Conversion of Fishery Waste to Proteases by Streptomyces speibonae and Their Application in Antioxidant Preparation
, Chien Thang Doan 2, Van Bon Nguyen 3, Anh Dzung Nguyen 3 and San-Lang Wang 4,5,*Round 1
Reviewer 1 Report
The introduction does not explain why the effects of the various chemical was assessed as part of the study, nor why radical scavenging activity was examined etc etc
Much of the English language is grammatically incorrect or difficult to understand. Take for example this mega-sentence that is in the Abstract:
"Furthermore, the antioxidant activity of the hydrolysates obtained from the hydrolysis of the head, viscera, and meat of tuna fish; the head, viscera, and meat of tilapia fish; the head, meat, and shell of shrimp; squid pen; crab shell; and soybean using papain, bromelain, and S. speibonae TKU048 crude-enzyme cocktail as the catalysts was also assessed and, interestingly, the crude-enzyme cocktail of S.speibonae TKU048 could efficiently enhance the 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals scavenging activities of all tested proteinaceous materials.
The overall focus of the manuscript is unclear - whether it is to assess the protein digestion capabilities of proteases from one Streptomyces strain, or assess the ideal pH and and temperature range for this strain, or to assess its potential to break down various seafood waste. A bit more structure in the manuscript would be very helpful for the reader.
The introduction makes various claims about the extraction of bioactives for human use from fish waste - there are some significant issues here around the types of processing conditions and materials that such bioactives destined for human consumption normally need to acheive - e.g., GMP standards - most fish waste would not achieve such standards from the outset.
The first sentence of the introduction appears to state that the inappropriate disposal of fish waste creates detrimental health issues, but it also creates significant environmental issues.
2.1 Materilas - spelt incorrectly
2.5 - what were the temperature and pH series that were tested?
2.7 - all of these substrates would have different concentrations of protein available for enzymes - so how is it possible to assess substrate "specificity" by this methodology?
3.1 - first two sentences are more methods presented in the results section - i.e., they are in the wrong place in the mansucript.
The quality of the gel images (Fig. 3) are poor and if these were in my lab I would be rerunning them, e.g., Fig 3b & c are very poor.
Figures 6 & 7 are microscopic and very difficult to view.
The conclusions of the paper don't cover the full gambit of the results of the study.
The supplementary materials are appropriate.
Overall, I found this manuscript poorly structured and difficult to follow. Most importantly the rationale for the various research elements is not well articulated at all in the introduction or in the conclusions. This is not helped by the relatively poor quality of English language. The overall impression of the study is that the availability of the Streptomyces led to opportunistic laboratory assessment of various protease assays, without any well structure rationale or progression from undertaking these laboratory assays.
Author Response
Reviewers' comments:
Reviewer #1:
The introduction does not explain why the effects of the various chemical was assessed as part of the study, nor why radical scavenging activity was examined, etc etc
Reply: Thank you very much for suggestions. More information was added in introduction section.
Much of the English language is grammatically incorrect or difficult to understand. Take for example this mega-sentence that is in the Abstract:
"Furthermore, the antioxidant activity of the hydrolysates obtained from the hydrolysis of the head, viscera, and meat of tuna fish; the head, viscera, and meat of tilapia fish; the head, meat, and shell of shrimp; squid pen; crab shell; and soybean using papain, bromelain, and S. speibonae TKU048 crude-enzyme cocktail as the catalysts was also assessed and, interestingly, the crude-enzyme cocktail of S.speibonae TKU048 could efficiently enhance the 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals scavenging activities of all tested proteinaceous materials.
Reply: The revised manuscript has sent for English proofreading (second time).
The overall focus of the manuscript is unclear - whether it is to assess the protein digestion capabilities of proteases from one Streptomyces strain, or assess the ideal pH and and temperature range for this strain, or to assess its potential to break down various seafood waste. A bit more structure in the manuscript would be very helpful for the reader.
Reply: The manuscript was revised as comments.
The introduction makes various claims about the extraction of bioactives for human use from fish waste - there are some significant issues here around the types of processing conditions and materials that such bioactives destined for human consumption normally need to acheive - e.g., GMP standards - most fish waste would not achieve such standards from the outset.
The first sentence of the introduction appears to state that the inappropriate disposal of fish waste creates detrimental health issues, but it also creates significant environmental issues.
Reply: Introduction section was revised and more information was added.
2.1 Materilas - spelt incorrectly
Reply: It was corrected.
2.5 - what were the temperature and pH series that were tested?
Reply: In section 2.5, the temperature series were from 30 to 100oC (30 oC, 40oC. 50 oC, 60 oC, 70 oC, 80 oC, 90 oC and 100 oC); and range of pH values were 3.6-10.6 (pH = 3.6; 4.0; 5.0; 5.6; 5.8; 6.0; 7.0; 7.4; 7.2; 8.0; 9.0; 9.2; 10; and 10.6).
2.7 - all of these substrates would have different concentrations of protein available for enzymes - so how is it possible to assess substrate "specificity" by this methodology?
Reply: Thank you very much for suggestions. All of these substrates in the substrate specificity section (2.7 and 3.4) were deleted.
3.1 - first two sentences are more methods presented in the results section - i.e., they are in the wrong place in the mansucript.
Reply: They were revised.
The quality of the gel images (Fig. 3) are poor and if these were in my lab I would be rerunning them, e.g., Fig 3b & c are very poor.
Reply: Figure 3b and 3c were updated a new one.
Figures 6 & 7 are microscopic and very difficult to view.
Reply: Figure 6 and Figure 7 were adjusted.
The conclusions of the paper don't cover the full gambit of the results of the study.
Reply: Many thanks for comments. The conclusions section was revised.
One more time, we would like to pay our deeply thanks for your careful and valuable comments for enhancement of the quality of the manuscript.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The article was well written and scientifically constructed.
The entire article is about the production of proteases using Streptomyces speibonae.
But attached reports say that Streptomyces speibonae are chitin degrading and used for production of chitinase;
https://www.mdpi.com/2073-4360/13/18/3048/htm
https://www.mdpi.com/2073-4360/11/10/1600
Need justification with proper references and scientific input.
Author Response
Reviewers' comments:
Reviewer #2:
The article was well written and scientifically constructed.
The entire article is about the production of proteases using Streptomyces speibonae.
But attached reports say that Streptomyces speibonae are chitin degrading and used for production of chitinase;
https://www.mdpi.com/2073-4360/13/18/3048/htm
https://www.mdpi.com/2073-4360/11/10/1600
Need justification with proper references and scientific input.
Reply: Thank you very much for suggestions. There are two references, including “Production of Thermophilic Chitinase by Paenibacillus sp. TKU052 by Bioprocessing of Chitinous Fishery Wastes and Its Application in N-acetyl-D-glucosamine Production, https://www.mdpi.com/2073-4360/13/18/3048/htm”, and “An Exochitinase with N-Acetyl-β-Glucosaminidase-Like Activity from Shrimp Head Conversion by Streptomyces speibonae and Its Application in Hydrolyzing β-Chitin Powder to Produce N-Acetyl-d-Glucosamine, https://www.mdpi.com/2073-4360/11/10/1600”.
Above references reported that Streptomyces speibonae TKU048 has been used to utilize wastes for the production of chitinase. However, S. speibonae TKU048 expresses proteolytic activity, that still not report. More information was added in manuscript.
One more time, we would like to pay our deeply thanks for your careful and valuable comments for enhancement of the quality of the manuscript.
Author Response File: Author Response.docx
Reviewer 3 Report
The present work reports the recovery of fishery wastes for protease production. The article is interesting and valuable for the industry.
It is well written and organized, although minor issues should be assessed.
1. Please check that the species names are all in italics throughout the text.
2. In Figure 5, the relative activity % goes to 800%? What is the control? Is this % relative to this control? Please clarify.
3. In Table 2 the relative activity is related to what? Please clarify and add that information to the caption.
4. In Figure 6, please indicate what was used as the control.
5. What was the yield of recovery of the proteases? Is this method economically viable? What quantity of fish waste can be recovered using this method? How easy would this method be applied in the industry?
Author Response
Reviewers' comments:
Reviewer #3:
The present work reports the recovery of fishery wastes for protease production. The article is interesting and valuable for the industry.
It is well written and organized, although minor issues should be assessed.
- Please check that the species names are all in italics throughout the text.
Reply: Thank you very much for your comments. They were revised.
- In Figure 5, the relative activity % goes to 800%? What is the control? Is this % relative to this control? Please clarify.
Reply: In Figure 5, the addition of SDS could greatly enhance the activity of the TKU048 crude enzyme cocktail (738.80%). The protease activity without added chemicals (control) was defined as 100%. More information was added.
- In Table 2 the relative activity is related to what? Please clarify and add that information to the caption.
Reply: In Table 2, the protease activity toward casein was set as 100% relative activity. More information was added.
- In Figure 6, please indicate what was used as the control.
Reply: In Figure 6, a reaction solution consisting of 1% (w/v) of each proteinaceous material, 103±7.79 U/mL proteolytic activity of each enzyme (bromelain, papain, and crude-enzyme cocktails from B. licheniformis TKU004, P. macerans TKU029, P. mucilaginosus TKU032, Paenibacillus sp. TKU042, S. thermocarboxydus TKU045, and S. speibonae TKU048) was incubated at 60oC for 15 h.
Control was a reaction solution consisting of 1% (w/v) of each proteinaceous material in phosphate buffer (100 mM, pH = 5.8). Its mean enzyme was replaced by phosphate buffer.
- What was the yield of recovery of the proteases?
Reply: After protein precipitation by ammonium sulfate, proteases activity yield was 54.3%.
Is this method economically viable?
Reply: In this study, tuna heads (1% w/v), a low cost source, was only used as the carbon/nitrogen nutrition for culturing bacteria, could promise production inexpensive proteolytic enzymes via fermentation with S. speibonae TKU048.
What quantity of fish waste can be recovered using this method?
Reply: Thank you very much for your kind consideration.
We attended to determine the recovery quantity of tuna heads by measuring their dry cell weight after fermentation by S. speibonae TKU048. However, our study used tuna head powder as the sole C/N source to produce protenolytic enzymes. Both of tuna head powder and Streptomyces biomass may not been separated (settled down together) even though different centrifugation speeds have been tried. Namely, it is difficult to perform a method to exactly determine the recovery quantity of tuna heads.
How easy would this method be applied in the industry?
Reply: Although this study was just performed in the laboratory, we hope it could be applied in the industry with the wide scale. According our knowledge, abundant and readily available source of tuna head could be reused and fermentation method has been used in many industry fields. Maybe they will be advantages when applied in the industry.
One more time, we would like to pay our deeply thanks for your careful and valuable comments for enhancement of the quality of the manuscript.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The article is recommended to be accepted in the present form.
Author Response
Detailed response to academic editor’s comments
Manuscript ID: fishes-1719409
The Original Title: Conversion of Tuna Head Waste to Proteases by Streptomyces speibonae and Their Application in Antioxidant Preparation
Dear Academic Editor,
Thank you for your time and effort, as well as your excellent suggestions for refining the readability and impact of the manuscript. We have gone through all the suggestions cautiously and made the revisions accordingly; and all amended parts have been typed in blue in the revised manuscript. Finally, we would like to express our thanks to your comments and suggestions again. You certainly have served to improve the quality of this paper.
Looking forward to hearing from you.
Yours Sincerely,
San-Lang Wang
Professor, Life Sciences Development Center/Department of Chemistry, Tamkang University, Taiwan
Editor’s Comments:
The manuscript "Conversion of Tuna Head Waste to Proteases by Streptomyces speibonae and Their Application in Antioxidant Preparation" can be accepted for publication, although there are still some inaccuracies that need to be corrected. They are reported herein
Line 197 a previous study instead of an earlier study
Reply: It was revised.
Line 217 was instead of were
Reply: It was revised.
Line 226 different concentration instead of differentconcentration
Reply: It was revised.
Line 269 erase “above”
Reply: It was revised.
lines 285-287 please substitute with "Among several temperatures screened (30-100 oC), the crude-enzyme cocktail of S. speibonae TKU048 was found to be optimally active at 70 oC . The activity slightly decreased to 88.69±2.71% at 80 oC and 70.94±2.52% at 90 oC."
Reply: It was revised.
Line 288 substitute when with since
Reply: It was revised.
Line 292 previous instead of earlier
Reply: It was revised.
Fig. S1 erase which
Reply: It was revised.
Fig. S2 containing instead of contained
Reply: It was revised.
One more time, we would like to pay our deeply thanks for your careful and valuable comments for enhancement of the quality of the manuscript.
Author Response File: Author Response.docx
