Hemp Seed Oil Extraction and Stable Emulsion Formulation with Hemp Protein Isolates

Round 1

Reviewer 1 Report

Generally, the manuscript have interesting but poorly expressed experimental work: the paper cannot be considered for publication in its actual format.

First of all, I suggest to add line number because it is very difficult for reviewers suggesting the exact line for the corrections

 

Abstract

Cannabis sativa in italics

 

Introduction

“The hemp plant… pharmaceutical industries.” Please add references

 

“Tocopherols… blood vessels.” Please add references

 

“The cold-pressing extraction… is also present.” Please add references

 

What is the SC-CO₂? You before named supercritical CO₂ as SFE-CO₂. Are the same? Please correct

 

“Based on some works of literature… be of high quality [10].” what are these articles? Please add more than one literature paper

 

“This type of protein… as foams or emulsions”. Please check references. I think that you have to format better

 

“Other works of literature… the stability of the emulsions [10]”. As previously stated, you stated that others works were performed but you add only one reference. Please check

 

“Another possibility… solid-like consistency”. Please check these sentences because are confusing and unclear

 

Materials and methods

Please reformat. It is very difficult read and correct this part because the formatting is completely missing: all subsections are joined

 

Cannabis sativa in italics

 

“About 30 grams… same grain size.” In this sentence, I do not understand “the same time”. What does it mean?

 

“…they were put in a backer without any solvent and pre-treated before extraction with an ultrasound bath…”

I don't know if I understand correctly, did you sonicate the hemp seeds not in solution? How did you do? The advantage of using ultrasound lies in intensification of extraction efficacy by increasing the penetration of a solvent into the plant cells via cavitation and consequently obtaining a higher extraction yield.

 

The research design of oil extraction is really badly described, is not clear and confusing. Please rewrite

 

 

Results and discussion

Please check SC-CO₂ or SFE-CO₂. Use always the same abbreviation

 

Comparing the soxhlet and supercritical CO₂ method (with and without ultrasound), the best oil yield resulted with soxhlet extraction. I strongly suggest to stress the positive advantages using supercritical CO₂ method in order to support your choice

 

“The emulsions were prepared…the possible reduction of the antiradical capacity [4]”.

I don’t understand: the best oil yield resulted with soxhlet extraction but you suggest to use supercritical CO₂ method. For this reason you try to improve oil yield applying ultrasound pre-treatment but, at least, to prepare emulsion you used the hemp oil extracted by supercritical fluid CO₂ without using the ultrasound pre-treatments because of the possible reduction of the antiradical capacity. All this does not make sense. Why did you carry out the pre-treatments if after you don't use the oil extracted with them?

 

In subsections “Droplet size distribution of emulsions”, “ζ-potential measurements”, and “Rheology of emulsions” the comparison with literature data is scarce or completely missing. Please add references and compare you results with them.

 

Moreover, in all paper the potential application are not present. How can this formulation be utilized? In which field can it be applied? Please add in order to complete your research

 

Author Response

Reviewer 1

Comments and Suggestions for Authors

Generally, the manuscript have interesting but poorly expressed experimental work: the paper cannot be considered for publication in its actual format.

We apologize for the low quality of the writing. We have deeply revised the paper according to your comments and suggestions, we hope that the quality of the current version is now suitable for publication.

First of all, I suggest to add line number because it is very difficult for reviewers suggesting the exact line for the corrections

Thanks to the reviewer for the suggestion, he is right. In the new version, there are line numbers.

 

Abstract

Cannabis sativa in italics

We amended the manuscript according to the reviewer’s suggestions.

Introduction

“The hemp plant… pharmaceutical industries.” Please add references

According to the reviewer’s comment, we added the references.

 

“Tocopherols… blood vessels.” Please add references

According to the reviewer’s comment, we added the references.

 

“The cold-pressing extraction… is also present.” Please add references

According to the reviewer’s suggestion, we added the reference.

 

What is the SC-CO2? You before named supercritical CO2 as SFE-CO2. Are the same? Please correct

We are grateful to the reviewer for the observation. We have used two acronyms but in effect, we should have only used one. We amended the manuscript and used only SFE-CO2.

 

“Based on some works of literature… be of high quality [10].” what are these articles? Please add more than one literature paper

According to the reviewer’s suggestion, we added other references.

 

“This type of protein… as foams or emulsions”. Please check references. I think that you have to format better

We amended the manuscript.

 

“Other works of literature… the stability of the emulsions [10]”. As previously stated, you stated that others works were performed but you add only one reference. Please check

According to the reviewer’s suggestion, we added other references.

 

“Another possibility… solid-like consistency”. Please check these sentences because are confusing and unclear

Thanks for the suggestion, we have rephrased the sentence

 

Materials and methods

Please reformat. It is very difficult read and correct this part because the formatting is completely missing: all subsections are joined

According to the reviewer’s suggestion, we have reformatted the manuscript.

 

Cannabis sativa in italics

Thanks, we fixed it.

 

“About 30 grams… same grain size.” In this sentence, I do not understand “the same time”. What does it mean?

Thanks for the question. The sentence was not very clear. We have therefore rewritten the sentence, we hope it will now be clearer.

 

“…they were put in a backer without any solvent and pre-treated before extraction with an ultrasound bath…”

I don't know if I understand correctly, did you sonicate the hemp seeds not in solution? How did you do? The advantage of using ultrasound lies in intensification of extraction efficacy by increasing the penetration of a solvent into the plant cells via cavitation and consequently obtaining a higher extraction yield.

We sonicate the grounded seeds directly by putting them into the backer according to the procedure suggested in the literature (da Porto et al., 2015). The grounded seeds are fresh and they have a residual humidity that can help to obtain cavitation and the subsequent penetration of the supercritical fluid. The reviewer is true, generally, the sonication involves the use of a solvent. However, this also causes an osmotic effect, so the solvent should then be removed before carrying out the extraction by SFE-CO₂ as also suggested by (Li et al., 2021). Then, in light of the necessity to add a further step, we preferred to follow the procedure of Da Porto (2015) (da Porto et al., 2015). At present, we are investigating the procedure with a solvent during the sonication step.

 

The research design of oil extraction is really badly described, is not clear and confusing. Please rewrite

According to the reviewer’s suggestion, we have rewritten the section. We hope that now is clearer.

 

Results and discussion

Please check SC-CO2 or SFE-CO2. Use always the same abbreviation

Thanks, we have amended the text.

Comparing the soxhlet and supercritical CO2 method (with and without ultrasound), the best oil yield resulted with soxhlet extraction. I strongly suggest to stress the positive advantages using supercritical CO2 method in order to support your choice

We are grateful to the reviewer for the suggestion. We have inserted the advantages of using SFE-CO2 at the end of the section “Comparison of SFE-CO2 extraction and solvent extraction”.

 

The emulsions were prepared…the possible reduction of the antiradical capacity [4]”.

I don’t understand: the best oil yield resulted with soxhlet extraction but you suggest to use supercritical CO2 method. For this reason you try to improve oil yield applying ultrasound pre-treatment but, at least, to prepare emulsion you used the hemp oil extracted by supercritical fluid CO2 without using the ultrasound pre-treatments because of the possible reduction of the antiradical capacity. All this does not make sense. Why did you carry out the pre-treatments if after you don't use the oil extracted with them?

 

We are grateful to the reviewer for the observations because now, we hope, we have better explained our decision. The use of organic solvents, as in the case of Soxhlet, has as a disadvantage the possible presence of toxic residues and generally, solvent extraction is used to know the maximum yield. This is the reason for the use of the SFE-CO2 extract.

Moreover, we use the extract by SFE-CO2 without using the ultrasounds because preprocessing with sonication the oil yield increase but there is the presence of chlorophyll in the extract, even if in our case the colour is a light green compared with the hemp oil obtained by cold pressing as also reported by (da Porto et al., 2012). This was the main reason. Then, we thought that probably, according also to your observations, it is better to explain the real practical reason and we canceled the previous sentence and added a phrase to explain how we use the oil from SFE-CO2 in our applications.

 

In subsections “Droplet size distribution of emulsions”, “ζ-potential measurements”, and “Rheology of emulsions” the comparison with literature data is scarce or completely missing. Please add references and compare you results with them.

We are grateful to the reviewer for the observations on the literature comparison. We have amended the section adding references and comparing our results with other similar systems obtained with hemp protein isolate or gellan gum.

 

Moreover, in all paper the potential application are not present. How can this formulation be utilized? In which field can it be applied? Please add in order to complete your research

We are grateful to the reviewer for the observation. We have added the potential application at the end of the emulsion section.

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Baldino et al studied oil extraction the protein recovery from hemp seeds. It can be a valuable study in the field of food production. However, I have some comments;

The main concerns:

title: It can be improved as one sentence. 

language: There are some typos and grammatical errors. Please check.

The introduction is so long and does not use recent references. Please shorten it and use some new references.

Write all trade names in the materials method section.

What is the reason for different sonication times?

Page 3, About 30 g of hemp seeds... it is not correct. about is not proper for a scientific study.

Which basic method has been used for the preparation of emulsion eg. reverse method? pH method? or ...? 

What software has been used for image analysis of emulsion droplets?

Please mention the details for zeta potential determination.

references are old.

 

Author Response

Reviewer 2

Comments and Suggestions for Authors

Baldino et al studied oil extraction the protein recovery from hemp seeds. It can be a valuable study in the field of food production. However, I have some comments;

 

The main concerns:

title: It can be improved as one sentence.

Thank you for the suggestion, indeed, in the hope of being clear about the purpose of the research we were certainly long-winded. We have summarized it in a shorter form

 

language: There are some typos and grammatical errors. Please check.

We are grateful to the reviewer we have checked the paper.

 

The introduction is so long and does not use recent references. Please shorten it and use some new references.

According to the reviewer’s suggestion, we have shortened the introduction and inserted new references.

 

Write all trade names in the materials method section.

According to the reviewer’s suggestion, we have written all trade names.

 

What is the reason for different sonication times?

We are grateful to the reviewer for the question. Ultrasound pre-treatment of grounded hemp seeds was carried out at different times to investigate the possibility of increasing the oil yield without altering the lipid profile.

We have added two lines in the materials and methods section to clarify.

 

Page 3, About 30 g of hemp seeds... it is not correct. about is not proper for a scientific study.

Thanks for the observation, we have adjusted writing the mean value and the standard deviation

 

Which basic method has been used for the preparation of emulsion eg. reverse method? pH method? or ...?

The production of emulsions followed a typical direct homogenization method. Two steps of homogenization were carried out (McClements, 2004): in the first step, the two phases were mixed at high shear rates with a rotor-stator device (UT T50, IKA, Germany, tool S50N G45F), whereas, the second step carried out with a gentle mixing done with a magnetic stirrer (AREX Heating Magnetic Stirrer, Velp Scientifica, Italia) at room temperature conditions completed the emulsions stabilization. The pH of samples was not varied, nor reverse methods were used.

Thanks to your observation we have modified the section inserting when above.

What software has been used for image analysis of emulsion droplets?

Particle Size Distribution (PSD) was obtained with a statistical analysis of data measured thanks to the contrast phase optical microscope (MX5300H, MEIJI, Japan, magnification 20X for all samples). The image analysis was performed with the software Dhs Particle Analysis (dhs image database, Germany) in which the equivalent circular area diameter of droplets, properly coloured with different colours according to their sizes, was calculated.

According to the reviewer’s observation, we have added the software specifications.

 

Please mention the details for zeta potential determination.

According to the reviewer’s suggestion, we have amended the text.

 

references are old.

According to the reviewer’s suggestion, we have added new references.

 

References:

da Porto, C., Decorti, D., & Tubaro, F. (2012). Fatty acid composition and oxidation stability of hemp (Cannabis sativa L.) seed oil extracted by supercritical carbon dioxide. Industrial Crops and Products, 36(1), 401–404. https://doi.org/https://doi.org/10.1016/j.indcrop.2011.09.015

da Porto, C., Natolino, A., & Decorti, D. (2015). Effect of ultrasound pre-treatment of hemp (Cannabis sativa L.) seed on supercritical CO2 extraction of oil. Journal of Food Science and Technology, 52(3), 1748–1753. https://doi.org/10.1007/s13197-013-1143-3

Li, X., Shi, J., Scanlon, M., Xue, S. J., & Lu, J. (2021). Effects of pretreatments on physicochemical and structural properties of proteins isolated from canola seeds after oil extraction by supercritical-CO2 process. LWT, 137, 110415. https://doi.org/https://doi.org/10.1016/j.lwt.2020.110415

McClements, D. J. (2004). Food emulsions: Principles, practices, and techniques: Second edition. In Food Emulsions: Principles, Practices, and Techniques, Second Edition.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Some minor comments:

I suggest not to use abbreviation in the title. You use HPI only in the title: in text HPI is not present and non-specified.

 

Line 130: CO₂ not CO2

 

Line 146: CH₃Cl? Are you sure? Maybe is it CHCl₃?

 

Lines 273-282: please add references

Author Response

Reviewer 1

Thanks to the reviewer, we have adjusted the paper according to your suggestions.

Some minor comments:

I suggest not to use abbreviation in the title. You use HPI only in the title: in text HPI is not present and non-specified.

 

Thanks for the suggestion, we have deleted the abbreviation in the title.

 

Line 130: CO2 not CO2

 

We have adjusted

 

Line 146: CH3Cl? Are you sure? Maybe is it CHCl3?

 

According to the reviewer’s comment, we checked the text and amended it.

 

Lines 273-282: please add references

According to the reviewer’s comment, we added the references.

Author Response File: Author Response.pdf

Reviewer 2 Report

Great. Thanks for good modification process. It can be accepted in the current form.

Author Response

Thank you very much