Antioxidant and Antimicrobial Properties of Selected Red Seaweeds from Central Portugal

Round 1

Reviewer 1 Report

The paper needs some corrections to avoid syntax errors and to meet the journal publication requirements. Please consider the following comments:

Line 109: “The healthy, clean biomass was dried in a ventilated oven (25 °C, 48 h).” This temperature may be suitable for the development of microbial germs!!*?

Line 115 (g/mL)  and line 122 (100 mg.mL-1) please make units unform /mL or ml-1 and do not use both.

Line 123: The section “2.3. Antioxidant Activity” should be placed before or after the section “2.4. Antimicrobial Activity »

Figure 1:  please make sure that letters are correctly added in the different histogram to show the significant differences  based on the Tukey HSD. Same comment for figure 2.

Line 331 / 336Other wrote Ceramium ciliatum and C. ciliatum, please use the full lain name in the first appearance in the text and in the other part use C. ciliatum. The Same comment for other species.

The conclusion seems to long and includes a lot of details that belong to the discussion section.

Please write the reference using a uniform style based on the journal guidelines, some journals names in the reference list are written in full and others as abbreviation.

 

 

 

Author Response

We thank you for your time and feedback, which certainly helps us improve our work. We assessed your comments one by one, being all of them highlighted in the manuscript in yellow. We are also describing in this cover letter all the changes we have done, for easiness of tracking and interpretation. Every change pertaining references is not signalled since the reference manager removes text highlights, but they were also made whenever applicable. In the current letter, all text in italic and blue is quoted from you, and we replied below each comment.

The paper needs some corrections to avoid syntax errors and to meet the journal publication requirements. Please consider the following comments:

Line 109: “The healthy, clean biomass was dried in a ventilated oven (25 °C, 48 h).” This temperature may be suitable for the development of microbial germs!!*?

Reply: Indeed it is, but to prevent the development of germs we would be forced to adopt significantly higher temperatures, and will certainly compromise a range of temperature-sensible compounds. For example, water-soluble pigments are known to be highly unstable at higher temperatures, and are present in appreciable quantities in red macroalgae, and are also known to possess appreciable bioactivity. Other compounds with bioactive abilities may also be temperature-sensible, and we did not wish to compromise their integrity. The best we could do was thoroughly and carefully clean and wash our seaweed before carefully drying it.

Line 115 (g/mL) and line 122 (100 mg.mL-1) please make units unform /mL or ml-1 and do not use both.

Reply: We did as you suggested.

Line 123: The section “2.3. Antioxidant Activity” should be placed before or after the section “2.4. Antimicrobial Activity »

Reply: As per suggestion of Reviewer #2, we now have “2.3. Antioxidant Activity”, “2.4. Total Phenolic Compound Assay (TPC)” and “2.5. Antimicrobial Activity”.

Figure 1:  please make sure that letters are correctly added in the different histogram to show the significant differences  based on the Tukey HSD. Same comment for figure 2.

Reply: The letters are now correctly added for Figure 1 (indeed letterbox omitted the second letter whenever there was more than one letter in a bar). Figure 2 was also checked, but no issues were found with the letters.

Line 331 / 336 Other wrote Ceramium ciliatum and C. ciliatum, please use the full lain name in the first appearance in the text and in the other part use C. ciliatum. The Same comment for other species.

Reply: We did as you suggested, and now the full latin name appears whenever it first appears in a major section (e.g. Introduction, Discussion, etc).

The conclusion seems too long and includes a lot of details that belong to the discussion section.

Reply: We did as you suggested, by moving one paragraph indeed belongs in the Discussion (specifically in the end of section 4.2.). Please let us know if you believe the Conclusion is still too long and needs further trimming.

Please write the reference using a uniform style based on the journal guidelines, some journals names in the reference list are written in full and others as abbreviation.

Reply: We did as you suggested.

 

Thank you,

The Authors

Reviewer 2 Report

Type: Articles

Title: Antioxidant and Antimicrobial Properties of Selected Red Seaweeds from Central Portugal

Authors: Marta V. Freitas * , Leonardo G. Inácio , Ana Ruas , Isabela A. Silva , Teresa Mouga * , Leonel Pereira , Clélia Afonso

This manuscript looks scientific soundness, but there are minor things that need to be corrected by the author through revision.

1. Antioxidant properties using only ABTS Assay? why not also do with DPPH, etc? it would reinforce the title "Antioxidant properties" and why not show the EC/IC₅₀ of its antioxidant activity.

2. in "2.3.2. Total Phenolic Compound Assay (TPC)" should be "2.4 Total Phenolic Compound Assay (TPC)" not part of measuring antioxidant activity. although TPC may contribute as an antioxidant, it is not appropriate to say that it is part of measuring antioxidant activity.

3. Conclusion is too long. Please concised the conclusion.

Author Response

We thank you for your time and feedback, which certainly helps us improve our work. We assessed your comments one by one, being all of them highlighted in the manuscript in green. We are also describing in this cover letter all the changes we have done, for easiness of tracking and interpretation. Every change pertaining references is not signalled since the reference manager removes text highlights, but they were also made whenever applicable. In the current letter, all text in italic and bolt blue is quoted from you, and we replied below each comment.

This manuscript looks scientific soundness, but there are minor things that need to be corrected by the author through revision.

Comment 1. Antioxidant properties using only ABTS Assay? why not also do with DPPH, etc? it would reinforce the title "Antioxidant properties" and why not show the EC/IC50 of its antioxidant activity.

We chose the ABTS assay since this method always worked very well for us, when performing it with the aqueous extract concentrations we adopted (100 mg/ml). On the other hand, the DPPH assay never worked out for us, as the aqueous extracts heavily precipitated when adding the DPPH – this is because ABTS assay is carried out in aqueous conditions, so is quite suitable for our extracts, while DPPH is more sensitive to apolar compounds, conventionally performed in etanol/metanol conditions, and therefore works better these extracts. Also, ABTS assay is more useful than DPPH assay for detecting the antioxidant activity in a variety of food sources, that are high-pigmented and hydrophylic such as fruits and vegetables (source: Comparison of ABTS/DPPH assays to measure antioxidant capacity in popular antioxidant-rich US foods - ScienceDirect) and the same may happen for macroalgae extracts as well.

Nevertheless, as the ressuspension in water was not working out for most species when performing the DPPH, we tried other solvents to ressuspend our extracts, and to prepare the DPPH solution: etanol, metanol, phosphate buffer and acetonitrile, etc. but nothing was preventing the extracts to precipitate in the wells, and therefore, further optimization needs to be done, in order to ensure that no precipitate forms during the assay and therefore express detectable radical scavenging activity. Adopting lower concentrations of extracts would likely lead to no activity, as pointed out by other colleagues, who have tried DPPH with far lower concentrations, with no success. We are considering, however, adopting the DPPH method with ethanolic extracts, as well as trying other antioxidant methods.

2. in "2.3.2. Total Phenolic Compound Assay (TPC)" should be "2.4 Total Phenolic Compound Assay (TPC)" not part of measuring antioxidant activity. although TPC may contribute as an antioxidant, it is not appropriate to say that it is part of measuring antioxidant activity.

Reply: We agree, and therefore we did as you suggested, by changing “2.3. Antioxidant Activity” to “2.3. ABTS Radical Scavenging Activity Assay”, and by changing "2.3.2. Total Phenolic Compound Assay (TPC)" to "2.4 Total Phenolic Compound Assay (TPC)". The subsequent numbers for subtitles were changed accordingly.

3. Conclusion is too long. Please concised the conclusion.

Reply: We concised the Conclusion by moving and entire paragraph to the end of section 4.2., as Reviewer #1 felt the conclusion was also too long, and had details that belonged to the discussion. Please let us know if you believe the Conclusion is still too long, and we will cut it further.

Thank you,

The Authors

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The  paper seems well revised according to previous comments. I have no further corrections, the paper can be considered for publication