Combination of Mechanical/Physical Pretreatments with Trypsin or Pancreatin on Arthrospira platensis Protein Degradation
Round 1
Reviewer 1 Report
The present manuscript “Combination of mechanical/physical pretreatments with trypsin 2 or pancreatin on Arthrospira platensis protein degradation”, treats with interest the effect of mechanical/ physical and enzymatic pretreatments on the A. platensis protein hydrolysis. We can observe the seriousness with which the authors treated the experiment. However, a number of mentions should be made:
Comment
In the article the approach used to evaluate the protein degradation was assessed through SDS-PAGE gels and the evaluation of coefficient of protein degradation (CPD) methods.
I think that you should use other chromatographic method like High-performance liquid chromatography (HPLC) or LC-MS chromatochraphy to identify and quantify aminoacid after protein degradation.
Also I think it’s crucial to determine the amino acid after protein hydrolysis to compare the composition of all the fraction after the mechanical and enzymatic treatment.
· Lines 95, 96 and 97 were confused in the table 1, please rectify it.
· Authors should add all the unites in table 1.
· Authors should give the mean values and standard deviations in table 1.
· Line 147 « 3.1. Effect of pretreatments and enzymes on A. platensis protein concentration » Authors should change this title as you have mechanical/physical pretreatments and other enzymatic treatment.
· Authors should give all the standard deviations in table 2.
· Line 180, there is a mistake, title 3.2 not 3.1
· Lines 195 -204 were confused in table 3 (The same problem with Table 4).
· Authors should give the yield of protein extraction by the different treatments used.
Author Response
Reviewer 1
- The present manuscript “Combination of mechanical/physical pretreatments with trypsin 2 or pancreatin on Arthrospira platensis protein degradation”, treats with interest the effect of mechanical/ physical and enzymatic pretreatments on the A. platensis protein hydrolysis. We can observe the seriousness with which the authors treated the experiment. However, a number of mentions should be made.
Reply: Thank you for your comments; we tried to address all of them, as described below.
- In the article the approach used to evaluate the protein degradation was assessed through SDS-PAGE gels and the evaluation of coefficient of protein degradation (CPD) methods. I think that you should use other chromatographic method like High-performance liquid chromatography (HPLC) or LC-MS chromatography to identify and quantify aminoacid after protein degradation. Also I think it’s crucial to determine the amino acid after protein hydrolysis to compare the composition of all the fraction after the mechanical and enzymatic treatment.
Reply: We agree that it would be interesting to identify and quantify the amino acids after protein degradation, but we did not perform it because the only difference in total peptides measured in OPA assay was a decrease of its concentration with extrusion (please, see Table 2 now added to the manuscript between lines 163 and 167, page 5). However, in forthcoming studies, we aim to explore the increase of total peptides and determine which amino acids were increased with the enzymes, mostly pancreatin, after using pre-treatments.
- Lines 95, 96 and 97 were confused in the table 1, please rectify it.
Reply: It is now rectified.
- Authors should add all the units in table 1.
Reply: All the units are now added in table 1.
- Authors should give the mean values and standard deviations in table 1.
Reply: We only have information about the mean values of A. platensis chemical composition (and not about standard deviations). These values were provided by Allmicroalgae company, as now specified between lines 88 and 89, page 2.
- Line 147 « 3.1. Effect of pretreatments and enzymes on A. platensis protein concentration » Authors should change this title as you have mechanical/physical pretreatments and other enzymatic treatment.
Reply: The title was changed, accordingly.
- Authors should give all the standard deviations in table 2.
Reply: We present the standard error of means, instead of standard deviations, in table 2 (now in table 3) because there was homogeneity of variance for the majority of parameters after testing with Levene´s test (line 147, page 4).
- Line 180, there is a mistake, title 3.2 not 3.1
Reply: The titles in Results section were modified, accordingly, together with the addition of a new subsection between lines 151 and 161, page 4.
- Lines 195 -204 were confused in table 3 (The same problem with Table 4).
Reply: The lines for Tables 3 and 4 were rectified.
- Authors should give the yield of protein extraction by the different treatments used.
Reply: The yield of protein extraction by the different treatments used is now specified between lines 198 and 202, page 6. The formula used to calculate it is also shown between lines 128 and 133, page 3. We did not present it in a Table because the main purpose of the present work was to evaluate the effect of treatments on algal protein degradation and not on protein extraction.
Reviewer 2 Report
Good work. The study would help in choosing the best methods to increase the bioavailability of algal feed for ruminants.
Revise the manuscript asper the comments given in the attached document
Comments for author File: 
Comments.pdf
Author Response
Reviewer 2
- Good work. The study would help in choosing the best methods to increase the bioavailability of algal feed for ruminants. Revise the manuscript asper the comments given in the attached document
Reply: Thank you for your comments; we tried to address all of them, as described below.
- Comment 1: The material and methods section must be modified.
- Line item No 75-76: Freeze drying method needs to be described clearly with sample size.
Reply: The freeze drying conditions, including sample size, are now more clearly described between lines 76 and 78, page 2.
- Line item No: 77: Heating method needs to be described clearly with sample size.
Reply: The heating method is now described.
- Line item No: 82-87: In the extrusion procedure, what was the % solids taken for treatment and what was the moisture /solids level achieved after drying?
Reply: The % solids taken for extrusion treatment was 94.1%, as added in line 107, page 3. The moisture /solids level achieved after drying was not specified by the company, although it is probably close to 0%.
- Basis for selecting the treatment conditions should be mentioned.
Reply: That aspect is now mentioned between lines 72 and 82, page 2.
- Comment 2: Table1 (Line item No: 93-98): References needs to be provided for all the methods followed to determine the chemical composition of Arthrospira platensis. The total chemical composition mentioned in the tables is exceeding 100% (close to 111.24%) and, the mentioned ash content (14.9%) is very high. Check the values once again and revise accordingly.
Reply: The references and a brief description of methods are now added between lines 114 and 120, page 3. Table 1 was modified to separate nutritional composition and pigment composition and, as the reviewer can verify, the sum of all the ingredients that are included in the nutritional composition of A. platensis is 100%. The values of ash were checked and are correct. Such high values are probably attributed to the culturing conditions.
- Comment 3: Since the enzymatic treatment was done with trypsin, the generic term pepsin should be replaced with trypsin throughout the manuscript.
Reply: Pepsin was replaced by trypsin throughout the manuscript (lines 17, 50-51 and 64).
- Comment4: The protein marker is not labelled in any of the figures. The concentration of total protein and protein fractions (mg/mL) of various pre-treatment are quantified based on SDS-PAGE. The cross validation should have been (Total protein) done with any of the standard methods like Bradford /Lowry.
Reply: The protein marker is now labelled in the figures. We did a cross validation for total protein using Bradford, which is now added between lines 180 to 195, page 4 and 5 (Material and Methods section) and between lines 205 and 215, page 5 and in Table 2 (Results section).
- Comment5: The bands for Extrusion + trypsin (Figure 2 b, ET + TP; Control) and Extrusion + pancreatin (Figure 3 a, ET + PC; Control) treatments looks almost similar. However, it has been discussed that, increased 18-26 kDa protein for Extrusion + pancreatin treatment. The results are nether visible in the gel nor in the table 2. Justify and revise accordingly.
Reply: We revised the results and, indeed, both the bands and the CPD values for Extrusion + trypsin and Extrusion + pancreatin are similar. The differences in the results are mainly due to a higher variability between replicates, which is reflected in SEM values (5.785 and 2.509 for 18-26 kDa protein fraction with pre-treatments combined with trypsin versus pancreatin). This aspect is now added 355 and 357, page 12.
- Comment 6: Feed moisture, and rotational speed of the screw are important to achieve higher shear force and higher cell lysis in the extrusion and these parameters are not mentioned in the article. What was the basis for selecting the followed conditions and mention the reason for not optimizing parameters.
Reply: These aspects are now added between lines 79 and 80, page 2, and 107 and 110, page 3. The basis for selecting the followed conditions was their adaptability to A. platensis biomass and to scale up the process to an industrial level. All the details about the procedure given by the company that performed alga extrusion are mentioned and more detailed in Material and Methods section.
- Comment7: There is a significant difference in the total protein obtained for each pre-treatment. To see the effect of enzyme, the enzyme dose should have been fixed on the available protein concertation in the supernatant, instead of sample volume (20 μg/mL). The higher hydrolysis observed for pancreatin + extrusion treatment could be due to the higher enzyme and lower protein. These results may mislead the selection of pre-treatment. Bring these points in the discussion and revise accordingly.
Reply: Thank you for your observation. That aspect is now considered between lines 420 and 423, page 14.
Round 2
Reviewer 1 Report
Comments and Suggestions of reviewer
The present manuscript “Combination of mechanical/physical pretreatments with trypsin 2 or pancreatin on Arthrospira platensis protein degradation”, treats with interest the effect of mechanical/ physical and enzymatic pretreatments on the A. platensis protein hydrolysis.
We can observe the seriousness with which the authors treated the experiment. I think that the manuscript has been sufficiently improved to warrant publication in Agriculture. My recommendation is to accept the manuscript in the present form.
