Proteins Extraction and Characterization in Spirulina Biomass: A Comparative Study of High-Pressure Homogenization and Alkaline Methods

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the submitted manuscript (foods-3963633-peer-review-v1), the authors compared the efficiency of one of the "classical" methods for extraction/isolation (alkaline pH) of phycobiliproteins from the cyanobacterium Spirulina and the newly introduced method, high-pressure homogenization (with different applied pressures). The obtained material was analyzed and characterized using various chemical, biochemical, and physicochemical methods. The obtained results convincingly indicate the advantages of high-pressure homogenization, and therefore suggest further optimization of the proposed procedure to obtain samples suitable for potential use in the food industry as well.

In short, an interesting, original, well-conceived study and written text with results adequately presented and discussed. Therefore, it is undoubtedly of interest to researchers, such as this reviewer, who work with vividly colored protein extracts of cyanobacteria and (red) algae in their studies.

However, additions to the existing text are necessary, in terms of specifying and supplementing some parts of the text, as well as correcting (a surprisingly large number, with an emphasis on the scientific quality of the manuscript) of technical imperfections.

Major criticism and suggestions:

1. Authors should be careful with terminology. Suppose commercial preparations of Spirulina are used, as in this study. In that case (in Materials and Methods & Results and Discussion sections), Limnospira platensis should be written, not Arthospira platensis (doi: 10.3390/foods13172762). Next, be careful when using the singular (from the title of the manuscript onwards) for the nouns protein or phycobiliprotein, i.e., when their plural must be used, since, of course, there is NOT just one unique protein in Spirulina.
2. Divide the introductory part of the text (Introduction section) into several logical units (paragraphs), so that the reader can go through the text more easily. In this part, briefly state the chemical (peptidoglycan) structure of the Spirulina cell wall (line 67).
3. State how the enthalpy change in the tested systems (lines 229 and 230) was calculated exactly (literary quote?), based on the (total) protein content.
4. Since it is stated that all parameters were performed in triplicate (line 252), include the standard deviations in Figure 3 (line 337). Are there really statistically significant differences for all points (e.g., between B and C)?
5. Reference number 17 is not listed in the text; it should be included somewhere in the middle of the Introduction section. For a considerable number of references, it is not stated where they are mentioned in the manuscript. It appears that the problem lies in their export from the appropriate program. Anyway, in so many places throughout the document can be found "Error! Reference source not found": lines 35, 46, 269, 323, 335, 336, 354, 357, 401, 413, 433, 446, 488, 489, 494, 496, 513, 520, 538! Please arrange the references properly and completely standardize them (according to the instructions for authors) at the end of the manuscript (from line 582).

Minor suggestions:

1. If the term "alkaline extraction" is already used (e.g., line 12), there is no need to replace it with the (more imprecise) "chemical method" (e.g., line 24).
2. Uniformize the writing of the range of values throughout the text of the manuscript. It is undoubtedly more correct to use "em dash" everywhere (as in line 524) than "en dash" (in many places, e.g., line 33).
3. There is no need to introduce the same terms or acronyms in the text repeatedly. Once (outside the manuscript Abstract) is enough. This remark applies, for example, to the common name for Arthrospira platensis, which is Spirulina (at least 3-4 times mentioned), followed by HPP, PBP(s), EP, EAI, ESI, and so on. Carefully review and refine the text, removing repetitions.
4. Sporadically, for no apparent reason, units next to numbers are put in italics (e.g., line 225). Please correct carefully.
5. Uniformize how the temperature values ​​are written (in degrees Celsius), with or without a space between the number and the unit.
6. In at least a dozen places, there is no space between the number and the symbol for pressure or (mostly) molarity of the solution - from line 84 (50MPa) onwards (lines 113, 118, 121, 135, 139, etc.). Correct everything carefully.
7. Remove excess text on line 572 (no human subjects).

Author Response

Manuscript ID: foods-3963633: Proteins Extraction and Characterization in Spirulina Biomass: A Comparative Study of High-Pressure Homogenization and Alkaline Methods.

We gratefully acknowledge the Reviewer for the thorough and careful examination of our manuscript. We have considered all comments/suggestions and revised the paper accordingly. Additions and changes in the manuscript are marked in red.

Reviewer 1

Major criticism and suggestions:

1. Authors should be careful with terminology. Suppose commercial preparations of Spirulina are used, as in this study. In that case (in Materials and Methods & Results and Discussion sections), Limnospira platensis should be written, not Arthospira platensis (doi: 10.3390/foods13172762). Next, be careful when using the singular (from the title of the manuscript onwards) for the noun’s protein or phycobiliprotein, i.e., when their plural must be used, since, of course, there is NOT just one unique protein in Spirulina.

We appreciate the reviewer’s observation. In accordance with the suggestion, we have replaced Arthrospira platensiswith Limnospira platensis throughout the manuscript (Lines 10, 50, 76, 105). Additionally, we have carefully reviewed the text to correct the use of singular and plural forms for the terms protein and phycobiliprotein wherever appropriate (Lines 2; 10; 17; 26; 47; 92; 94; 115; 171; 172; 174; 190; 193; 336; 380; 384; 387; 456; 462).

1. Divide the introductory part of the text (Introduction section) into several logical units (paragraphs), so that the reader can go through the text more easily. In this part, briefly state the chemical (peptidoglycan) structure of the Spirulina cell wall (line 67).

The Introduction has been reorganized into several logical paragraphs to improve clarity and readability (Lines 42, 55, 66, 86). In addition, the following text has been added in the Introduction (Lines 68–72): “The cell wall of Spirulina consists of four main layers (L-I—L-IV), including an inner fibrillar β–1,2–glucan layer and a peptidoglycan layer providing structural rigidity. This multilayered network is externally covered by a sheath of acidic polysaccharides and glycoproteins, which limits solvent permeability and contributes to the high mechanical strength of the cell wall [20].”

 

1. State how the enthalpy change in the tested systems (lines 229 and 230) was calculated exactly (literary quote?), based on the (total) protein content.

According to reviewer’s comment the equation used for the calculation of enthalpy change has been reported in the revised manuscript (Lines 232–237).

1. Since it is stated that all parameters were performed in triplicate (line 252), include the standard deviations in Figure 3 (line 337). Are there really statistically significant differences for all points (e.g., between B and C)?

Statistical analysis confirmed that the differences among the samples, including those between samples B and C, are significant (p < 0.05). In addition, the resolution of the Figure 3 (line 343) has been increased to improve the readability of the graph and the visualization of the error bars.

1. Reference number 17 is not listed in the text; it should be included somewhere in the middle of the Introduction section. For a considerable number of references, it is not stated where they are mentioned in the manuscript. It appears that the problem lies in their export from the appropriate program. Anyway, in so many places throughout the document can be found "Error! Reference source not found": lines 35, 46, 269, 323, 335, 336, 354, 357, 401, 413, 433, 446, 488, 489, 494, 496, 513, 520, 538! Please arrange the references properly and completely standardize them (according to the instructions for authors) at the end of the manuscript (from line 582).

All reference citations have been carefully checked and corrected throughout the manuscript. The entire reference list has been standardized according to the journal’s formatting guidelines.

Minor suggestions:

1. If the term "alkaline extraction" is already used (e.g., line 12), there is no need to replace it with the (more imprecise) "chemical method" (e.g., line 24).

As suggested, the terminology has been revised to ensure consistency, and the term “alkaline extraction” is now used throughout the manuscript (lines 4; 15; 21; 24; 96; 114; 115; 149; 152; 156; 256; 266; 269; 272; 284; 290; 297; 304; 308; 318; 346; 363; 366; 371; 393; 397; 478; 483; 528; 537).

1. Uniformize the writing of the range of values throughout the text of the manuscript. It is undoubtedly more correct to use "em dash" everywhere (as in line 524) than "en dash" (in many places, e.g., line 33).

All numerical ranges have been carefully checked, and the notation has been uniformed throughout the manuscript, using a consistent dash symbol according to the authors’ suggestions (Lines 33, 58, 64, 68, 83, 86, 225, 248, 276, 284, 318, 336, 343, 391, 443, 451, 478, 523, 539)

1. There is no need to introduce the same terms or acronyms in the text repeatedly. Once (outside the manuscript Abstract) is enough. This remark applies, for example, to the common name for Arthrospira platensis, which is Spirulina (at least 3-4 times mentioned), followed by HPP, PBP(s), EP, EAI, ESI, and so on. Carefully review and refine the text, removing repetitions.

The manuscript has been carefully revised following the Reviewer's suggestion.

1. Sporadically, for no apparent reason, units next to numbers are put in italics (e.g., line 225). Please correct carefully.

The manuscript has been carefully revised (lines 169, 170, 190, 191, 195, 201, 209, 213, 228, 241).

1. Uniformize how the temperature values are written (in degrees Celsius), with or without a space between the number and the unit.

Temperature values have been standardized across the entire text (Lines 119, 123, 137, 139, 184, 196).

1. In at least a dozen places, there is no space between the number and the symbol for pressure or (mostly) molarity of the solution - from line 84 (50MPa) onwards (lines 113, 118, 121, 135, 139, etc.). Correct everything carefully.

All the missing space between number and symbol for pressure or molarity have been carefully checked and corrected throughout the manuscript (Lines 117, 122, 126, 140, 144, 181, 196, 210, 241).

1. Remove excess text on line 572 (no human subjects).

Done

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1- Introduction: please add emulsifying, rheological and thermal properties behavior of protein extracted from spirulina

2- Add which economy the traditional methods or the HPH methods

3- Please add the missing references.

4-Line 120, add the volume of alkaline solution

5-line 129, add the time of homogenization 

6- How do you calculate the initial protein in Spirulina biomass?

7- Identfy SDS

8- In figure 4: At which pH measured the solubility of each method measured?

9- Figure 5, not clear

 

Author Response

Manuscript ID: foods-3963633: Proteins Extraction and Characterization in Spirulina Biomass: A Comparative Study of High-Pressure Homogenization and Alkaline Methods.

We gratefully acknowledge the Reviewer for the thorough and careful examination of our manuscript. We have considered all comments/suggestions and revised the paper accordingly. Additions and changes in the manuscript are marked in red.

Reviewer 2

• Introduction: please add emulsifying, rheological and thermal properties behaviour of protein extracted from Spirulina

We thank the reviewer for the suggestion. However, we would like to point out that the introduction already includes references and descriptions related to the emulsifying, rheological, and thermal behavior of Spirulina protein extracts. Specifically, the last paragraph of the Introduction outlines that the study evaluates these properties. Furthermore, relevant literature (e.g., Magpusao et al.[24], Shkolnikov Lozober et al. [25], Giannoglou et al. [21]) is cited to contextualize the importance of these functional properties. For these reasons, we believe that no further modifications are required in the Introduction section.

• Add which economy the traditional methods or the HPH methods

The economic comparison between traditional alkaline extraction and HPH methods has been included in the Conclusions section (Lines 556 - 557)

• Please add the missing references.

All reference citations have been carefully checked and corrected throughout the manuscript.

• Line 120, add the volume of alkaline solution

The volume of the alkaline solution varied depending on the mass of precipitate obtained during extraction. Therefore, it was not possible to report a fixed volume, as NaOH 0.5 M was gradually added until the pH 7 was reached.

• Line 129, add the time of homogenization 

In high-pressure homogenization (HPH), processing time depends on both the applied pressure and the volume of the suspension, and it is not a controlled parameter. As the pressure increases, the flow rate of the suspension typically decreases, resulting in longer processing times. However, since the system operates in continuous mode, the duration is variable and does not represent a standardized input parameter. Instead, as is commonly done in HPH studies, we reported the number of passes through the homogenizer (in our case, two passes), which is a consistent and reproducible processing variable across all tested conditions.

• How do you calculate the initial protein in Spirulina biomass?

The initial protein content in the Spirulina biomass was determined using the Kjeldahl method, following the official AOAC procedure. The total nitrogen content was measured and converted to protein by applying a nitrogen-to-protein conversion factor (NTP) of 6.25, which is commonly used for microalgal biomass.

• Identfy SDS

The acronym “SDS” has been clarified by adding its full name, sodium dodecyl sulfate (Line 213).

• In figure 4: At which pH measured the solubility of each method measured?

As reported at line 183, the solubility of the supernatants obtained using the two extraction methods was assessed at pH 7. This information has been added to the caption of Figure 4 (Line 363) for greater clarity.

• Figure 5, not clear

The resolution of Figure 5 and the axes labels and legends has been improved (Line 439).

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors, congratulations on your work. It is very well written and presents an interesting dataset regarding protein recovery from Spirulina. Minor corrections are required before its publication.

Errors in citations throughout the text should be corrected.

The introduction is well-written and presents the gaps regarding the use of Spirulina to obtain proteins.

Material and Methods - Why didn't the authors experimentally confirm the proximate composition of the biomass?

Figure 1 - Use precipitate instead of pellets to agree with Table 1. Revise it in the manuscript.

Why were the concentrations and pHs different used to evaluate the solubility of proteins obtained by different methods? iten 2.5

Item 3 - Use Results and Discussion

Line 275 - Use italic for species. Revise it throughout the text.

Improve the resolution of Figures 5, 6 and 7. Make the legends and axes sharper.

Why haven't the proteins been identified in order to determine their contribution to the molecular weight? This characterization would help in the discussion of thermal properties. Is it possible to perform this analysis?

In conclusion, I recommend that the authors maintain the same order presented throughout the manuscript. That is, extraction, characterization, and techno-functional properties.

 

 

 

 

 

 

 

 

 

 

Comments on the Quality of English Language

It should be revised. 

Author Response

Manuscript ID: foods-3963633: Proteins Extraction and Characterization in Spirulina Biomass: A Comparative Study of High-Pressure Homogenization and Alkaline Methods.

We gratefully acknowledge the Reviewer for the thorough and careful examination of our manuscript. We have considered all comments/suggestions and revised the paper accordingly. Additions and changes in the manuscript are marked in red.

Reviewer 3

1. Errors in citations throughout the text should be corrected.

All reference citations have been carefully checked and corrected throughout the manuscript.

2. The introduction is well-written and presents the gaps regarding the use of Spirulina to obtain proteins.

We sincerely thank the reviewer for the positive feedback and appreciation of our work.

3. Material and Methods - Why didn't the authors experimentally confirm the proximate composition of the biomass?

The proximate composition of the Spirulina biomass was experimentally verified prior to use. The results obtained were consistent with the values declared by the supplier, with only minimal deviations attributable to analytical variability. Therefore, the manufacturer’s composition data were considered representative and were used throughout the study.

4. Figure 1 - Use precipitate instead of pellets to agree with Table 1. Revise it in the manuscript.

The term pellet has been replaced with precipitate throughout the manuscript to ensure consistency with Table 1 (Lines 151, 287/288, 293, 297, 317, 320, 322, 349, 379, 447, 483, 496, 544).

5. Why were the concentrations and pHs different used to evaluate the solubility of proteins obtained by different methods? Item 2.5

Preliminary tests performed on the supernatants at the same concentration as the precipitates (0.5% w/v) resulted to be below the sensitivity limit of the analytical method used. This was due to the lower amount of total proteins in the supernatants compared to the precipitates. Therefore, the mass concentration of the samples was increased to allow for an accurate determination of protein solubility.

6. Item 3 - Use Results and Discussion

The heading has been revised to Results and Discussion (Line 264).

7. Line 275 - Use italic for species. Revise it throughout the text.

Following the reviewers’ suggestions, the modifications have been applied throughout the manuscript (lines 10, 50, 76, 105)

8. Improve the resolution of Figures 5, 6 and 7. Make the legends and axes sharper.

The resolution of Figures 5, 6, and 7 has been improved.

9. Why haven't the proteins been identified in order to determine their contribution to the molecular weight? This characterization would help in the discussion of thermal properties. Is it possible to perform this analysis?

We thank the reviewer for this valuable suggestion. Unfortunately, our department currently does not have the instrumentation required to perform protein identification and molecular weight distribution analyses. We have already contacted external research groups and specialized laboratories to carry out this characterization; however, the analyses are still in progress, and the results are not yet available. Once obtained, they will be considered for future work to complement the present study.

10. In conclusion, I recommend that the authors maintain the same order presented throughout the manuscript. That is, extraction, characterization, and techno-functional properties.

We have reviewed and revised the organization of the sections and conclusions to ensure consistency with the recommended sequence: extraction, characterization, and techno-functional properties. We believe the current structure reflects a logical and coherent progression and aligns with the reviewer’s recommendation.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Accept in present form