The Production Optimization of a Thermostable Phytase from Bacillus subtilis SP11 Utilizing Mustard Meal as a Substrate
and Shakila Nargis Khan 1,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsOverall, quite an interesting article цшер useful material from a practical point of view. I liked the part related to the use of statistical methods.
One fundamental question to the authors: how can more than 1000 mg of product be obtained from 1 g of raw material (1000 mg)? In addition to violating the law of conservation of matter, the average phosphorus content in grain crops is from 250 to 350 mg per g (depending on the crop), however the authors were able to obtain a yield of the product from 800 to 1900 mg per g of the substrate....
Author Response
Comment 1: One fundamental question to the authors: how can more than 1000 mg of product be obtained from 1 g of raw material (1000 mg)? In addition to violating the law of conservation of matter, the average phosphorus content in grain crops is from 250 to 350 mg per g (depending on the crop), however the authors were able to obtain a yield of the product from 800 to 1900 mg per g of the substrate....
Response 1: Thank you very much for your valuable comment. We mentioned the unit in “microgram”(µg), As thousand microgram converts into one milligram so our results suggest that it can only release approximately 2000 “microgram” or 2 “mg” of phosphate per gram (1000 mg) of crop residues. However, your comment has made us to have a deeper look into the matter and it is noticed that, we mistakenly mentioned the average speed of phosphate release as 1956 µg, but it would be 978 µg. This error has been corrected in the abstract and this is not appeared elsewhere in the manuscript. Thank you very much.
Reviewer 2 Report
Comments and Suggestions for AuthorsThank you very much for the opportunity to review the manuscript entitled: ‘Production Optimisation of a Thermostable Phytase from Bacillus subtilis SP11 Utilising Mustard Meal as a Substrate’.
The article deals with the scientifically and economically interesting topic of phytate degradation. The theoretical introduction provides a sufficiently good introduction to the subject. The bibliography has been selected correctly by the authors. The materials and methods are structured correctly, but there are some minor imperfections in this section that can be easily corrected (all described in detail below). The extensive statistical analysis, which allowed the identification of critical factors influencing phytase production, is particularly noteworthy. The conclusions reflect the results presented.
All the imperfections I have noticed are listed below. After minor revision, the manuscript should be published.
Lines 48-49: Please add units of the global fitase market (tonnes, dollars?).
Line 51: Please provide an appropriate bibliography for this sentence, as I find it somewhat debatable: ‘Also, the long transportation hinders the enzyme's shelf life and activity.’ What does ‘long transportation’ mean (days or years?) Perhaps this sentence could be reworded to better reflect reality?
Lines 53 and 55: Two sentences begin with ‘So.’ I suggest changing this.
Line 56: Perhaps ‘Placket-Burman’ would be better, as it comes from the names?
Line 60? At the beginning of the methodology description, please describe, at least in basic terms (place of origin/producer/harvest season?), the raw materials used as a source of carbon for cultivation (rice bran, wheat bran, soybean meal, corn meal, mustard meal, linseed meal and sesame meal). Later in the manuscript (line 175), the authors also describe the use of Ca2+, Cu2+, Mg2+, Fe2+, Zn2+, Co2+, 175 Mn2+ and Fe3+ ions. As you do not explain in what form these ions were added to the solution, I suggest adding this information to the ‘Materials and methods’ section.
Line 63: Please specify the temperature range for ‘cool conditions’ in brackets.
Line 67: Can the notation of powers in dilutions be corrected (so that the power is written in superscript)?
Line 69: This is a general request for the entire text that the authors standardise the notation of the litre abbreviation, for example, L?
Line 128: Please specify what the percentages mean when determining the inoculum size, was it inoculum volume (v/v %)? If so, please specify the cell concentration (CFU/mL? or g DM/mL?) in the inoculum.
Line 137: This comment applies to the entire text – please ensure that the summary formulas of compounds are written using superscripts and subscripts
Author Response
Comment 1: Lines 48-49: Please add units of the global fitase market (tonnes, dollars?).
Response 1: Thank you very much for your observation. It is in fact in dollars and this has been added in line 48-49.
Comment 2: Line 51: Please provide an appropriate bibliography for this sentence, as I find it somewhat debatable: ‘Also, the long transportation hinders the enzyme's shelf life and activity.’ What does ‘long transportation’ mean (days or years?) Perhaps this sentence could be reworded to better reflect reality?
Response 2: The idea has been generated from our experience gathered from the laboratory inhouse experiments. Generally, during shipping and storage, enzyme loses its shelf-life and activity due to temperature fluctuation, moisture and the normal ageing process. Although there is no specific article for detailed reference, a popular pharma “HUVEPHARMA” (that sells many enzymes) said on their website “The current global situation includes massive delays in sea freight transport, as well as the common logistic problems faced in countries without a solid customs structure. These factors lead to disruption in the availability and quality of feed additives that require control of temperatures. It is particularly important to consider delivery time with regard to the shelf life of the enzyme to ensure optimal enzyme activity. In the case of liquid enzymes, shelf life is typically one year”
https://technicalnewsletters.huvepharma.com/articles/solving-enzyme-application-problems-in-aquaculture-with-huvematic/?utm_source=chatgpt.com
As we failed to find specific reference regarding this, we think it is better to omit the sentence. Therefore, Line 51 is deleted.
Comment 3: Lines 53 and 55: Two sentences begin with ‘So.’ I suggest changing this.
Response 3: Thank you very much for your suggestions. The sentences are corrected according to your suggestion.
Comment 4: Line 56: Perhaps ‘Placket-Burman’ would be better, as it comes from the names?
Response 4: Yes. Thank you for your suggestion. This has been corrected accordingly.
Comment 5: Line 60: At the beginning of the methodology description, please describe, at least in basic terms (place of origin/producer/harvest season?), the raw materials used as a source of carbon for cultivation (rice bran, wheat bran, soybean meal, corn meal, mustard meal, linseed meal and sesame meal). Later in the manuscript (line 175), the authors also describe the use of Ca2+, Cu2+, Mg2+, Fe2+, Zn2+, Co2+, 175 Mn2+ and Fe3+ ions. As you do not explain in what form these ions were added to the solution, I suggest adding this information to the ‘Materials and methods’ section.
Response 5: Thank you for mentioning a very important point.
The raw materials used were bought from a local shop of Dhaka City.
The salts are CaCl2.2H2O, CuSO4.5H2O, MgSO4.7H2O, FeSO4.7H2O, ZnSO4.7H2O. It was a mistake mentioning the Co2+ and Fe3+ as we didn’t test them (can be seen in the graph).
Comment 6: Line 63: Please specify the temperature range for ‘cool conditions’ in brackets.
Response 6: The temperature was -4oC. It is corrected in this line.
Comment 7: Line 67: Can the notation of powers in dilutions be corrected (so that the power is written in superscript)?
Response 7: Thank you for pointing out. It is corrected in the manuscript.
Comment 8: Line 69: This is a general request for the entire text that the authors standardise the notation of the litre abbreviation, for example, L?
Response 8: All the small letters (l) have been changed to capital letter “L” for the notation of Liter
Comment 9: Line 128: Please specify what the percentages mean when determining the inoculum size, was it inoculum volume (v/v %)? If so, please specify the cell concentration (CFU/mL? or g DM/mL?) in the inoculum.
Response 9: Yes, the inoculum sizes were in-fact in (% v/v) and the colony counts were not done. We rather used overnight grown culture (20 hours). The information has been added.
Comment 10: Line 137: This comment applies to the entire text – please ensure that the summary formulas of compounds are written using superscripts and subscripts
Response 10: Thank you very much for your critical observations. This is corrected in the manuscript.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThank you for making corrections to the manuscript. In my opinion, the authors have made significant improvements to the text, which makes it suitable for publication.
