Fructooligosaccharides (FOS) Production by Microorganisms with Fructosyltransferase Activity
and Mercedes G. López 2,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe study reviewed the fructosyltransferases from microbial sources. The review covers a clear and focused topic, which is important as a useful and detailed reference for potential readers. The authors have strong academic backgrounds to prepare this review and the critical review adequately summarize the well-known background as well as the challenges in this field.
For further improvement:
(1) Reorganize the abstract. Too much introduction is summarized in abstract, some is repetitive with main context (the first sentence); key information is lacked, as the feature and advantage of microbial fructosyltransferases, representative and star fructosyltransferases which will have brilliant future in practical application.
(2) The authors already published plant derived fructosyltransferases. So Section 4.1 is unnecessary and can be moved to the introduction part.
(3) EC codes should be added for all major enzymes covered in this review.
Author Response
Responds to reviewers’ comments:
All changes made by the reviewers’ suggestions are highlighted in RED in the manuscript. We are uploading a Highlighted version and an Unhighlighted version.
REVIEWER 1
Thank you very much for all your comments, they have been essential to enrich the work. Below are the improvements made.
Comment 1) Reorganize the abstract. Too much introduction is summarized in abstract, some is repetitive with main context (the first sentence); key information is lacked, as the feature and advantage of microbial fructosyltransferases, representative and star fructosyltransferases which will have brilliant future in practical application.
Response: The abstract was reorganized, the features and advantages of microbial fructosyltransferase were described, as well as their future potential.
Comment 2) The authors already published plant derived fructosyltransferases. So Section 4.1 is unnecessary and can be moved to the introduction part.
Response: The section 4.1 “Fructosyltransferase activity in plants” was deleted, part of the information was used to complement the section fructan diversity.
Comment 3) EC codes should be added for all major enzymes covered in this review.
Response: EC codes were added for all the enzymes the first time that they were mentioned.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript discusses methods of obtaining fructooligosaccharides mainly in microbial cultures. It is worth particularly emphasizing that the tables include the cultivation conditions conducive to the formation of these saccharides. This publication seems to be within the scope of journal. However, it seems that the authors had not attached the final version of publication but earlier version. For this reason, it needs several corrections to be more acceptable for publication.
1. The title of paper should be changed because the publication devotes a lot of space to plants or microorganism cultivation conditions conducive to the synthesis of fructooligosaccharides, and not only to the isolated enzymes themselves.
2. When the authors discuss the benefits of taking fructooligosaccharides, please add recent publication Costa, G. T., Vasconcelos, Q. D., Aragão, G. F. Fructooligosaccharides on inflammation, immunomodulation, oxidative stress, and gut immune response: a systematic review. Nutrition Reviews, 2022, 80(4), 709-722.; Guerra, L., Ureta, M., Romanini, D., Woitovich, N., Gómez-Zavaglia, A., Clementz, A. Enzymatic synthesis of fructooligosaccharides: From carrot discards to prebiotic juice. Food Research International, 2023, 170, 112991.
3. In the publication, there is lacks references to many recent works, e.g.: Braga, A., Gomes, D., Rainha, J., Cardoso, B. B., Amorim, C., Silvério, S. C., ... Rodrigues, L. R. Tailoring fructooligosaccharides composition with engineered Zymomonas mobilis ZM4. Applied microbiology and biotechnology, 2022, 106(12), 4617-4626.; Lekakarn, H., Bunterngsook, B., Jaikaew, P., Kuantum, T., Wansuksri, R., Champreda, V. Functional Characterization of Recombinant Endo-Levanase (LevBk) from Bacillus koreensis HL12 on Short-Chain Levan-Type Fructooligosaccharides Production. The Protein Journal, 2022, 41(4-5), 477-488.; Chu, J., Tian, Y., Li, Q., Liu, G., Yu, Q., Jiang, T., He, B. Engineering the β-fructofuranosidase Fru6 with promoted transfructosylating capacity for fructooligosaccharide production. Journal of Agricultural and Food Chemistry, 2022, 70(31), 9694-9702.; Canatar, M., Tufan, H. N. G., Ünsal, S. B. E., Koc, C. Y., Ozcan, A., Kucuk, G., ... Turhan, I. Inulinase and fructooligosaccharide production from carob using Aspergillus niger A42 (ATCC 204447) under solid-state fermentation conditions. International Journal of Biological Macromolecules, 2023, 125520.
4. Line 95: it should be „Dactylis glomerata L.” instead of „Dactylis Glomerata L.”.
5. Line 109: It should be „(1.70×103 kDa)” instead of „(1.70x103KDa)”.
6. In Table 1, please add examples of plants producing these fructanes.
7. Line 122 please add information specifically about what toxic compounds may be released.
8. Consistently, each enzyme should have an EC number next to it when it appears in the text for the first time, not just the selected ones. In turn, there is no need to repeat the EC number on lines 180, 181 if it appears earlier on lines 146 and 147.
9. Line 281: it should be „Brachybacterium sp.” istead of „Brachybacterium sp.”.
10. Line 282: it should be „Lactobacillus reuteri” instead of „Lb. reuteri”.
11. Lines e.g. 285, 291, 495, 506: it should be „Gram-negative”, „Gram-positive”. Please check carefully the whole manuscript and correct evident mistake.
12. Line 290: it should be „Brenneria sp.” instead of „Brenneria sp.”.
13. In table 2, instead of the summary formula of the compound (C6H14N207), there should be its name. Additionally, in table 2, table 3, table 4, table 5 it should be „sucrose” instead of „sucrase”.
14. Line 303: in text please explain what the abbreviation „IBSBa” and „IBSBb” means respectively.
15. Line 322: it should be „(ftf)” instead of „(ftf)”.
16. Line 579: it should be „Ni-NTA agarose” instead of „Ni2+–nitrilotriacetic acid”, because Ni-NTA agarose is a matrix used in affinity chromatography for the purification of recombinant proteins, where histidine residues bind to free positions in the coordination sphere of immobilized nickel ions.
Comments for author File: 
Comments.pdf
Author Response
Responds to reviewers’ comments:
All changes made by the reviewers’ suggestions are highlighted in RED in the manuscript. We are uploading a Highlighted version and an Unhighlighted version.
REVIEWER 2
Thank you very much for all your comments, they have been essential to enrich our manuscript. Below you can find the corrections we applied as requested by you.
Comment 1) The title of paper should be changed because the publication devotes a lot of space to plants or microorganism cultivation conditions conducive to the synthesis of fructooligosaccharides, and not only to the isolated enzymes themselves.
Response: The title was modified to “Fructoligosaccharides (FOS)-Production by microorganisms with Fructosyltransferase Activity”. In addition, we tried to rest importance to plants, moving section 4.1 “Fructosyltransferase activity in plants” to complement the section related to "fructan diversity".
Comment 2) When the authors discuss the benefits of taking fructooligosaccharides, please add recent publication:
- Costa, G. T., Vasconcelos, Q. D., Aragão, G. F. Fructooligosaccharides on inflammation, immunomodulation, oxidative stress, and gut immune response: a systematic review. Nutrition Reviews, 2022, 80(4), 709-722.;
- Guerra, L., Ureta, M., Romanini, D., Woitovich, N., Gómez-Zavaglia, A., Clementz, A. Enzymatic synthesis of fructooligosaccharides: From carrot discards to prebiotic juice. Food Research International, 2023, 170, 112991.
Response: The suggested reference “Costa et al., 2022” was included in Line 66: “affect favorably the immune system, increasing the IgA secretion, decreasing proinflammatory cytokines, and having an antioxidant effect 17, 20-23]”. In addition, the work done by “Guerra et al., 2023.” was added in Line 68: “FOS improve mouthfeel, texture, taste, shelf-life of food products, and can be used as low-calorie sugar substitutes.”
Comment 3) In the publication, there is lacks references to many recent works, e.g.:
- Braga, A., Gomes, D., Rainha, J., Cardoso, B. B., Amorim, C., Silvério, S. C., ... Rodrigues, L. R. Tailoring fructooligosaccharides composition with engineered Zymomonas mobilisApplied microbiology and biotechnology, 2022, 106(12), 4617-4626.;
- Lekakarn, H., Bunterngsook, B., Jaikaew, P., Kuantum, T., Wansuksri, R., Champreda, V. Functional Characterization of Recombinant Endo-Levanase (LevBk) from Bacillus koreensisHL12 on Short-Chain Levan-Type Fructooligosaccharides Production. The Protein Journal, 2022, 41(4-5), 477-488.;
- Chu, J., Tian, Y., Li, Q., Liu, G., Yu, Q., Jiang, T., He, B. Engineering the β-fructofuranosidase Fru6 with promoted transfructosylating capacity for fructooligosaccharide production. Journal of Agricultural and Food Chemistry, 2022, 70(31), 9694-9702.;
- Canatar, M., Tufan, H. N. G., Ünsal, S. B. E., Koc, C. Y., Ozcan, A., Kucuk, G., ... Turhan, I. Inulinase and fructooligosaccharide production from carob using Aspergillus nigerA42 (ATCC 204447) under solid-state fermentation conditions. International Journal of Biological Macromolecules, 2023, 125520.
Response: Thank you very much for the references, the suggested recent works focused in fructosyltansferase activity were included in Lines 599 and 605. The works for levanase and inulinase were consulted and have excellent information, however we decided to exclude them for FOS production because they are centred on the fructan polymers degradation.
Comment 4) Line 95: it should be “Dactylis glomerata L.” instead of “Dactylis Glomerata L.”.
Response: The correction was done in line 101.
Comment 5) Line 109: It should be (1.70×103 kDa)” instead of „(1.70x103KDa)”.
Response: The correction was done in line 114.
Comment 6) In Table 1, please add examples of plants producing these fructanes.
Response: The Table 1 was modified, and a column was added including plants examples.
Comment 7) Line 122 please add information specifically about what toxic compounds may be released.
Response: The information was added in line 140.
Comment 8) Consistently, each enzyme should have an EC number next to it when it appears in the text for the first time, not just the selected ones. In turn, there is no need to repeat the EC number on lines 180, 181 if it appears earlier on lines 146 and 147. The EC number was reported for the other enzymes and deleted when repeated
Response: EC number was added for all the enzymes and removed when was necessary, for example in line 252.
Comment 9) Line 281: it should be “Brachybacterium sp.” instead of “Brachybacterium sp.”.
Response: The correction was done in line 294.
Comment 10) Line 282: it should be “Lactobacillus reuteri” instead of “Lb. reuteri”.
Response: The correction was done in line 295.
Comment 11) Lines e.g. 285, 291, 495, 506: it should be “Gram-negative”, “Gram-positive”. Please check carefully the whole manuscript and correct evident mistake.
Response: The correction was done in the whole manuscript.
Comment 12) Line 290: it should be “Brenneria sp.” instead of “Brenneria sp.”.
Response: The correction was done in line 303.
Comment 13) In table 2, instead of the summary formula of the compound (C6H14N207), there should be its name. Additionally, in table 2, table 3, table 4, table 5 it should be „sucrose” instead of „sucrase”.
Response: The first correction was done in table 2, and the second one in tables 2, 3, 4 and 5.
Comment 14) Line 303: in text please explain what the abbreviation “IBSBa” and “IBSBb” means respectively.
Response: The correction was done in line 339.
Comment 15) Line 322: it should be „(ftf)” instead of „(ftf)”.
Response: The correction was done in line 335.
Comment 16 Line 579: it should be “Ni-NTA agarose” instead of “Ni2+–nitrilotriacetic acid”, because Ni-NTA agarose is a matrix used in affinity chromatography for the purification of recombinant proteins, where histidine residues bind to free positions in the coordination sphere of immobilized nickel ions.
Response: The correction was done in line 594.
