectra in Supplementary materials, and not to limit oneself to optical density at one wavelength. This is especially true for interactions with metal ions. In some cases, a shift in the maximum of optical density can be observed, leading to a change in the color of the solution and indicating the interaction of the components. However, for one wavelength, the optical density can remain the same (the isosbestic point is the point where the optical density of the substance before and after the reactions coincide) and considering only one wavelength, one can make an incorrect conclusion about the stability/instability of the compound.

 

Response: Figure 1, Panel A, also shows the spectral change observed within the thermal degradation of the pigment. Absorption spectra clearly display a monomorphic pattern, with no expressive peak shift nor presence of secondary peaks. To better address this issue, this text was added to the revised manuscript.

 

Therefore, I recommend that the authors rethink the experiments they obtained, taking into account the understanding of the chemistry of the key dye. There are also two technical comments:

 

Response: we thank the reviewer’s comments and hope that the presented responses, as well as the manuscript revisions, to be able to clarify some aspects of the original manuscript that led him(her) to this opinion.

 

• Reviewer Comments: Some text may have been lost – line 23: “keywords: … endophytic fungus-” (the line ends with a “-”)

Line 28 “he growing…” (the sentence does not start with a capital letter)

Response: Thank you for pointing this out. The errors pointed out have been resolved in the revised manuscript.

 

• Reviewer Comments: All figures (figure 1 – figure 4) should be redone. This may be a problem with the final document, but they are not displayed correctly in the final document. The entire range of values should be displayed on both the Y and X axis.

 

Response: We realized that the graphs format in the PDF version, created at the submission to the journal's website has been altered. This error was addressed in the revised version. We apologize for this.

 

• Reviewer Comments: it is necessary to format the list of references in accordance with the rules for authors, including adding DOI for many references.

 

Response: Thank you for your observation. The missing reference details have been verified in the revised manuscript.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The entire graphs and some missing information was entered by authors. Now, the manuscript is on good way towards acceptation. However, revisions are still necessary since the text is unclear at some places.

1. The sentence introduced by authors in lines 65-67 is unfortunately not clear. Since I asked for this revision, I guess that "it" means "Pseudofusicoccum adansoniae" but this fungus is only introduced in line 69. Therefore, the lines 65-70 have to be reorganized.

1a. Line 65: "Pseudofusicoccum adansoniae" is an isolated and identified fungus. So please, cite literature data on this fungus.

1b. Lines 64-65: a sentence or a part of a sentence is still needed to make the transition with the general literature overview on fungi in lines 48-64. What was the purpose of studying the pigment of "Pseudofusicoccum adansoniae"? To extent the range of available pigments? To remedy to a particular drawback of other pigments? Please, specify.

2. Lines 348-349. Of course, a "Discussion" section is needed in the manuscript. My point was that the reader is missing a discussion whether the outcome of the study is meeting the initial expectations. Is the pigment of "Pseudofusicoccum adansoniae" a promising alternative or a promising complement to other pigments?

Author Response

Reviewer 2

The entire graphs and some missing information was entered by authors. Now, the manuscript is on good way towards acceptation. However, revisions are still necessary since the text is unclear at some places.

1. The sentence introduced by authors in lines 65-67 is unfortunately not clear. Since I asked for this revision, I guess that "it" means "Pseudofusicoccum adansoniae" but this fungus is only introduced in line 69. Therefore, the lines 65-70 have to be reorganized.

• Reviewer comments: Line 65: "Pseudofusicoccum adansoniae" is an isolated and identified fungus. So please, cite literature data on this fungus.

Response: Thank you for pointing this out. The reference Pavlic et al., (2008) was inserted to contextualize the data.

• Reviewer comments: Lines 64-65: a sentence or a part of a sentence is still needed to make the transition with the general literature overview on fungi in lines 48-64. What was the purpose of studying the pigment of "Pseudofusicoccum adansoniae"? To extent the range of available pigments? To remedy to a particular drawback of other pigments? Please, specify.

Response: Thank you for the suggestion. For better understanding and contextualization, the paragraph has been re-edited.

“Although Pseudofusicoccum adansoniae has already been isolated and characterized [33], there is a significant gap in the literature regarding the production and characterization of its pigments. Studying these compounds is relevant to broaden the sources of natural dyes, especially those with potential applicability and safety, contributing to overcome limitations observed in other available fungal pigments.”

• Reviewer comments: Lines 348-349. Of course, a "Discussion" section is needed in the manuscript. My point was that the reader is missing a discussion whether the outcome of the study is meeting the initial expectations. Is the pigment of "Pseudofusicoccum adansoniae" a promising alternative or a promising complement to other pigments?

Response: We appreciate the suggestion. The paragraph has been rewritten to clarify the importance of studying the pigment obtained. “The pigment of Pseudofusicoccum adansoniae represents a promising alternative to existing natural dyes, as it exhibits reasonable stability at acidic pH, controlled light resistance, lack of cytotoxicity, and a distinctive burgundy color that is still scarcely explored among fungal pigments.”

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have significantly improved the quality of the figures and references, but at present they are unable to improve the quality of the material from a chemical point of view.
Despite this, the article can be published.

Author Response

Thank you for taking the time to review the work.