Combined Effects of Defatted Hydrolyzed Collagen from Salmon Skin and Vitamin C on Proliferation and Migration of Human Fibroblast Cell

Round 1

Reviewer 1 Report

Comments

1. In the introduction section, authors should discuss in more detail why collagen hydrolyzate was chosen in the study, and not native high-molecular collagen. This is important, and it has been discussed in the scientific literature of recent years.

2. You should change the phrase: After thawing, cut skins were pretreated with 0.05 mol/L NaOH (1:10, w/v) and subsequently washed using running water as detailed by Nilsuwan et al. [9]. The resulting skins were mixed with 0.05 mol/L citric acid (1:10, w/v) for 1 h, and subsequently neutralized by washing with running water." Neutralization occurs as a result of adding acid, not water.

3. In the paragraph 2.3.” Isopropanol present in HC pellet was removed with the aid of blowing air at 25 °C for 30 min. How was the absence of isopropanol controlled? Why exactly 30 minutes, and not more and not less?

4. The main aim of the work is formulated in the introduction: Thus, the purposes of this study were

1) to characterize molecular weight distribution and amino acids composition of HC and

 2) to elucidate the combined effects between HC and Vit C on human fibroblast cell proliferation and wound healing.

The first point of the aim of the work is not reflected in the Conclusions section of the research.

5. In Figure 1, you should remove the values ​​"0.0 and "0.00" on the x-axis. Two values are not indicated at the intersection of the coordinate axes.

Author Response

Response to reviewer 1

Comments and Suggestions for Authors

*********Thank you for the invaluable comments and suggestions. All queries have been responded and the corrections have been made as highlighted in green.

Comments

1. In the introduction section, authors should discuss in more detail why collagen hydrolysate was chosen in the study, and not native high-molecular collagen. This is important, and it has been discussed in the scientific literature of recent years.

*********The additional information has been provided in introduction. Please see line 42-45.

2. You should change the phrase: After thawing, cut skins were pretreated with 0.05 mol/L NaOH (1:10, w/v) and subsequently washed using running water as detailed by Nilsuwan et al. [9]. The resulting skins were mixed with 0.05 mol/L citric acid (1:10, w/v) for 1 h, and subsequently neutralized by washing with running water." Neutralization occurs as a result of adding acid, not water.

*********For pretreatment, there were 2 major processes. The first step was pretreatment of skin with alkaline solution to remove non-collagenous protein and subsequently washed with running water until a neutral pH was gained. The second step was swelling process, in which the alkali-treated skin from the first step was soaked in citric acid solution. Thereafter, the swollen skin was washed with running water again to remove acid distributed in skin. Based on the processes used, washing process resulted in the removal of alkai or acid, leading to the neutral of pH of skins. We tried to avoid adding acidic/alkaline solution in adjusting pH since salt might be generated.

To avoid confusion, the methodology has been rewritten for clarity. Please see line 87-88, 90-91.

Thank you for invaluable comment.

3. In the paragraph 2.3.” Isopropanol present in HC pellet was removed with the aid of blowing air at 25 °C for 30 min. How was the absence of isopropanol controlled? Why exactly 30 minutes, and not more and not less?

*********The air blowing condition used in the present study was based on our previous work. HC pellet was spread on the tray as thin layer and blown with the aid of air at 25°C for 30 ​min until drying HC was obtained. The absence of isopropanol was determined by SPME GC-MS, in which the isopropanol was not found in the resulting HC pellet (Nilsuwan et al., 2021).

Drying more than 30 min was not necessary (as explained above for drying time) and higher energy was consumed. However, the drying with time less than 30 min was not enough to remove all the isopropanol. Thus, the appropriate time used was 30 min.

Reference

Nilsuwan, K.; Chantakun, K.; Chotphruethipong, L.; Benjakul, S. Development of hydrolysis and defatting processes for production of lowered fishy odor hydrolyzed collagen from fatty skin of sockeye Salmon (Oncorhynchus nerka). Foods. 2021, 10, 2257.

4. The main aim of the work is formulated in the introduction: Thus, the purposes of this study were

1) to characterize molecular weight distribution and amino acids composition of HC and

 2) to elucidate the combined effects between HC and Vit C on human fibroblast cell proliferation and wound healing.

The first point of the aim of the work is not reflected in the Conclusions section of the research.

*********We do agree with reviewer’s comment. The details associated with the first objective (size distribution and amino acid composition) have been added in ‘conclusions’. Please see line 412-413. Thank you for invaluable comment.

5. In Figure 1, you should remove the values ​​"0.0 and "0.00" on the x-axis. Two values are not indicated at the intersection of the coordinate axes.

*********The values ​​"0.0 and "0.00" are absorbance of peptides or proteins with aromatic rings of HC on the y-axis. For data on the x-axis, it is fraction no. of peptides collected. The data on the x-axis and the y-axis are different Each axis start from zero. The absorbance of peptides and proteins was reported in Y-axis on the left-hand side and right-hand side, respectively. Their values started from values of 0.0 and 0.00, respectively.

Fractions collected were reported in the x-axis, which started from the value of 0.

To provide the better understanding for readers, those values are kept in the present form.

Several studies also presented their data in the similar way (Li et al., 2022; Thamnarathip et al., 2016; Sun et al., 2021).

References

Li, L., Tian, Y., Zhang, S., Feng, Y., Wang, H., Cheng, X., Ma, Y., Zhang, R. and Wang, C., 2022. Regulating effect of mung bean peptide on prediabetic mice induced by high-fat diet based on gut microbiota. Frontiers in Nutrition, p.1074.

Thamnarathip, P., Jangchud, K., Nitisinprasert, S. and Vardhanabhuti, B., 2016. Identification of peptide molecular weight from rice bran protein hydrolysate with high antioxidant activity. Journal of Cereal Science, 69, pp.329-335.

Sun, X., Ruan, S., Zhuang, Y. and Sun, L., 2021. Anti-osteoporosis effect and purification of peptides with high calcium-binding capacity from walnut protein hydrolysates. Food & Function, 12(18), pp.8454-8466.

Reviewer 2 Report

The manuscript entitled "Combined effects of defatted hydrolyzed collagen from salmon skin and vitamin C on proliferation and migration of human fibroblast cell" by JLalita Chotphruethipong and colleagues investigate the combined effects of vitamin C (Vit C) and fish hydrolyzed collagen (HC) on proliferation and migration of human dermal fibroblast (HDF). The results indicated that the use of HC and Vit C could stimulate human dermal fibroblast proliferation, and the effectiveness was increased when HC and Vit C were used in combination. HC+Vit C could enhance proliferation and migration of HDF cells effectively. HC, Vit C and HC+Vit C augmented expression of p-Akt, p-FAK and p-p38 proteins associated with cell proliferation and migration. It can be published after making minor revision. Some suggestions have been listed as follow:

1. Line 117: Please refine the cell culture process.

2. Line 151: Please indicate the brand and item number of the antibody used.

3. Line 161: "p" should be italic.

Author Response

Response to reviewer 2

Comments and Suggestions for Authors

The manuscript entitled "Combined effects of defatted hydrolyzed collagen from salmon skin and vitamin C on proliferation and migration of human fibroblast cell" by Lalita Chotphruethipong and colleagues investigate the combined effects of vitamin C (Vit C) and fish hydrolyzed collagen (HC) on proliferation and migration of human dermal fibroblast (HDF). The results indicated that the use of HC and Vit C could stimulate human dermal fibroblast proliferation, and the effectiveness was increased when HC and Vit C were used in combination. HC+Vit C could enhance proliferation and migration of HDF cells effectively. HC, Vit C and HC+Vit C augmented expression of p-Akt, p-FAK and p-p38 proteins associated with cell proliferation and migration. It can be published after making minor revision. Some suggestions have been listed as follow:

*********Thank you for understanding our work. All queries have been responded and the corrections have been made and highlighted in yellow.

1. Line 117: Please refine the cell culture process.

*********Cell culture process has been provided. Please see line 119-123. Thank you for the insightful comment.

2. Line 151: Please indicate the brand and item number of the antibody used.

*********The information has been given in text as per the reviewer’s suggestion (please see line 155-157 and 160).

3. Line 161: "p" should be italic.

********* "p" has been presented as ‘italic’ following the reviewer’s suggestion. Please see line 167. Thank you for comment.