Chemical Characterization of Selected Algae and Cyanobacteria from Bulgaria as Sources of Compounds with Antioxidant Activity

Round 1

Reviewer 1 Report

AS per my observations, the manuscript is very poorly written. There is no novelty or interesting facts about this research. 

I am rejecting this manuscript for further processing.

Comments for author File: Comments.pdf

Author Response

Dear reviewer, we appreciate the time and effort that you have dedicated to providing your valuable feedback on our manuscript.

I don’t find any interesting or novel findings in this research article. So I am sorry to say that I am rejecting the manuscript for further processing. But I have noted some points, which should be implemented for modifications of the manuscript.

Comments from Reviewer 1

Comment 1. Line 28, narginine? Check spelling

Response: Agree. We check spelling and corrected as follows: naringenin

 

Comment 2. The algal biomass was subjected to “solo” freezing in a continuously operating fluidized bed apparatus for 20 minutes at -35°C until the raw materials reached a temperature of -18°C. At that low temperature due to cold stress, there must be some changes in the metabolic processes. I could not understand why these procedure was needed?

Response: Тhank you for the recommendation. The samples were stored at -18 C. Therefore, the description in the text of the article was corrected.

 

Comment 3. 2.2.2 Methods for evaluating the antioxidant activity, total and individual polyphenolic compounds, anthocyanins. Have you measured anthocyanin content?

Response: Thank you for the recommendation. The word anthocyanins was removed from the title of paragraph 2.2.

 

Comment 4. Table 4. Genus Ulv?

Response: Тhank you for the recommendation. This technical mistake was corrected as follows: Ulva

 

Comment 5. Figure 1. Are these microscopic structures? Then it is needed to mention magnification, dimension and all the other specification. Picture are very poorly taken.

Response: Yes, figure 1 was presented microscopic structures. We added the description of the microscope and its magnification (20x) in the section on materials and methods.

 

Comment 6. Figure 3 in the legends vanilla acids? Give the chemical name

Response:  Тhank you for the recommendation. We corrected figure 3, and the chemical names of phenolic acids were added.

 

Comment 7. How protein content was measured? There is no mention of that

Response: Тhank you for the recommendation. The description of the method for determination of protein content was added in the section materials and methods.

 

Comment 8. Despite the higher polyphenolic content of the algae Ulva intestinalis, which is very widespread in the Bulgarian waters, they show lower antioxidant activity compared to Cladophora spp. according to ORAC and HORAC. Why?

Response: Thank you for pointing this out. The possible explanation for the higher antioxidant potential of Cladophora spp. in compassion to Ulva intestinalis according to ORAC and HORAC methods could be explained with higher values of some flavonoids (especially catechins), not with total phenolic content. The detailed profile of flavonoids is shown in figure 4. It was demonstrated that Cladophora spp. contained catechin - 28 mg/100g, while in Ulva intestinalis, this flavonol is entirely missing. Another phenolic component with higher content in Cladophora is ellagic acid, with more than three times higher content than Ulva intestinalis. There both phenolics (catechin and ellagic acid) possessed solid antioxidant potential. The ORAC and HORAC method measures the hydrogen atom donating ability of antioxidants. The ORAC and HORAC method measures hydrogen atom donating ability of antioxidants, therefore the presence of catechin in Cladophora spp could be the reason for the higher antioxidant potential of this age in comparison to Ulva. [1]

[1] Roy MK, Koide M, Rao TP, Okubo T, Ogasawara Y, Juneja LR. ORAC and DPPH assay comparison to assess antioxidant capacity of tea infusions: relationship between total polyphenol and individual catechin content. Int J Food Sci Nutr. 2010 Mar; 61(2):109-24. doi: 10.3109/09637480903292601. PMID: 20109129.

Comment 9. Line 422, 423 the name of the metabolites are misspelled several times.

Response: Thank you for pointing this out. We revised all names in the manuscript and made appropriate corrections.

 

Comment 10. What amino acids were determined? There is no mention of that.

Response: They were not determined for all investigated samples, and the results were omitted; the method also was removed from the paper.

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

1. I don't think the authors have given the correct sources in Table 3. I have downloaded Reference 48, which is just a short article introducing the extraction of plants.

2. I do not deny that Marine algae are worthy of investigation, but due to the mistakes in Table 3, I cannot find a suitable reason to focus specifically on Spirulina and Chlorella.

3. I think the authors give a good description of the results of the tests, but I suggest that they compare these results with other published Marine algae.

Author Response

Review 2

Dear reviewer, we appreciate the time and effort that you have dedicated to providing your valuable feedback on our manuscript.

 

Comments from Reviewer 2

 

Comment 1. I don't think the authors have given the correct sources in Table 3. I have downloaded Reference 48, which is just a short article introducing the extraction of plants.

Response:  Thank you for pointing this out. We agree with this comment. Therefore, we have replaced citation 48 with the correct one in Table 3 [56.    Ghaeni, M.; Roomiani, L. Review for Application and Medicine Effects of Spirulina Microalgae. Journal of Advanced Agricultural Technologies 2016, 3, 114-117.].

 

Comment 2. I do not deny that Marine algae are worthy of investigation, but due to the mistakes in Table 3, I cannot find a suitable reason to focus specifically on Spirulina and Chlorella.

Response: Agree. We removed Chlorella from the text as Table 3 only applies to Spirulina, thus emphasizing this point.

 

Comment 3. I think the authors give a good description of the results of the tests, but I suggest that they compare these results with other published Marine algae.

Response: We agree with this and have incorporated your suggestion throughout the manuscript. The authors reviewed other literature sources and added new comparisons.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript contains interesting data on the metabolite profile of selected algae of the Bulgarian Black sea region. It can be considered for the publication, however not in the present form. Improvement required as listed below:

 “The total phenol content in the tested samples varies from 2662.4 mg/100g to 267.7 308 mg/100g” – past tense should be used for the experiments that have already been finished. Please take this into consideration throughout the whole manuscript, which also should be checked in terms of English language correctness.

„p” for p-coumaric shall be written using italics

Since the following sentence is placed in the discussion, a suitable table with the comparative results on the polyphenolic content in the samples prepared using different methods for conservation and storage, is required: “Lyophilization of Spirulina platensis reduces the amount of rutin (Table 7).”

The discussion section should be more focused on the reflections of the results with the relation to the available data from the literature. In the present form, large part of this section consists of the repetition of the results. It should be also emphasized whether the polyphenolic and photosynthetic pigments’ content found in algae is significantly higher compared to other natural sources or the composition is maybe more complex and thus may be of high interest.

The comparison of the results in terms of the methods of the samples preparation should be discussed not only reported. It seems to be an important issue for the potential application of this particular biomaterial.

Conclusion section is also a repetition of the results. Changes required.

Author Response

Review 3

 

The manuscript contains interesting data on the metabolite profile of selected algae of the Bulgarian Black sea region. It can be considered for the publication, however not in the present form. Improvement required as listed below:

 

Comments from Reviewer 3

 

Comment 1: “The total phenol content in the tested samples varies from 2662.4 mg/100g to 267.7 308 mg/100g” – past tense should be used for the experiments that have already been finished. Please take this into consideration throughout the whole manuscript, which also should be checked in terms of English language correctness.

Response: Agree. We've revised the manuscript and made appropriate adjustments.

 

Comment 2: p” for p-coumaric shall be written using italics

Response: Agree. It was corrected.

 

Comment 3: Since the following sentence is placed in the discussion, a suitable table with the comparative results on the polyphenolic content in the samples prepared using different methods for conservation and storage, is required: “Lyophilization of Spirulina platensis reduces the amount of rutin (Table 7).”

Response: Thank you for this suggestion. Therefore, the sentence has been removed from the thread. We have completely revised the Table 8 and introduced appropriate discussion and comparisons with literature data.

 

Comment 4: The discussion section should be more focused on the reflections of the results with the relation to the available data from the literature. In the present form, large part of this section consists of the repetition of the results. It should be also emphasized whether the polyphenolic and photosynthetic pigments’ content found in algae is significantly higher compared to other natural sources or the composition is maybe more complex and thus may be of high interest.

Response: Thank you for this recommendation. We have avoided the repetition you noticed, adding new references to compare our results and emphasize this point.

 

Comment 5: The comparison of the results in terms of the methods of the samples preparation should be discussed not only reported. It seems to be an important issue for the potential application of this particular biomaterial.

Response: Thank you for this comment. The seaweed was harvested from the Bulgarian Black Sea at 2 meters in June 2021. Sampling again from the same regions will not give similar results, as the environmental conditions have changed.

We purchased fresh Spirulina from the bioreactor, but out of scientific curiosity, we also decided to purchase the convection-dried biomass of the same cyanobacterium offered to track whether there is a difference in the studied parameters under different storage conditions.

We do not have such a convection drying system, which is too expensive for most university facilities to develop for research. Therefore, in future research, we may consider a partnership with the team from the bioreactor and make a thorough study of the influence of individual methods on the level of individual parameters and for algae - marine or freshwater - from uncontrolled environments.

We can discuss the effect of lyophilization and convection drying only on the Spirulina samples, as it is grown in the bioreactor from which we bought the fresh biomass.

• Lyophilization of Spirulina platensis had a small positive effect on polyphenol content and antioxidant activity determined by the HORAC method.
• The process of lyophilization of Spirulina platensis did not affect the content of quercetin but led to a decrease in the values of quercetin-3-β-glycoside, kaempferol, naringenin by nearly three times.
• The protein content in Spirulina platensis dried by lyophilizations is almost 20% lower than the protein content in the convection dried sample. Therefore, the lyophilization process did not positively affect the preservation of the protein content of the Spirulina platensis samples.

 

Comment 6: Conclusion section is also a repetition of the results. Changes required.

Response: Agree. We have changed the conclusion.

 

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

The manuscript has been improved.

Author Response

Thank you for giving me the opportunity a revised draft of my manuscript titled “Phytochemical characterization of selected algae and cyanobacteria from Bulgaria as sources of compounds with antioxidant activity “to Applied Science. We appreciate the time and effort that you and the reviewers have dedicated to providing your valuable feedback on our manuscript. We are grateful to the reviewers for their insightful comments on my paper.