Improving Effects of Laccase-Mediated Pectin–Ferulic Acid Conjugate and Transglutaminase on Active Peptide Production in Bovine Lactoferrin Digests

Round 1

Reviewer 1 Report

In general, the manuscript has got a good structure, writing, and interesting results. However, the discussion section is limited and don’t apport relevance to results obtained. Mainly with the possible of the laccase effect on the results. It's for that, is recommended include this and enrich the discussion.

Other observations are:

Page 3, Line 105-109: it’s recommended to explain detailly this point.

Page 6, Line 187-194. It’s considered that the authors should include a discussion of the differences observed between both treatments, which would be related to the biological efficiency in a potential application.

Page 12, Line 319. It’s recommended to include laccase characteristics (source, organism productor, if it is a laccase with high redox potential, etc.).

Author Response

Point 1: In general, the manuscript has got a good structure, writing, and interesting results. However, the discussion section is limited and don’t apport relevance to results obtained. Mainly with the possible of the laccase effect on the results. It's for that, is recommended include this and enrich the discussion.

Response 1: Discussion section has been expanded, including the effects of pectin structural feature on the TPC of PP-FA conjugates and the laccase effect on the conjugation reaction, PF-encapsulated complex, and the resultant digests. Please refer to p. 12-13, lines 281-310. (in the revised manuscript with Track changes-All markup)

Point 2: Page 3, Line 105-109: it’s recommended to explain detailly this point.

Response 2: The paragraph has been revised, added more explanations, and shifted to the first paragraph in Discussion section. Please refer to p. 12, lines 281-294.

Point 3: Page 6, Line 187-194. It’s considered that the authors should include a discussion of the differences observed between both treatments, which would be related to the biological efficiency in a potential application.

Response 3: Discussion about the differences between treatments has been added in p. 13, lines 304-310. The detected peptide profiles in Figure 5 concern active and nonactive peptides and cannot be directly linked to potentially active peptide fragments shown in Table 2. For illustrating their biological efficiency, more investigations on the isolation, characterization, and biological activities of the peptide fragments are needed in the future.

Point 4: Page 12, Line 319. It’s recommended to include laccase characteristics (source, organism productor, if it is a laccase with high redox potential, etc.).

Response 4: laccase information indicated was obtained from the official website of Sigma-Aldrich Co. (https://www.sigmaaldrich.cn/CN/zh/search/laccase?focus=products&page=1&perpage=30&sort=relevance&term=laccase&type=product). It is sorry for that no more precise information about laccase from Aspergillus sp. can be reached and indicated.

Reviewer 2 Report

The manuscript “Improving Effects of Laccase-Mediated Pectin-Ferulic Acid Conjugate and Transglutaminase on Active Peptides Production in Bovine Lactoferrin Digests” by Xing et al. describes the identification of active peptides with two methods: encapsulation of laccase-mediated pectin-ferulic acid conjugate (PF) and TG treatment.

The authors presented a scientifically sound rationale manuscript, discussed the results in detail but lack detailed descriptions in the discussions.

 To improve the manuscript:

 -I suggest broadening the discussions

-indicate the amount (percentage/yield) of each peptide identified by UPLC-MS-MS

- the YLGSRYLT peptide contains the GSRY moiety which is indicated as a potential inhibitor of SARS-CoV-2. Several peptides have been identified with different methods as potential inhibitors of SARS-CoV-2, the authors verified whether the sequences identified in their work are common to peptides derived from proteins of SARS-CoV-2 or to antimicrobials against SARS-CoV-2?

- the quality of figure 5 and figure 6 needs to be improved.

-Also, the authors should explain why might they be important? Could they be used in diagnostics or therapy? Or as nutraceuticals??

Author Response

Point 1: I suggest broadening the discussions

Response 1: Discussion section has been expanded, including the effects of pectin structural feature on the TPC of PP-FA conjugates and the laccase effect on the conjugation reaction, PF-encapsulated complex, and the resultant digests. Please refer to p. 12-13, lines 281-310. (in the revised manuscript with Track changes-All markup)

 

Point 2: indicate the amount (percentage/yield) of each peptide identified by UPLC-MS-MS

Response 2: The total purified peptide yield has been added in p. 7, lines 222-224; and p. 17, lines 469-472.

 

Point 3: the YLGSRYLT peptide contains the GSRY moiety which is indicated as a potential inhibitor of SARS-CoV-2. Several peptides have been identified with different methods as potential inhibitors of SARS-CoV-2, the authors verified whether the sequences identified in their work are common to peptides derived from proteins of SARS-CoV-2 or to antimicrobials against SARS-CoV-2?

Response 3: The active peptide sequence of anti-SARS-CoV-2 activity is reported GSRY for bLf peptide [34], different from those (ALMPHIR, IPAVFK) for goat milk beta-lactoglobulin [Çakir, Okuyan, Şener, Tunali-Akbay, Eur. J. Pharmacol. 2021, 891, 173781]. The related reports are quite limited up to date, not yet sufficient to conclude whether the sequence GSRY is common for peptides from diverse proteins. Because that peptide of anti-SARS-CoV-2 activity is not the topic of our study, no verification is made in the revised manuscript.

 

Point 4: the quality of figure 5 and figure 6 needs to be improved.

Response 4: The quality of Figures 5 & 6 has been improved and revised. Please refer to p. 8 and 12.

 

Point 5: the authors should explain why might they be important? Could they be used in diagnostics or therapy? Or as nutraceuticals??

Response 5: Related descriptions have been added in Discussion (p. 13, lines 335-338) and Conclusion (p. 17, line 514-516).

Round 2

Reviewer 1 Report

In general, the document has has got good structure with interesting results. Also, the authors tried attending all observations were done.