Nutritional Composition and Safety Parameters of Mealworms (Tenebrio molitor) Reared on Substrates Derived from By-Products

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors present the manuscript with the title „Nutritional composition and safety parameters of mealworms (Tenebrio molitor) reared on substrates derived from by-products”. The yellow mealworm is the larval form of the mealybug, Tenebrio molitor, and it is presented in the literature as a promising source of protein for human consumption with the required fat and essential amino acids. Thus, the aim of the study was to find the most suitable products that could be used in the mass production of larvae as the main substrate. In my opinion, the article is very rigorously prepared, the references are appropriate and the results may be of practical use in the future, particularly in the context of alternative nutrition promoted globally.

I have some comments, particularly for the methodology:

1.      Abbreviations for AA and FA have already been defined on lines 24 and 25. It is mandatory to use these abbreviations everywhere in the manuscript. Please, check each line regarding this aspect (eg line 102, line 317 etc.). Instead, I recommend that a short information (2-3 sentences) be inserted here to highlight the original aspects of the article and which informational gap in the literature is covered by the present results.

2.      Lines 124-129, this information is a conclusion, and this should not be found in the introduction.

3.      Page 5, lines 179-188, the sub-section Determination of protein and fat content: It is true that these methods are the standardized ones, but considering the high level of the study, are those methods the most appropriate? Please mention if these methods have certain limitations. Also, I recommend introducing a brief description of the analytical steps followed.

4.      Page 6, lines 193-216 Determination of AA: How the procedure for the extraction was decided? If it was previously published, please cite the source, if it is an adaptation of a method previously published by other authors, but with small/big changes, I also recommend citing the source. Also, was this method previously validated in terms of reproducibility, precision, sensitivity, limit of detection, limit of quantification. From my point of view, for high accuracy, using only the calibration curve, without prior validation, is not enough.

5.      Page 6, lines 218-229 Determination of FAs: How the procedure for the extraction was decided? If it was previously published, please cite the source, if it is an adaptation of a method previously published by other authors, but with small/big changes, I also recommend citing the source. Also, was this method previously validated in terms of reproducibility, precision, sensitivity, limit of detection, limit of quantification. From my point of view, for high accuracy, using only the calibration curve, without prior validation, is not enough.

6.      Page 6, lines 231-236, the sub-section Determination of peroxide value: It is true that this method is the standardized one, but is this method the most accurate? Please mention if this method has limitations. Also, I suggest to present a brief description of the analytical steps followed.

7.      Page 7, line 253. Was this determination performed in triplicate for each sample? This aspect should be mentioned here.

8.      Page 9, Line 306, Figure 2: Avoid overlapping text/numbers in the figure. Example: columns 1S and 2S.

9.      Table 1, Table 2, Table 3. – Move these tables to individual horizontal pages. Also, put the letters as superscript.

10.   Page 10, line 369: Figure 3 (3 fig.)

11.   Page 11, Line 393, Figure 3: Avoid overlapping text/numbers in the figure. Example: columns 1S and 2S.

12.   Page 16, line 584-591. Table 5: Respect the size of the text and the font, according to the template.

13.   Page 16, lines 608-610. Thus, the results have showed that both content of FAs and AA in larvae depended on the substrate on which they were reared. In my opinion, this conclusion was a hypothesis known/intuitive from the beginning of the study. It would be necessary to highlight which is the most suitable substrate, considering the values from the present study. This would be the conclusion...

14.   Page 16, line 610, This study… - move this phrase immediately before presenting the ideas for future research.

15.   Conclusion: I recommend introducing a strong conclusion that emphasizes the usefulness of the results in the short term and in the long term, respectively.

Author Response

Dear reviewer,

I would like to express my gratitude for your valuable comments and feedback. Your insights have been instrumental in enhancing the quality and clarity of the manuscript. Your time and expertise are greatly appreciated, and I am thankful for the constructive suggestions that have contributed to the improvement of the paper.

Thank you once again for your thoughtful and thorough review.

Best regards,

Authors

 

Comments:

The authors present the manuscript with the title „Nutritional composition and safety parameters of mealworms (Tenebrio molitor) reared on substrates derived from by-products”. The yellow mealworm is the larval form of the mealybug, Tenebrio molitor, and it is presented in the literature as a promising source of protein for human consumption with the required fat and essential amino acids. Thus, the aim of the study was to find the most suitable products that could be used in the mass production of larvae as the main substrate. In my opinion, the article is very rigorously prepared, the references are appropriate and the results may be of practical use in the future, particularly in the context of alternative nutrition promoted globally.

I have some comments, particularly for the methodology:

1. Abbreviations for AA and FA have already been defined on lines 24 and 25. It is mandatory to use these abbreviations everywhere in the manuscript. Please, check each line regarding this aspect (eg line 102, line 317 etc.). Instead, I recommend that a short information (2-3 sentences) be inserted here to highlight the original aspects of the article and which informational gap in the literature is covered by the present results.

 Answer: Thank You for Your comments, we made corrections.

2. Lines 124-129, this information is a conclusion, and this should not be found in the introduction.

 Answer: Thank You for Your comments, we have removed the last lines of the introduction with the conclusions.

3. Page 5, lines 179-188, the sub-section Determination of protein and fat content: It is true that these methods are the standardized ones, but considering the high level of the study, are those methods the most appropriate? Please mention if these methods have certain limitations. Also, I recommend introducing a brief description of the analytical steps followed.

 Answer:  Thank You for Your comments,  there is no regulated method specifically for mealworms. Therefore, the fat and protein determination test is based on the meat. Determination of fat in larvae according to the meat standard was chosen due to the similar nutritional declaration, affinity and close fat content of larvae and meat. The standard is intended for meat and meat products, there are no other restrictions. And the determination of protein is done according to the feed standard, although in principle the determination of protein does not differ significantly in different matrices, the coefficient for conversion from nitrogen to protein can only vary. We have added the methodology of the article.

4. Page 6, lines 193-216 Determination of AA: How the procedure for the extraction was decided? If it was previously published, please cite the source, if it is an adaptation of a method previously published by other authors, but with small/big changes, I also recommend citing the source. Also, was this method previously validated in terms of reproducibility, precision, sensitivity, limit of detection, limit of quantification. From my point of view, for high accuracy, using only the calibration curve, without prior validation, is not enough.

Answer: The test was carried out following COMMISSION REGULATION (EC) No 152/2009 of 27 January 2009, tested using different solvents and extracted several times to check if one time is enough. Derivatization procedure was performed according to (https://doi.org/10.1016/j.ab.2021.114423 ) with some modifications. Also, this methodology has already been used in our other study and is published (Juknienė et al.): https://www.mdpi.com/2076-3417/12/24/12984. Thank you for your comments, we have added the methodology of the article.

5. Page 6, lines 218-229 Determination of FAs: How the procedure for the extraction was decided? If it was previously published, please cite the source, if it is an adaptation of a method previously published by other authors, but with small/big changes, I also recommend citing the source. Also, was this method previously validated in terms of reproducibility, precision, sensitivity, limit of detection, limit of quantification. From my point of view, for high accuracy, using only the calibration curve, without prior validation, is not enough.

 Answer: Thank you for your observations. We used the same research process as in the article written and published together with co-authors (Juknienė et al.): https://www.mdpi.com/2076-3417/12/24/12984 . Fats were extracted with n-hexane. We would like to note that it was not a validation, but a verification, since the ISO standard was used, the reproducibility was evaluated under the conditions of the internal reproducibility of the tightness, it corresponds to the limits specified in the method standard. We have added methodologies based on your observations.

6. Page 6, lines 231-236, the sub-section Determination of peroxide value: It is true that this method is the standardized one, but is this method the most accurate? Please mention if this method has limitations. Also, I suggest to present a brief description of the analytical steps followed.

 Answer: The determination of peroxides according to ISO 27107:2008 is a reliable, standartised and universal method for the quantitative assessment of peroxides in vegetable and animal fats using potentiometric endpoint determination. This standard offers a systematic approach to assess the oxidative stability and quality of fats and oils, which are essential components in various food products and industrial applications. The methot is applicaple to all animal and vegetable fats and oils with peroxide values from 0 meq to 30 meq of active oxygen per kilogram. Compliance with ISO 27107:2008 helps food manufacturers, suppliers, and regulatory authorities meet quality and safety requirements related to the oxidative stability of fats and oils in food products. The method outlined in ISO 27107:2008 is widely utilized in the food industry, as well as in research and development, to assess the shelf life, storage conditions, and suitability of fats and oils for various applications. We have added the methodology of the article.

7. Page 7, line 253. Was this determination performed in triplicate for each sample? This aspect should be mentioned here.

 Answer: Thank you for your observation. Yes, it was repeated three times, each sample, so we supplemented the methodologies.

8. Page 9, Line 306, Figure 2: Avoid overlapping text/numbers in the figure. Example: columns 1S and 2S.

 Answer: Thank You for Your comments, we have corrected it.

9. Table 1, Table 2, Table 3. – Move these tables to individual horizontal pages. Also, put the letters as superscript.

Answer: Thank You for Your comments, we have corrected it.

10. Page 10, line 369: Figure 3 (3 fig.)

 Answer: Thank You for Your comments, we have corrected it.

11. Page 11, Line 393, Figure 3: Avoid overlapping text/numbers in the figure. Example: columns 1S and 2S.

 Answer: Thank You for Your comments, we have corrected it.

12. Page 16, line 584-591. Table 5: Respect the size of the text and the font, according to the template.

 Answer: Thank You for Your comments, we have corrected it.

13. Page 16, lines 608-610. Thus, the results have showed that both content of FAs and AA in larvae depended on the substrate on which they were reared.In my opinion, this conclusion was a hypothesis known/intuitive from the beginning of the study. It would be necessary to highlight which is the most suitable substrate, considering the values from the present study. This would be the conclusion...

 Answer: Thank you, we have added a sentence with emphasis on a specific sample.

14. Page 16, line 610, This study - move this phrase immediately before presenting the ideas for future research.

 Answer: Thank You for Your comments, we have corrected it.

15. Conclusion: I recommend introducing a strong conclusion that emphasizes the usefulness of the results in the short term and in the long term, respectively.

 Answer: Thank You for Your comments, we have corrected it.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The study is very impressive. The experimental design is clear, the data are detailed, the presentation is accurate, the application is strong, and it meets the journal's development objectives. Some suggestive comments for the authors in the Results and Discussion section are as follows:

 

(1) L297-299. When referring to protein content, are the results in the literature inconsistent with the units of your results and can they be used as a comparison?

 

(2) The quality of figure 1 ,2 and 3 needs to be improved, e.g. removing the background would be better.

 

(3) 3.2. Composition of amino acids Why are all the references italicized? Please check.

 

(4) L379. There is a 14% difference, does the author have an explanation?

 

 

Author Response

Dear reviewer,

I would like to express my gratitude for your valuable comments and feedback. Your insights have been instrumental in enhancing the quality and clarity of the manuscript. Your time and expertise are greatly appreciated, and I am thankful for the constructive suggestions that have contributed to the improvement of the paper.

Thank you once again for your thoughtful and thorough review.

Best regards,

Authors

Comments:

The study is very impressive. The experimental design is clear, the data are detailed, the presentation is accurate, the application is strong, and it meets the journal's development objectives. Some suggestive comments for the authors in the Results and Discussion section are as follows:

 (1) L297-299. When referring to protein content, are the results in the literature inconsistent with the units of your results and can they be used as a comparison?

 Answer: Yes, it was not appropriate to compare these data. To compare the amount of protein in grams per 100 g of food and in percentages, the formula should be used:

​ ; %=54,9

So, comparing the data, we would have: 54.9 g/100g protein equals 54.9% protein. 53.08% protein equals 53.08 g/100g protein. Thus, in the text, parentheses indicate that this corresponds to 54.9%.

(2) The quality of figure 1 ,2 and 3 needs to be improved, e.g. removing the background would be better.

  Answer: Thank You for the suggestion, the background has been removed.

(3) 3.2. Composition of amino acids Why are all the references italicized? Please check.

  Answer: Thank You for the suggestion, we corrected the text.

(4) L379. There is a 14% difference, does the author have an explanation?

 Answer: Both the research methods and the substrate used may have had an influence: 100 percent. oats in the aforementioned research study, while in our study we used bran, as well as brewer's yeast and carrots. Based on your observations, we have added possible options to the article: ‘‘One of the hypotheses for the difference in fat content between our study and the aforementioned study, and what could have influenced it, is that different research methods, larval treatments, and substrates were used.‘‘