Comparison of Analytical Methods for Determining Methylesterification and Acetylation of Pectin
Round 1
Reviewer 1 Report
To Authors:
In the manuscript of Y. Yu, et al. "Comparison of analytical methods for determining methylesterification and acetylation of pectin", a comparative study on determination of methyl and acetyl ester groups in a single sample of citrus pectin by three different methods (titration, FT-IR, and HPLC) is reported. Three values of degree of methylation (DM) were obtained: 47.0%, 44.0%, and 48.0 %, respectively.
Referee’s notes. General.
- The novelty of this study looks doubtful. Indeed, determinations of DM and DAc in pectins are protocolized (for example: Melton L.D.; Smith, B.G., Determining the degree of methylation and acetylation of pectin, Current Protocols in Food Analytical Chemistry, 2001, 00 (1), E3.4.1—E3.4.6, doi: 10.1002/0471142913.fae0304s00, or, earlier standard protocol: Food Chemical Codex, 2nd Ed., Natl. Res. Council, Washington DC, 1972, p. 580). Comparative studies concerning determination of DM of pectin were done previously, for example: O. Shulga, et al., Method of pectin esterification determination degree by titrated acidity, Ukrainian Food J., 2020, 9, 383—393 (methods: NMR, FT-IR, titration); Luzio, G.A.; Cameron, R.G.; Determination of degree of methylation of food pectins by chromatography. J. Sci. Food Agric., 2013, 93, 2463—2469. (methods: IEC, AEC, colorimetric, conductivity). See also Meller-Maatsch, J. et.al, J. Agric. Food Chem., 2014, 62, 9081—9087). What is new in the submitted research? — It must be stressed, if it is really done.
- There is no statistics. Reproducibility of the data is not discussed: how the data are repeatable (at least, date-to-date reproducibility should be checked and confidence intervals should be given).
- Lines 182—195, Subsection 3.3. How the areas of peaks A(1630) and A(1740) were measured? Please, show it in Figure 2. The choice of zero and separation lines (i.e., integration procedure) is important.
- Line 272—273. “…neither the titration nor FT-IR methods can be used to determine DAc content.” This is not so evident: in IR spectrum of native pectin, COOMe and OAc ester carbonyl bands coincide to each other, so the value of DM determined by FT-IR should be overestimated due to acetyl groups. The reported data (44 %) are lower than other ones. Why? See note 3 above.
Minor. 1. “…Hollow fiber membrane…” Why “hollow” is capitalized? — It is not a brand name.
- Line 150. “”m” represents the weight average molecular weight…”: what does it mean? Some words are excessive.
- Lines 114—128. How the samples for FT-IR were prepared: as pectin (neat) thin pellets or KBr tablets? “Previously described”: — where? A reference should be given.
- Line 282, Table 2, column “HPLC”. “A little complex” — maybe, “complicated” or “laborious”? Below: HOAc, not HAc.
- Line 326. Ref. [7]. Should be: Maurique, G.D.; Lajolo, F.M.
- Line 366, Ref. [21]. Should be Grasdalen, H.; Barkoy, O.E.; Larsen, B.
The manuscript can be published after major revision and repeated review as a regular paper.
Comments for author File: 
Comments.pdf
Author Response
Please see the attachment.
Author Response File: 
Author Response.docx
Reviewer 2 Report
This manuscript is pretty informative and quite well written. I only have a few questions and remarks.
RGI usually acetylated and it is present to a small extent in citrus pectin.
Need a reference for the phenyl-3-methyl-5-pyrazolone method
Line 94 What does methanol sulfate “replaced” mean? Did the pectin really dissolve in the methanolic HCl?
Line 116 We need a reference for “previously described”.
The equation for the DM and DAc don’t make sense to me. Why is’ “m” represents the weight average molecular weight of HG pectin to be determined’, included? You don’t determine this, nor is it relevant to the determination.
I do not understand lines 218 to 223. Did you not remove the polymer before the HPLC analysis? Why refer to a C18 column pectin retention?
In he HPLC profiles do you have any idea what the peak at 6.55 minutes might be?
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
Review pectins
This paper concerns an important problem concerning the characterisation of pectins. It means the determination of the degrees of methylation DM and of acetylation DAc.
The paper is clearly written and discussed. It may be usefull to characterise pectins.
I would like to get more informations as follows :
-in relation II, which is the meaning of the number 176 ?? m is not determined if « the “m” represents the weight average molecular weight of HG pectin to be determined » ? How to determine DM and Dac ?? There is no molecular weight determination in this paper.
-please determine HG the first time it is used.
To conclude, I estimate that this pape ris able to be published after the correction and answer tomy questions.
Comments for author File: Comments.pdf
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 4 Report
The presented study compares analytical methods for the determination of esterification of pectin. It is acceptable for publishing after minor revisions if several issues are considered and modified carefully by the authors. Moreover, in some parts of this article, several redundancies of terms make it heavy to read.
There are specific problems as follows:
Introduction:
Page 1, line 29 = Please change “methanol” with “methyl group” for DM description as made for DAc description.
Page 1, line 32 = The authors report that DM and DAc affect biological activities, repeating it in lines 36-37. Please correct
Material and methods:
Paragraph 2.3 = Please add the volumes used for acid and basic hydrolysis and the pH of these reactions. Furthermore, the authors should define HG acronym.
In line 95, they report that solution has been stored for four days. Has it been stirred? Please correct in case.
Paragraph 2.4 = Please specify that V1 and V2 values are listed in Table 1
Page 3, line 134 = Please change “correction” with “corrected”.
Page 4, lines 128-129 = Please specify the number of scans.
Page 4, line 130 = Please state the concentration of NaOH solution.
Results and discussion:
Paragraph 3.2 = Do CP and CP-HM-HG contain acetyl groups? This latter should not contain them as esterification has been conducted in MeOH/H2SO4, not for alkali saponification.
Page 6, line 220 = Please change O-6 with carboxyl function. Moreover, the oxygen atoms should be in italic font.
Page 7, line 242 = Please change “simply” and “conveniently” respectively with “simple” and “convenient”
Page 8, lines 271-278 = Please report the data in histograms.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
- The novelty of the study is not approved. “Few literature” — that’s not true: there are a lot of publications concerning DM and DAc of pectins of various origin with critical discussion of advantages and limitations of the applied methods (above, I have presented a small no. of materials). As for HPLC method, authors’ modifications (in comparison to S. Levigne et al., Ref. [17]) are as follows: 1. 0.2 M sulfuric acid was used for neutralization of NaOH instead of Maxi-clean IC-H device (Alltech). 2. “we did not remove pectin before HPLC analysis, as pectin will not be reserved on C18 column”. As for point 1, this is not something new and advantageous : G.A.Luzio & R.G. Cameron (J. Sci. Food Agric., 2013, 93, 2463) used 0.1 M nitric acid, G. Limbert et al. used 4 M HCl to stop de-esterification of pectin (Carbohydr. Res., 2000, 327, 293). Strong, mineral acid is an evident reagent, and counter anion is not sufficient. Point 2. The last suggestion is not so evident. Where pectin is, if it “not be reserved”? I guess, it was adsorbed on a pre-column. Is it a real advantage?
- Points 2 & 3: accepted.
- Point 4: Just what I want to ask again: why? Really, the difference looks low but (statistically) significant (FT-IR: 44.0 +/- 0.54 % in comparison to titration 47.0 +/- 0.63 % and HPLC 48.0 +/- 0.43 %). For critical analysis of FT-IR for determination of DM see A. Fellah, et al., Carbohydr. Polymers, 2009, 78, 847-853 (not cited in the manuscript).
- For other notes, see attached file.
Author Response
Response to Reviewers
Dear editor and reviewers:
Thank you very much for your constructive comments. Based on your suggestions, we have made the corresponding revisions to our manuscript. The following is our itemized list of the changes that have been made.
Reviewer 1
Point 1: The novelty of the study is not approved. “Few literature” — that’s not true: there are a lot of publications concerning DM and DAc of pectins of various origin with critical discussion of advantages and limitations of the applied methods (above, I have presented a small no. of materials). As for HPLC method, authors’ modifications (in comparison to S. Levigne et al., Ref. [17]) are as follows: 1. 0.2 M sulfuric acid was used for neutralization of NaOH instead of Maxi-clean IC-H device (Alltech). 2. “we did not remove pectin before HPLC analysis, as pectin will not be reserved on C18 column”. As for point 1, this is not something new and advantageous : G.A.Luzio & R.G. Cameron (J. Sci. Food Agric., 2013, 93, 2463) used 0.1 M nitric acid, G. Limbert et al. used 4 M HCl to stop de-esterification of pectin (Carbohydr. Res., 2000, 327, 293). Strong, mineral acid is an evident reagent, and counter anion is not sufficient. Point 2. The last suggestion is not so evident. Where pectin is, if it “not be reserved”? I guess, it was adsorbed on a pre-column. Is it a real advantage?
Response: Thank you for your advice and we agree with your comment that a lot of references concerning determination of DM and DAc of pectins have been published. We have revised the Introduction section and cited more references you referred before [17, 23-25]. Our study further verified some conclusions reported in these literatures. For the HPLC method, we have added the references you referred in the manuscript (Ref 24; line 237-248). Compared with G.A.Luzio & R.G. Cameron (J. Sci. Food Agric., 2013, 93, 2463), our method saved more time both in sample pre-treatment and in HPLC procedure. For point 2, we wanted to express that pectin could be eluted very quickly from the column (before 5 min), which does not affect the detection of MeOH and HAc.
Point 2: Just what I want to ask again: why? Really, the difference looks low but (statistically) significant (FT-IR: 44.0 +/- 0.54 % in comparison to titration 47.0 +/- 0.63 % and HPLC 48.0 +/- 0.43 %). For critical analysis of FT-IR for determination of DM see A. Fellah, et al., Carbohydr. Polymers, 2009, 78, 847-853 (not cited in the manuscript).
Response: We have detected DM of CP by FT-IR again, and recalculated DM of 47.6 %. (Line 214)
The reference of Carbohydr. Polymers, 2009, 78, 847-853 has been cited in Ref 15.
