Potential Prebiotic and Anti-Obesity Effects of Codium fragile Extract
and Byungjae Ahn 1,*Round 1
Reviewer 1 Report
Dear Editor, dear authors,
The article “Potential Prebiotic and Anti-obesity Effects of Polysaacharides from Codium fragile” by Oh et al. focusses on an aqueous polysaccharide extract from a green alga and shows positive effects on bacterial growth of two probiotic strains as well as inhibition of adipocyte differentiation.
Overall, the aim of the article is of interest and suitable for publication in a journal like “Applied Sciences”. The part showing prebiotic activity is very interesting and adds to the field.
Despite of this, I have major concerns on accepting the article in its current form and strongly recommend very intensive revision.
Major points:
- In my point of view, the title stating “anti-obesity effects” is very strong and not clearly supported by the data. For me, the authors should explain clearly why they chose these two biological activities. I also wondered why the strong hypothesis of “anti-obesity effect” is only covered in the discussion part in 8 (!) lines. Beside in line 64, you don’t mention “in vitro” throughout the manuscript. The discussion on that topic is descriptive, a repetition from the results part and completely lacks discussion of other literature on that topic.
- I don’t understand why your conclusion was “PCF inhibited adipocyte differentiation by inducing differentiation-related factors” (ll. 305-307) when you wrote a few lines earlier (ll. 300-301) “…blocked expression PPARγ, C/EBPα and Fas…”. Could it be a mistake in writing?
- Especially, bioavailability of PCF should be taken into account and should be discussed. How can this macromolecular mixture be present in pre-adipocyte tissue by oral uptake (as it would be necessary for prebiotic activities)? Furthermore, the authors showed fermentation by bacterial strains. Is the fermented PCF also active on adipocyte-differentiation? Your statement in lines 62-65 fosters the question why you have not tested a fermented extract of PCF.
- I have to mention that I am no expert in adipocyte research. For me, a justification of the cell line 3T3-L1 should be given. This fibroblast cell line seems to be a controversially discussed cell line, because of a high number of influence factors on the differentiation process to adipocyte-like cells. Was it only because of the availability and simplicity of the “cocktail method”?
- It is not clear for me why the authors tested and plotted pH value and acidity. In my understanding pH is a measure to quantify the acidic or alkaline character of a solution. This is also visible in Figures 2 and 3, where the graphs are very similar, but opposing. Maybe I misunderstood your statement, but isn’t it redundant to mention that the acidity is high, when the pH value is low?
- I wondered why you have not additionally used mixtures of bacterial strains and not only separate strains (e.g. l. 89) in the same medium. As the human gut is not inhabited by individual strains but complex mixtures of strains, this would strengthen your argument of prebiotic effects. Maybe your results can be enhanced with synergistic effects by that.
- Table 1 is labelled “structural characterization of PCF”. In my understanding, it doesn’t contain “structural” data but only “compositional” data. Structure would include linkage-types of the monosaccharides and sulfate groups. I don’t understand which % is stated in “monosaccharide composition (%)". Is it molar-%, mass-% or area-% from the analysis? Why can the combined percentages of monosaccharides in one sample be more than 100%: Fuc + Rha + Ara + Gal + Glc + Man + Xyl = 5.69 + 5.12 + 11.30 + 81.09 + 77.45 + 28.48 + 4.49 = 213.62 % (!)
- I missed detailed references on Codium (fragile) polysaccharides. You referenced review articles focusing on algal polysaccharides in a very general manner (e.g. 6, 14). There are good original articles covering your exact species and providing detailed structural data, like Estevez et al. (2009), Ciancia et al. (2007) or Love and Percival (1964). These articles are just a few hits be searching databases for Codium I strongly recommend to strengthen the discussion in that point. For example, the statement in ll. 252+253 is unreferenced and could be enhanced by additional articles.
- In lines 67-73 you described the extraction procedure. For me, the separation step of extract and algal material is missing (l. 72). Have you concentrated the sample (l. 72) without separation?
- Why is the blank control in lines 128+129 done by using methanol? You wrote that “the fermented broth was replaced by methanol”. Why not by (unfermented) MRS broth?
- In my view, it would add to Table 2 if you give the abbreviation code of the culture collections (you mentioned them in ll. 85+86) for each species. You should think about adding an additional column with either “probiotic” or “pathogenic” for each bacterial strain. This would add to the ease of reading for non-experts.
- You wrote that in addition to the prebiotic effects “a concomitant decrease in pathogenic microbes” (l. 303) happened. I do not find any OD600 value for the blank control growth. In Table 2 there are values for the pathogenic bacteria in ranges from 0.59-0.72. I do not understand this as an absolute decrease and would only conclude slower growth unless no OD600 blank is given. Therefore it seems to be a “relative decrease” in a hypothetical mixture and not a direct negative effect on pathogenic bacterial growth.
Minor points:
- Line 39: “algae” is plural. You used it as singular form, which is “alga”. Maybe you mean “Codium fragile is a species of green algae”?
- Line 80: “HP-AEC” is usually written “HPAEC”
- Line 99: a redundant “and” before “were cultured”.
- Line 114: a greek “rho” is misplaced here. It should be a “p” for “para-“ in italics.
- Please use always company names, city, state and country (e.g. lines 111, 103-104) like you used it in line 95-96.
- Please carefully check for typos (e.g. line 103-104: “Mettler Toredo” should be “Mettler-Toledo”)
- Figure 1 and 2 have exactly the same label. Please correct!
Best regards!
Author Response
Major point
Point 1: In my point of view, the title stating “anti-obesity effects” is very strong and not clearly supported by the data. For me, the authors should explain clearly why they chose these two biological activities. I also wondered why the strong hypothesis of “anti-obesity effect” is only covered in the discussion part in 8 (!) lines. Beside in line 64, you don’t mention “in vitro” throughout the manuscript. The discussion on that topic is descriptive, a repetition from the results part and completely lacks discussion of other literature on that topic.
Response 1: (Line15, 71,-73, 53-57 and 322-335 of the revised manuscript)We appreciate the reviewer’s valuable comments that convincing data on anti-obesity effects from in vitro experiments should be supplemented. We have added a further discussion of the anti-obesity effects obtained from in vitro experiments and described the mechanisms involved in both functions as suggested by the reviewer.
Point 2:I don’t understand why your conclusion was “PCF inhibited adipocyte differentiation by inducing differentiation-related factors” (ll. 305-307) when you wrote a few lines earlier (ll. 300-301) “…blocked expression PPARγ, C/EBPα and Fas…”. Could it be a mistake in writing?
Response 2:(Line 322-335 and 340 of the revised manuscript) We appreciate the reviewer’s valuable comments that the conclusion of western blotting results should be supplemented. We have edited the discussion of the results so that the western blotting results can be interpreted accurately as suggested by the reviewer.
Point 3:Especially, bioavailability of PCF should be taken into account and should be discussed. How can this macromolecular mixture be present in pre-adipocyte tissue by oral uptake (as it would be necessary for prebiotic activities)? Furthermore, the authors showed fermentation by bacterial strains. Is the fermented PCF also active on adipocyte-differentiation? Your statement in lines 62-65 fosters the question why you have not tested a fermented extract of PCF.
Response 3:As a prebiotic, PCF is absorbed into the body through oral ingestion.The substrate specificity of the selected lactic acid bacteria for prebiotic evaluation of PCF was confirmed, suggesting that it can be used as a prebiotic material. Although the evaluation of synergistic biotics that improves the physiologically active function after fermentation has not been made yet, it is expected to become a good research topic in the future. Also, although PCF has an anti-obesity effect, it is necessary to check whether fermented PCF has an anti-obesity effect.
Point 4:I have to mention that I am no expert in adipocyte research. For me, a justification of the cell line 3T3-L1 should be given. This fibroblast cell line seems to be a controversially discussed cell line, because of a high number of influence factors on the differentiation process to adipocyte-like cells. Was it only because of the availability and simplicity of the “cocktail method”?
Response 4:In many studies, the cell line 3T3-L1, which exhibits significant morphological and biochemical changes, has been mainly used in experiments related to obesity improvement and prevention. The related papers are as follows.
1)Kim et al.,The protective effects of steamed ginger on adipogenesis in 3T3-L1 cells and adiposity in diet-induced obese mice. Nutr. Res. Pract.2021, 15, 279-293. https://doi.org/10.4162/nrp.2021.15.3.279
2) Karunakaran et al., Anti-Obesity and Lipid Lowering Activity of Bauhiniastatin-1 is Mediated Through PPAR-γ/AMPK Expressions in Diet-Induced ObeseRat Model. Front. Pharmacol.2021, 12, 704074. https://doi.org/10.3389/fphar.2021.704074
3) Park, et al., TheAntiobesity Effects of Buginawa in 3T3-L1 Preadipocytesand in a Mouse Model of High-Fat Diet-Induced Obesity. 2019, BioMed. Res. Int. https://doi.org/10.1155/2019/3101987
4) Dayarathne, et al., Restoration of the adipogenic gene expression by naringenin and naringin in 3T3-L1 adipocytes. J. Vet. Sci. 2021, 22, e55. https://doi.org/10.4142/jvs.2021.22.e55
5) Kim, et. al., Flavonoids from Acer okamotoanum Inhibit Adipocyte Differentiation and Promote Lipolysis inthe 3T3-L1 Cells.Molecules.2020, 25, 1920. https://doi: 10.3390/molecules25081920.
Point 5:It is not clear for me why the authors tested and plotted pH value and acidity. In my understanding pH is a measure to quantify the acidic or alkaline character of a solution. This is also visible in Figures 2 and 3, where the graphs are very similar, but opposing. Maybe I misunderstood your statement, but isn’t it redundant to mention that the acidity is high, when the pH value is low?
Response 5:PCF is used as a carbon source for lactic acid bacteria, and through fermentation, short-chain fatty acids such as acetic acid are produced to lower the pH. Acidity is confirmed by measuring acidity while neutralizing using a base that can react with hydrogen ions generated when the pH is lowered. Therefore, the lower the pH, the more acid is produced and the higher the acidity.
Point 6:I wondered why you have not additionally used mixtures of bacterial strains and not only separate strains (e.g. l. 89) in the same medium. As the human gut is not inhabited by individual strains but complex mixtures of strains, this would strengthen your argument of prebiotic effects. Maybe your results can be enhanced with synergistic effects by that.
Response 6:In this study, PCF prebiotics were evaluated using representative lactic acid bacteria. In the future, we plan to research new biotics that produces synergistic effects using complex strains depicting the human gut and tools such as Next Generation Sequencing.
Point 7:Table 1 is labelled “structural characterization of PCF”. In my understanding, it doesn’t contain “structural” data but only “compositional” data. Structure would include linkage-types of the monosaccharides and sulfate groups. I don’t understand which % is stated in “monosaccharide composition (%)". Is it molar-%, mass-% or area-% from the analysis? Why can the combined percentages of monosaccharides in one sample be more than 100%: Fuc + Rha + Ara + Gal + Glc + Man + Xyl = 5.69 + 5.12 + 11.30 + 81.09 + 77.45 + 28.48 + 4.49 = 213.62 % (!)
Response 7:(Line 42 of the revised manuscript) We appreciate the reviewer’s valuable comments on the meaning of the terms in Table 1. title and the correction of the monosaccharide composition units. We have editedthe term used incorrectly in Table 1. title and the unit of monosaccharide composition as suggested by the reviewer.
Point 8:I missed detailed references on Codium (fragile) polysaccharides. You referenced review articles focusing on algal polysaccharides in a very general manner (e.g. 6, 14). There are good original articles covering your exact species and providing detailed structural data, like Estevez et al. (2009), Ciancia et al. (2007) or Love and Percival (1964). These articles are just a few hits be searching databases for Codium I strongly recommend to strengthen the discussion in that point. For example, the statement in ll. 252+253 is unreferenced and could be enhanced by additional articles.
Response 8:(Line 263-268 of the revised manuscript) We appreciate the reviewer’s valuable comments regarding the request for a reference of articles covered with the detailed structural data of C. fragile. We have discussed the structural data of C. fragileas suggested by the reviewer.
Point 9:In lines 67-73 you described the extraction procedure. For me, the separation step of extract and algal material is missing (l. 72). Have you concentrated the sample (l. 72) without separation?
Response 9:We concentrated the sample without separation.
Point 10:Why is the blank control in lines 128+129 done by using methanol? You wrote that “the fermented broth was replaced by methanol”. Why not by (unfermented) MRS broth?
Response 10:When measuring DPPH activity, the blank to which no sample has been added is measured using a stable alcohol. (When measuring DPPH activity, the blank was taken as methanol) After fermentation, PCF was compared with glucose as a control.
Point 11:In my view, it would add to Table 2 if you give the abbreviation code of the culture collections (you mentioned them in ll. 85+86) for each species. You should think about adding an additional column with either “probiotic” or “pathogenic” for each bacterial strain. This would add to the ease of reading for non-experts.
Response 11:Table 2 format has been modified in response to review comments.
Point 12:You wrote that in addition to the prebiotic effects “a concomitant decrease in pathogenic microbes” (l. 303) happened. I do not find any OD600 value for the blank control growth. In Table 2 there are values for the pathogenic bacteria in ranges from 0.59-0.72. I do not understand this as an absolute decrease and would only conclude slower growth unless no OD600 blank is given. Therefore it seems to be a “relative decrease” in a hypothetical mixture and not a direct negative effect on pathogenic bacterial growth.
Response 12:Prebiotics inhibit the growth of pathogenic bacteria. Therefore, we compared the relative growth inhibition of pathogenic bacteria using glucose as a comparative carbon source for PCF and galactooligosaccharide, a conventional prebiotic material.
Minor point
Point 1:Line 39: “algae” is plural. You used it as singular form, which is “alga”. Maybe you mean “Codium fragile is a species of green algae”?
Response 1:(Line 42 of the revised manuscript) We appreciate the reviewer’s valuable comments regarding the minor spell check. We have fixed typos as suggested by the reviewer.
Point 2:Line 80: “HP-AEC” is usually written “HPAEC”
Response 2:(Line 88 of the revised manuscript) We appreciate the reviewer’s valuable comments regarding the minor spell check. We have fixed typos as suggested by the reviewer.
Point 3:Line 99: a redundant “and” before “were cultured”.
Response 3:(Line 108 of the revised manuscript) We appreciate the reviewer’s valuable comments regarding the minor spell check. We have fixed typos and run a spellcheck as suggested by the reviewer.
Point 4:Line 114: a greek “rho” is misplaced here. It should be a “p” for “para-“ in italics.
Response 4:(Line 123 of the revised manuscript) We appreciate the reviewer’s valuable comments regarding the minor spell check. We have fixed typos as suggested by the reviewer.
Point 5:Please use always company names, city, state and country (e.g. lines 111, 103-104) like you used it in line 95-96.
Response 5:(Line 112-113 and 120-122 of the revised manuscript) We appreciate the reviewer’s valuable comments regarding the minor spell check. We have fixed typos as suggested by the reviewer.
Point 6:Please carefully check for typos (e.g. line 103-104: “Mettler Toredo” should be “Mettler-Toledo”)
Response 6:(Line 112 of the revised manuscript) We appreciate the reviewer’s valuable comments regarding the minor spell check. We have fixed typos as suggested by the reviewer.
Point 7:Figure 1 and 2 have exactly the same label. Please correct!
Response 7:(Figure 2 of the revised manuscript) We appreciate the reviewer’s valuable comments regarding the error that the titles of Figure 1 and 2 are the same. We have edited the title of Figure 2.
Author Response File: 
Author Response.docx
Reviewer 2 Report
A really relevant and important exploration of the role of algal material in food processes. Thank you for the general introduction, perhaps the authors could look at some more recent papers about the potential utilisation of algal polysaccharides and food uses and nutritional basis. For instance discussions from recent papers exploring this link and illustrating the diversity of polysaccharides and the impact of proteins and oils which may also be of use in improving healthly food applications such as
Zhu, Y., Zhao, X., Zhang, X., Liu, H. and Ao, Q. (2021), Amino acid, structure and antioxidant properties of Haematococcus pluvialis protein hydrolysates produced by different proteases. Int. J. Food Sci. Technol., 56: 185-195. https://doi.org/10.1111/ijfs.14618
Hu, Q., Yin, X., Li, H., Wang, X., Jiang, Z., Li, L., Ni, H., Li, Q. and Zhu, Y. (2021), Characterisation of a novel laminarinase from Microbulbifer sp. ALW1 and the antioxidant activity of its hydrolysates. Int. J. Food Sci. Technol., 56: 4129-4138. https://doi.org/10.1111/ijfs.15041
The methodology section is appropriate. Please just check the statistical analysis on the figures to ensure that the statistical differences are accurate and appropriate.
The discussion is appropriate. It may be worth discussiong some recent research evaluating the fate of phenolic and other compounds during digestion and how that relates to our understanding of human nutrition such as :
Huang, Z., Chen, Q., Hu, K., Zhang, R., Yuan, Y., He, S., Zeng, Q. and Su, D. (2021), Effects of in vitro simulated digestion on the free and bound phenolic content and antioxidant activity of seven species of seaweeds. Int. J. Food Sci. Technol., 56: 2365-2374. https://doi.org/10.1111/ijfs.14860
Afonso, C., Matos, J., Campos, A.M., Gomes, R., Delgado, I., Coelho, I., Castanheira, I., Bandarra, N.M. and Cardoso, C. (2021), Elemental composition and bioaccessibility of three insufficiently studied Azorean macroalgae. Int. J. Food Sci. Technol., 56: 330-341. https://doi.org/10.1111/ijfs.14647
However a very interesting paper. Thank you.
Author Response
Point 1: A really relevant and important exploration of the role of algal material in food processes. Thank you for the general introduction, perhaps the authors could look at some more recent papers about the potential utilisation of algal polysaccharides and food uses and nutritional basis. For instance discussions from recent papers exploring this link and illustrating the diversity of polysaccharides and the impact of proteins and oils which may also be of use in improving healthly food applications such as
Zhu, Y., Zhao, X., Zhang, X., Liu, H. and Ao, Q. (2021), Amino acid, structure and antioxidant properties of Haematococcus pluvialis protein hydrolysates produced by different proteases. Int. J. Food Sci. Technol., 56: 185-195. https://doi.org/10.1111/ijfs.14618
Hu, Q., Yin, X., Li, H., Wang, X., Jiang, Z., Li, L., Ni, H., Li, Q. and Zhu, Y. (2021), Characterisation of a novel laminarinase from Microbulbifer sp. ALW1 and the antioxidant activity of its hydrolysates. Int. J. Food Sci. Technol., 56: 4129-4138. https://doi.org/10.1111/ijfs.15041
Response 1: (Line 30-32 of the revised manuscript) We appreciate the reviewer’s valuable comments regarding the request for a reference of recent articles. We have discussed polysaccharides and proteins derived from marine algae for use in improving healthy food applications as suggested by the reviewer.
Point 2:The methodology section is appropriate. Please just check the statistical analysis on the figures to ensure that the statistical differences are accurate and appropriate.
Response 2:Statistical analysis of the figures confirmed their accuracy and relevance.
Point 3:The discussion is appropriate. It may be worth discussiong some recent research evaluating the fate of phenolic and other compounds during digestion and how that relates to our understanding of human nutrition such as :
Huang, Z., Chen, Q., Hu, K., Zhang, R., Yuan, Y., He, S., Zeng, Q. and Su, D. (2021), Effects of in vitro simulated digestion on the free and bound phenolic content and antioxidant activity of seven species of seaweeds. Int. J. Food Sci. Technol., 56: 2365-2374. https://doi.org/10.1111/ijfs.14860
Afonso, C., Matos, J., Campos, A.M., Gomes, R., Delgado, I., Coelho, I., Castanheira, I., Bandarra, N.M. and Cardoso, C. (2021), Elemental composition and bioaccessibility of three insufficiently studied Azorean macroalgae. Int. J. Food Sci. Technol., 56: 330-341. https://doi.org/10.1111/ijfs.14647
Response 3:(Line 312-315 of the revised manuscript)We appreciate the reviewer’s valuable comments regarding the request for a reference of recent articles. We have discussed studies evaluating the fate of phenolic and other compounds during digestion and how that relates to our understanding of human nutrition.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Dear editor, dear authors,
thank you for providing the revised version of “Potential Prebiotic and Anti-obesity Effects of Polysaccharides from Codium fragile” by Oh et al. After review of the initially submitted manuscript, I recommended reconsideration after major revision.
In the revised version, the authors extended the discussion part in a way that is appropriate and which adds to the manuscript (part of a number of major points in my prior comments). They also clarified a lot of minor points from the first review round and strengthened the manuscript with 11 additional references.
The authors’ responses leave a number of open and (in my view) unsatisfactorily answered points. In the following, I would like to respond again (in blue) to the major points numbered according to the authors responses.
Point 3:Especially, bioavailability of PCF should be taken into account and should be discussed. How can this macromolecular mixture be present in pre-adipocyte tissue by oral uptake (as it would be necessary for prebiotic activities)? Furthermore, the authors showed fermentation by bacterial strains. Is the fermented PCF also active on adipocyte-differentiation? Your statement in lines 62-65 fosters the question why you have not tested a fermented extract of PCF.
Response 3:As a prebiotic, PCF is absorbed into the body through oral ingestion.The substrate specificity of the selected lactic acid bacteria for prebiotic evaluation of PCF was confirmed, suggesting that it can be used as a prebiotic material. Although the evaluation of synergistic biotics that improves the physiologically active function after fermentation has not been made yet, it is expected to become a good research topic in the future. Also, although PCF has an anti-obesity effect, it is necessary to check whether fermented PCF has an anti-obesity effect.
Reviewer’s Response 3: I think there might be a misunderstanding. I don’t question the prebiotic character of your applied sample (PCF). Regarding that point, your response is absolutely right. My question aims to clarify the influences on pre-adipocyte tissues you proposed. You have a complex sample ingested orally. Therefore, it is present in the intestinal system. How can this sample influence pre-adipocyte tissues? This is only possible if the sample as a whole or in part is available in the systemic circle. I would like to see a clear explanation in the text.
Point 4:I have to mention that I am no expert in adipocyte research. For me, a justification of the cell line 3T3-L1 should be given. This fibroblast cell line seems to be a controversially discussed cell line, because of a high number of influence factors on the differentiation process to adipocyte-like cells. Was it only because of the availability and simplicity of the “cocktail method”?
Response 4:In many studies, the cell line 3T3-L1, which exhibits significant morphological and biochemical changes, has been mainly used in experiments related to obesity improvement and prevention. The related papers are as follows.
Reviewer’s Response 4: Thank you for that explanation. I think it would add to the manuscript if you give a justification of the cell line in the manuscript and also reference the mentioned literature.
Point 5:It is not clear for me why the authors tested and plotted pH value and acidity. In my understanding pH is a measure to quantify the acidic or alkaline character of a solution. This is also visible in Figures 2 and 3, where the graphs are very similar, but opposing. Maybe I misunderstood your statement, but isn’t it redundant to mention that the acidity is high, when the pH value is low?
Response 5:PCF is used as a carbon source for lactic acid bacteria, and through fermentation, short-chain fatty acids such as acetic acid are produced to lower the pH. Acidity is confirmed by measuring acidity while neutralizing using a base that can react with hydrogen ions generated when the pH is lowered. Therefore, the lower the pH, the more acid is produced and the higher the acidity.
Reviewer’s Response 5:
Your response revealed that my assumption was right.
You wrote: “Therefore, the lower the pH, the more acid is produced and the higher the acidity.” This is part of the definition of pH.
In the manuscript, you wrote “The acidity was determined by measuring the amount of 0.1N NaOH required to adjust the pH to 8.2.”
Why is it necessary to give both, the pH and the acidity? In my point of view, it is redundant to give both plots. Both are showing the same results. Please remove one of the plots or provide a clear explanation why it is necessary to keep both (as an answer to the reviewer’s comment and also in the text)
Point 9:In lines 67-73 you described the extraction procedure. For me, the separation step of extract and algal material is missing (l. 72). Have you concentrated the sample (l. 72) without separation?
Response 9:We concentrated the sample without separation.
Reviewer’s Response 9: This statement changes a lot in the evaluation of the results! You can not write about PCF (polysaccharides from Codium fragile) anymore. If you have not separated the algae from the extract, you have in fact an algal suspension. To call an algal suspension with 21.4 % carbohydrates (combined monosaccharide composition data, Table 1) or 34.9 % carbohydrates (neutral sugar determination), respectively, “polysaccharides” is misleading. Please revise the name and clearly indicate that you used no extract, but an algal suspension.
Point 10:Why is the blank control in lines 128+129 done by using methanol? You wrote that “the fermented broth was replaced by methanol”. Why not by (unfermented) MRS broth?
Response 10:When measuring DPPH activity, the blank to which no sample has been added is measured using a stable alcohol. (When measuring DPPH activity, the blank was taken as methanol) After fermentation, PCF was compared with glucose as a control.
Reviewer’s Response 10: Thank you for the explanation. This is methodically correct as most protocols share this. I just wondered why you have not tested MRS broth as a blank control. What you did (methanol as control) is in fact no “blank control” but a “negative control”. A blank control is not intended to be always negative, but should be used to determine the background value. Please correct that in the Materials and Methods section.
Point 12:You wrote that in addition to the prebiotic effects “a concomitant decrease in pathogenic microbes” (l. 303) happened. I do not find any OD600 value for the blank control growth. In Table 2 there are values for the pathogenic bacteria in ranges from 0.59-0.72. I do not understand this as an absolute decrease and would only conclude slower growth unless no OD600 blank is given. Therefore it seems to be a “relative decrease” in a hypothetical mixture and not a direct negative effect on pathogenic bacterial growth.
Response 12:Prebiotics inhibit the growth of pathogenic bacteria. Therefore, we compared the relative growth inhibition of pathogenic bacteria using glucose as a comparative carbon source for PCF and galactooligosaccharide, a conventional prebiotic material.
Reviewer’s Response 12: Thank you for your explanation. Is the ability of prebiotics to “inhibit the growth of pathogenic bacteria” a direct antibiotic effect? Please clarify and reference that clearly in the manuscript.
In conclusion, major questions remain unanswered or unsatisfactorily addressed in the manuscript. I see the motivation of the authors to improve the manuscript, but cannot recommend acceptance of the manuscript due to the serious open questions outlined above. Based on my evaluation, I recommend again “Reconsider after major revision”.
Best regards
Author Response
Please see attached file.
Author Response File: Author Response.docx
Round 3
Reviewer 1 Report
Dear Authors,
Thank you for revising your manuscript with the corrected title "Potential Prebiotic and Anti-obesity Effects of Codium fragile Extract". Your corrections increased the quality and significance of that manuscript. From my point of view, this improved version is appropriate for publication in the journal "Applied Sciences" and I therefore recommend acceptance.
Congratulations to such an interesting article!
Best regards
