Antioxidant, Collagenase Inhibitory, and Antibacterial Effects of Bioactive Peptides Derived from Enzymatic Hydrolysate of Ulva australis
Round 1
Reviewer 1 Report
The manuscript fits into the research area that Marine Drugs deals with and meets the general standards for publication. Then, I think the paper can be acceptable for publication after a minor revision.
1. All the indexes of Table 1 have not been fully discussed, especially the free amino acid components that the hydrolysate does not contain. Please confirm wether sample means dry powder of U. australis.
2. Two statements of line82- line84 contradict each other. Significant characters are not stated in Figure 1.
3. More latest references should be cited.
Author Response
Response to Reviewers
Reviewer 1
Comments and Suggestions for Authors
The manuscript fits into the research area that Marine Drugs deals with and meets the general standards for publication. Then, I think the paper can be acceptable for publication after a minor revision.
- All the indexes of Table 1 have not been fully discussed, especially the free amino acid components that the hydrolysate does not contain. Please confirm wether sample means dry powder of australis.
>Thanks, we try to explain the sample mean and free amino acid component. We improve on our weakness, and insert in the Table and text.
The samples used as powder of dried conditions. Thus modified into “powder of dried sample” at Table 1 and text. Also, free amino acid only measured at U. australis hydroysate .
>Unify the sample condition as “powder of a dried sample” and “hydrolysate” in the Table 1 and text. Since the objectives of this study is to screen the bioactive of peptides from the hydrolysate from U. australis, we didn’t analyze the free amino acid composition of the powder of a dried sample.
- Two statements of line82- line84 contradict each other. Significant characters are not stated in Figure 1.
>Figure 1a indicates the solubility of hydrolysate dissolved in pH 5.5 and 8.0 according to the amount of hydrolysate, whereas figure 1b indicates the effect of pH on the solubility of hydrolysate at 50 mg/mL. The figure 1b showed the 30% solubilities pH 5.5 and pH 8.0. The r results of the hydrolysate at 50 mg/mL at pH 5.5 and 8.0(Fig.1a) were equal to the results of pH dependence of Fig1b at 50 mg/mL. I think that the results not contradict each other. The sentence was corrected for clear explanation. The significance can be verified by the standard deviation in figures.
>Figure 1a and Figure 1b changed. Significant characters of p<0.01 inserted at legend of Figure 2 and stated at text.
Fig.1a and Fig1b were not changed. Fig 1a showed the effect of hydrolysate concentration on the solubility, while Fig.1b showed the effect of pH on solubility at 50 mg/mL. We stated the significant characts in the text.
- More latest references should be cited.
More latest references cited and “Pyeun, J.H.; Jeon, J.G. Seafood chemistry, Suhaksa, Seoul, South Korea., 1994; pp.283–355.” inserted.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This manuscript described the potential bioactivities of peptides derived from Ulva australis hydrolysate. Total antioxidant capacity (TAC), inhibition of collagenase, and antibacterial properties were observed from several fractions like #5 (SP5) and #7 (SP7). MALDI-TOF/MS and Q-TOF/MS/MS analyses revealed 42 promising peptides, and eight were chosen for molecular docking on target proteins and subsequently synthesized. Some of them displayed bioactivities with IC50 values of 0.84~13.23 mM. Overall, this manuscript is poorly organized and written. The reviewer does not recommend the publication on Marine Drugs due to the following concerns.
1) Language should be improved.
2) About the identification of the peptide fragments using MALDI-TOF/MS and Micro Q-TOF/MS/MS spectrometer, it looks like the differences between calculated MW and the experimental values are significant.
3) The resulting potency is too weak. If the authors are willing to develop nutraceuticals and cosmetics, more appropriate journals are supposed to choose. For drug discovery, these peptides are not good candidates.
4) For the synthesized peptides, more evidences should be provided to show their purity and validity.
Many grammar mistakes and format issues.
Author Response
Response to Reviewers
Reviewer 2
Comments and Suggestions for Authors
This manuscript described the potential bioactivities of peptides derived from Ulva australis hydrolysate. Total antioxidant capacity (TAC), inhibition of collagenase, and antibacterial properties were observed from several fractions like #5 (SP5) and #7 (SP7). MALDI-TOF/MS and Q-TOF/MS/MS analyses revealed 42 promising peptides, and eight were chosen for molecular docking on target proteins and subsequently synthesized. Some of them displayed bioactivities with IC50 values of 0.84~13.23 mM. Overall, this manuscript is poorly organized and written. The reviewer does not recommend the publication on Marine Drugs due to the following concerns.
1) Language should be improved.
> Language improved.
2) About the identification of the peptide fragments using MALDI-TOF/MS and Micro Q-TOF/MS/MS spectrometer, it looks like the differences between calculated MW and the experimental values are significant.
> The sequence of peptide fragments was identified by m/z value of mass list of SP7/S9 fraction from MALDI-TOF/MS and the sequence data base of proteins in Ulva pertusa (protein entry name: Q6T6H8, BOYGZ5 and) H9KVI6 using de novo sequencing. The sequence of peptide fragments was analyzed in Protein Works (Dajeon, Korea) using UHPLC- Q-TOF/MS/MS spectrometer. We display the mass list of MALDI-TOF/MS as supplement-1. Table 3 modified.
Supplement-1: Mass list of SP7/S9 fraction
m/z S/N Quality Fac. Res. Intens. Area
440.720 430 28516 4196 52744 7923
442.722 144 34867 4054 17679 2736
567.959 114 40201 4726 15507 2802
659.184 103 26048 5139 13966 2870
460.739 64 11084 4043 8008 1342
444.757 46 8533 3858 5766 952
476.725 45 4222 4305 5745 958
671.925 45 15773 4491 6123 1533
665.887 43 8439 4430 5834 1471
614.102 42 11106 4173 5745 1287
482.873 37 13049 3918 4717 852
523.930 35 9477 4036 4610 890
877.035 33 3595 4132 3850 1490
454.714 30 2328 4186 3709 620
892.999 30 1527 4340 3448 1351
655.949 29 7472 4800 3923 854
567.912 25 33818 1570 3825 2016
438.752 25 450 2516 3061 795
681.855 24 1202 4772 3247 837
687.896 23 3657 4620 3212 819
586.950 22 3443 4901 3023 611
649.919 20 4621 4343 2763 648
549.927 19 5164 4626 2586 470
456.863 19 766 4149 2448 399
861.075 19 1030 4472 2314 766
675.180 17 1126 4271 2365 570
593.005 15 2476 2232 3691 1549
598.092 15 1056 3555 2048 505
519.903 15 500 3704 2051 463
637.171 13 1249 3873 1779 457
464.838 12 588 3818 1554 273
908.975 10 893 4427 1170 467
856.993 9 611 4874 1111 386
439.818 9 26.1 4492 1163 180
522.883 8 199 3311 1117 282
464.863 6 154 2573 1597 414
488.901 5 171 1196 792 452
3) The resulting potency is too weak. If the authors are willing to develop nutraceuticals and cosmetics, more appropriate journals are supposed to choose. For drug discovery, these peptides are not good candidates.
>You are right. Macroalgae is not good resource for a functional activity of peptide. Most functional activity derived from macroalgae comes from polysaccharide, polyphenol, flavonoid and pigments etc. Protein content of Ulva spp. Represents between 5.8 and 24.8% of dry weight (Pyun and Jeon, 1994). However, macroalgae stand for a sustainable source of proteins that can be applied as ingredients for human and animal consumption (Echave et al., 2021: Marine drugs: doi.org/10.3390/md19090500). If new technologies are developed for pretreatment prior protein extraction, involving disruptive techniques of cell wall. Protein and hydrolysate from Ulva spp. are good source for human health.
Collagenase inhibitory activities of GPM from Alaska pollack skin, GPK from oyster hydrolysate, VDL and VICE from flounder skin were 3.92, 19.07, 13.46 and 83.35% at 1 mg/mL, respectively (Kim et al., 2022; J Korean Soc Food Sci Nutr; doi/org/10.3746/jkfn.2022.51.4.322). Therefore, IC 50 value (0.84 mM) of RDRF from Ulva australis for collagenase inhibition is lower than that of VICE from a flounder skin. IC50 value of RDRF against P. acnes was comparable to the that of salicylic acid. Salicylic acid of 0.5~1.0% was added to cleasing cream.
4) For the synthesized peptides, more evidences should be provided to show their purity and validity.
>Purity and molecular weight of the synthetic peptides were inserted in Materials and display it as supplement-2.
Supplement-2. Product specification of the synthesized peptides
|
Product name |
Peptide sequence |
Formula |
Molecular weight, Da |
Qty, mg |
Purity, % |
Lot No. |
|
FL-4 |
FDPL |
C24H34N4O7 |
490.54 |
200.0 |
97.22 |
P220714-MX1004266 |
|
TW-4 |
TGTW |
C21H29N5O7 |
463.48 |
200.0 |
97.07 |
P220701-MX1001219 |
|
HY-4 |
HAVY |
C23H32N6O6 |
488.53 |
200.0 |
95.14 |
P220701-MX1001220 |
|
HA-4 |
HVIA |
C20H34N6O5 |
438.52 |
200.0 |
95.39 |
P220701-MX1001221 |
|
NY-4 |
NRDY |
C23H34N8O9 |
566.56 |
200.0 |
97.62 |
P220701-MX1001222 |
|
LG-5 |
LPYPG |
C27H39N6O7 |
545.62 |
200.0 |
97.58 |
P220714-MX1004266 |
|
PF-4 |
PETF |
C23H32N4O8 |
492.52 |
200.0 |
97.45 |
P220714-MX1004267 |
|
RF-4 |
RDRF |
C26H40N10O27 |
592.64 |
200.0 |
97.08 |
P220714-MX1004268 |
Author Response File: Author Response.pdf
Reviewer 3 Report
This manuscript uses Ulva australis as the raw material, starting from the functional requirements of cosmetics such as antioxidant, anti collagen degradation, and anti acne, to explore the active protein peptides, which has certain research and application value. The author separated and identified peptide sequences using anion/cation adsorption chromatography, exclusion chromatography, and HPLC, and validated the efficacy and activity of the active peptide using bioinformatics technology and in vitro activity analysis. The research of the article is in line with the scope of this journal, and has certain innovation and reference significance. But there are still some issues that need to be clarified before the article can be accepted, as follows:
1) line 105, Please provide exclusion chromatography and high-performance liquid chromatography separation chromatograms.
2) line 113, Although no inhibition of NO production and cox-2 inhibitory activity were observed, experimental methods and results need to be provided as evidence.
3) Table 2. Please indicate the pooled fraction numbers for each samples(SP5, SP7, Q3, Q4......).
4) line 156. Please revise the PDB protein name error and incomplete expression in the first sentence of section 2.4.
5) line 158. Remove the space symbol from the protein name "6 WXV" in line 158.
6) Table 4. Are reported active peptide sequences set as positive controls? Using commercial or literature reported active peptide sequences as positive controls will have more reference value.
7)line 304. Does the percentage data here refer to the dry weight of protein?
8) line 337. Lack of experimental methods for cell culture and detection of NO release.
9) line 394. Please provide the Denovo identification results of the peptide sequence to demonstrate the reliability of sequence identification.
The English quality of the manuscript is acceptable.
Author Response
Response to Reviewers
Reviewer 3
Comments and Suggestions for Authors
This manuscript uses Ulva australis as the raw material, starting from the functional requirements of cosmetics such as antioxidant, anti collagen degradation, and anti acne, to explore the active protein peptides, which has certain research and application value. The author separated and identified peptide sequences using anion/cation adsorption chromatography, exclusion chromatography, and HPLC, and validated the efficacy and activity of the active peptide using bioinformatics technology and in vitro activity analysis. The research of the article is in line with the scope of this journal, and has certain innovation and reference significance. But there are still some issues that need to be clarified before the article can be accepted, as follows:
1) line 105, Please provide exclusion chromatography and high-performance liquid chromatography separation chromatograms.
Line105: Size exclusion chromatogram didn’t contain in the text because of much figure, Preparative RP-HPLC was not performed because RP-HPLC is performed in the stage of Q-TOF/MS/MS.
>Our results contained many tables and figure. Therefore, this data displayed as supplement data. Size exclusion chromatogram is following as:
Supplement-3. Size exclusion chromatogram of SP 7 fraction, and the 9-10 fractions,19-20 fractions and 21 fraction were pooled for further analysis.
Supplement-4. Size exclusion chromatogram of Q3 fraction, and the 20 fraction and 21-22 fractions and were pooled for further analysis
2) line 113, Although no inhibition of NO production and cox-2 inhibitory activity were observed, experimental methods and results need to be provided as evidence.
>Experimental methods for NO production and COX-2 inhibitory activity were inserted in “Materials and Methods’ section. Also, results were inserted in Table 2.
3) Table 2. Please indicate the pooled fraction numbers for each samples(SP5, SP7, Q3, Q4......).
>“SP5 and SP7 obtained from SP-Sepharose ion-exchange column chromatography. Q3, Q4, Q15, Q16, Q17 and Q23 obtained from Q-Sepharose ion exchange column chromatography.” inserted at “2.2. purification of bioactive peptides”.
4) line 156. Please revise the PDB protein name error and incomplete expression in the first sentence of section 2.4.
>iHFC was revised into 1HFC. The first sentence also was revised into “In order to screen in silico bioactive peptides, eighteen peptides (Table 4) were docked into the active sites of target proteins, including 6WXV (pdb code) for antioxidant, 1HFC (pdb code) for collagenase inhibition, and 3NX7 (pdb code), using CDOCKER ligand docking module of BIOVIA Discovery Studio (Dassault Systems, Walsham, MA, USA).”
5) line 158. Remove the space symbol from the protein name "6 WXV" in line 158.
>Thank you for your attention. We removed the space of “6 WXV.
6) Table 4. Are reported active peptide sequences set as positive controls? Using commercial or literature reported active peptide sequences as positive controls will have more reference value.
>All peptides in Table 4 come from the hydrolysate of U. australis. Commercial and the active peptides reported in literature didn’t use.
7)line 304. Does the percentage data here refer to the dry weight of protein?
>One percentage represents the ratio of protease to the precipitated protein from dried U.australis. 4.2 section was revised.
8) line 337. Lack of experimental methods for cell culture and detection of NO release.
>Experimental methods for cell culture and NO detection present in “4.8. Inhibitory activity of NO production”
9) line 394. Please provide the Denovo identification results of the peptide sequence to demonstrate the reliability of sequence identification.
>The sequence of peptide fragments was identified by comparison of m/z value of mass list of SP7/S9 fraction from MALDI-TOF/MS and the sequence data base of proteins in Ulva pertusa (protein entry name: Q6T6H8, BOYGZ5 and) H9KVI6. This data displayed as supplement data.
Supplement-1: Mass list of SP7/S9 fraction
m/z S/N Quality Fac. Res. Intens. Area
440.720 430 28516 4196 52744 7923
442.722 144 34867 4054 17679 2736
567.959 114 40201 4726 15507 2802
659.184 103 26048 5139 13966 2870
460.739 64 11084 4043 8008 1342
444.757 46 8533 3858 5766 952
476.725 45 4222 4305 5745 958
671.925 45 15773 4491 6123 1533
665.887 43 8439 4430 5834 1471
614.102 42 11106 4173 5745 1287
482.873 37 13049 3918 4717 852
523.930 35 9477 4036 4610 890
877.035 33 3595 4132 3850 1490
454.714 30 2328 4186 3709 620
892.999 30 1527 4340 3448 1351
655.949 29 7472 4800 3923 854
567.912(NRDY) 25 33818 1570 3825 2016
438.752 25 450 2516 3061 795
681.855 24 1202 4772 3247 837
687.896 23 3657 4620 3212 819
586.950 22 3443 4901 3023 611
649.919 20 4621 4343 2763 648
549.927 19 5164 4626 2586 470
456.863 19 766 4149 2448 399
861.075 19 1030 4472 2314 766
675.180 17 1126 4271 2365 570
593.005(RDRF) 15 2476 2232 3691 1549
598.092 15 1056 3555 2048 505
519.903 15 500 3704 2051 463
637.171 13 1249 3873 1779 457
464.838 12 588 3818 1554 273
908.975 10 893 4427 1170 467
856.993 9 611 4874 1111 386
439.818 (HVIA) 9 26.1 4492 1163 180
522.883 8 199 3311 1117 282
464.863(TGTW) 6 154 2573 1597 414
488.901(HAVY) 5 171 1196 792 452
Amino acid sequence of protein from U. australis (www.uniprot.org)
>tr|H9KVI6|H9KVI6_ULVPE Ferritin OS=Ulva pertusa OX=3120 PE=1 SV=1
AQEVTGMVFQPFSEVQGELSTVTQAPVTDSYARVEYHIECEAAINEQINIEYTISYVYHA
LHSYFARDNVGLPGFAKFFKEASDEEREHAHMLMDYQTKRGGRVELKPLAAPEMEFANDD
KGEALYAMELALSLEKLNFQKLQALQAIADKHKDAALCDFVEGGLLSEQVDAVKEHAVYV
SQLRRVGKGVGVYLLDQELGEEEA
Biological process: Iron storage
Ligand: Iron, metal-binding
>tr|B0YGZ5|B0YGZ5_ULVPE Ribulose bisphosphate carboxylase large chain (Fragment) OS=Ulva pertusa OX=3120 GN=rbcL PE=3 SV=1AFRMTPQPGVPAEEAGAAVAAESSTGTWTTVWTDGLTSLDRYKGRCYDIEPLGEDDQYIAYIAYPLDLFEEGSVTNLFTSIVGNVFGFKALRALRLEDLRIPPAYVKTFQGPPHGIQVERDKLNKYGRGLLGCTIKPKLGLSAKNYGRAVYECLRGGLDFTKDDENVNSQPFMRWRDRFLFVAEAIYKSQSETGEVKGHYLNATAGTCEEMMERGQFAKDLGVPIVMHDYITGGFTANTSLAHFCRASGLLLHIHRAMHAVIDRQRNHGIHFRVLAKILRMSGGDHLHSGTVVGKLXGEREITL
Biological process: Calvin cycle, carbon dioxide fixation, photorespiration, photosynthesis
Ligand: magnesium, metal-binding
>sp|Q6T6H8|LEC_ULVPE Lectin OS=Ulva pertusa OX=3120 GN=UPL1 PE=1 SV=1MINILHVIAGLALASVGVDARQVGVGADVLHAVENTIDSITGVEASHSALEVGGGITNTDNWETFAGLPLTGAIKVNDGNSVVHISAYFPEDRRGKYSYYAATSDELQKTVVFLFVVEDDGLLLQAVKNNAHYPVTNGMYLASHRYYPKDSKYEGMVRLMVHADPAKAVIWEFVTVGGKQYLKVKENRDYTALQIPRHHPRPG
>sp|P56274|PLAS_ULVPE Plastocyanin OS=Ulva pertusa OX=3120 GN=PETE PE=1 SV=1AQIVKLGGDDGSLAFVPSKISVAAGEAIEFVNNAGFPHNIVFDEDAVPAGVDADAISYDDYLNSKGETVVRKLSTPGVYGVYCEPHAGAGMKMTITVQ
Biological process: antiviral defense, electron transport, transport
Ligand: copper, metal-binding
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
1. Please provide the characterization data (1H NMR, 13C NMR, etc) for the synthsized peptides.
2. Figure 5 is not clear. Please provide high-quality picture.
Fine.
Author Response
Response to Reviewers
Reviewer 2
Comments and Suggestions for Authors
- Please provide the characterization data (1H NMR, 13C NMR, etc) for the synthesized peptides.
>Synthetic peptides were composed of L-amino acids, and synthesized by solid phase of three step-partially protected amino acid, the formation of peptide bond, and cleavage of the protecting groups. Finally, the synthetic peptide was purified by RP-HPLC. Conventionally, The purity and identification of synthetic peptides were identified by HPLC and m/z values of mass spectrometry, respectively (Gross and Caprioli, 2005. The encyclopedia of mass spectrometry, Elsevier, NY, pp.165-176).
In peptide research, NMR is usually used to verify specific interactions of the compound with protein and identification of the binding site on the protein (Moy et al., 2001. Analytical Chem, 73: 571-581).
We submit the example of peptide, RDRF, to verify the purity and m/z values of mass spectrum (Figure 1 and Figure 2).
Fig. 1. RP-HPLC chromatogram of peptide, RDRF, for identification of purity.
Fig. 2. Mass spectrum of peptide, RDRF, for identification of mass(m/z).
- Figure 5 is not clear. Please provide high-quality picture.
>Resolution of Figure 5 increased with 600 dpi to show high-quality picture. It submitted as figure file.
Author Response File: Author Response.pdf
