Valorization of Residual Babassu Mesocarp Biomass to Obtain Aroma Compounds by Solid-State Fermentation
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis manuscript describes the production of compounds useful in the flavor and aroma industries through the fermentation of agricultural waste products (babassu mesocarp) by fungal organisms (Trichoderma harzianum and Geotrichum candidum). The authors provide a introduction that sufficiently justifies the importance of the study and provides valuable context to the work. The materials and methods section provides sufficient detail such that this work could be reproduced by another laboratory. The authors describe the detailed analysis of the agricultural products used as solid supports, and the quantification of the compounds of interest produced from the fermentation processes. The authors provide valuable context by comparing their findings to other studies that have targeted the production of the same compounds using other agricultural waste products as solid supports. Some deeper context provided in the conclusion section would improve the quality of this manuscript.
Some editing/proofreading of English language in the manuscript is required prior to publication. Word choices occasionally make the meanings of statements unclear. Several formatting errors also occur throughout the manuscript.
Abstract: The word “hexanal” should not be capitalized (also in methods section).
It is unclear to this reviewer why this study chose to focus on the specific fungal organisms selected (Trichoderma harzianum and Geotrichum candidum), or the specific biomass source chosen (Babassu mesocarp). While the authors do describe the value of fungal organisms generally in this context, as well as the potential of this type of biomass as a solid support, they do not expand on why these particular selections were made. Are these organisms known to be particularly productive? Is Babassu mesocarp a particularly common or readily available farming byproduct? This should be described in more detail in the introduction.
The context and importance of using “defatted” and “degreasing” Babassu mesocarp should be described in more detail. It is unclear to this reviewer based on the text what the difference is between “MB-ENG” and “MB-DES”.
Introduction, Line 101: The word choice of “compassionate” makes the meaning of this sentence unclear.
Results and Discussion, Line 234: Formatting such as “…was also reported by [25]” is not correct. This should read as “”…was also reported by Liu, et. al. [25]”. This formatting is also used elsewhere and should be corrected throughout the manuscript.
Results and Discussion: Some chemical names (ie (NH4)2SO4) are not formatted correctly (subscripts). This needs to be corrected throughout the manuscript.
Figure 4: “distilled” is spelled incorrectly in A) and B)
Results and Discussion, Line 390: The terminology “slightly close” is unclear and should be reworded.
It is unclear from the conclusion section whether the optimal conditions identified in this study represent a viable means of commercially producing the compounds of interest. The authors should provide some comment as to the practical utility of this method, particularly if it necessary for glucose to be added to the agricultural waste product for optimal production. Although some general comment is provided, it is not supported by facts that convince the reader that this method has practical utility.
Comments on the Quality of English LanguageSome editing/proofreading of English language in the manuscript is required prior to publication. Word choices occasionally make the meanings of statements unclear. Several formatting errors also occur throughout the manuscript.
Author Response
Referee 1
This manuscript describes the production of compounds useful in the flavor and aroma industries through the fermentation of agricultural waste products (babassu mesocarp) by fungal organisms (Trichoderma harzianum and Geotrichum candidum). The authors provide a introduction that sufficiently justifies the importance of the study and provides valuable context to the work. The materials and methods section provides sufficient detail such that this work could be reproduced by another laboratory. The authors describe the detailed analysis of the agricultural products used as solid supports, and the quantification of the compounds of interest produced from the fermentation processes. The authors provide valuable context by comparing their findings to other studies that have targeted the production of the same compounds using other agricultural waste products as solid supports. Some deeper context provided in the conclusion section would improve the quality of this manuscript.
Some editing/proofreading of English language in the manuscript is required prior to publication. Word choices occasionally make the meanings of statements unclear. Several formatting errors also occur throughout the manuscript.
- Abstract: The word “hexanal” should not be capitalized (also in methods section).
Answer: Done.
- It is unclear to this reviewer why this study chose to focus on the specific fungal organisms selected (Trichoderma harzianum and Geotrichum candidum), or the specific biomass source chosen (Babassu mesocarp). While the authors do describe the value of fungal organisms generally in this context, as well as the potential of this type of biomass as a solid support, they do not expand on why these particular selections were made. Are these organisms known to be particularly productive? Is Babassu mesocarp a particularly common or readily available farming byproduct? This should be described in more detail in the introduction.
Answer: Dear referee, thank you for the observation. The microorganisms Trichoderma harzianum and Geotrichum candidum were selected for this work because of their ability to metabolize lignocellulosic biomass and produce high-value bioactive compounds. Among the various bioactive compounds that can be produced by these microorganisms are aroma compounds, which can also be produced using a lignocellulosic source rich in available nutritional constituents. In addition, these microorganisms are GRAS (generally recognized as safe) and their use in this process allows aroma compounds to be used in the cosmetics and food industry and gives the product a natural connotation. The choice of babassu mesocarp as biomass was based on the abundant availability of this waste in the environment, given the extractive activity of the babassu palm, and the extent to which this volume of waste produced and disposed of inappropriately has affected the environment. Our group has been working with lignocellulosic biomass to produce enzymes, including babassu mesocarp, which we decided to investigate for its potential in the production of aroma compounds. The bioproduction of flavors has also been explored by our group using other types of lignocellulosic waste and the efficiency of this waste as biomass has been demonstrated, also using T. harzianum and G. candidum for fermentation.
We've improved the explanation in the text.
- The context and importance of using “defatted” and “degreasing” Babassu mesocarp should be described in more detail. It is unclear to this reviewer based on the text what the difference is between “MB-ENG” and “MB-DES”.
- Answer: Dear referee, thank you for your question. Indeed it wasn't clear about the difference between the two biomasses. The purpose of this pre-treatment was to study the influence of the residual lipid and extractive components on obtaining aroma compounds and assimilating the residual BM during the fermentation process, identifying some type of metabolic inhibition or activation in the strains studied. To do this, part of the raw BM was subjected to a lipid extraction process using soxhlet, and the fraction obtained was called (DEF-BM). The in natura fraction was called (IN-BM). We have changed the nomenclatures throughout the text, and explain this procedure further in section 2.2 as well as in the introduction section.
- Introduction, Line 101: The word choice of “compassionate” makes the meaning of this sentence unclear.
Answer: Dear referee, thank you for the observation. We have changed the sentence.
- Results and Discussion, Line 234: Formatting such as “…was also reported by [25]” is not correct. This should read as “”…was also reported by Liu, et. al. [25]”. This formatting is also used elsewhere and should be corrected throughout the manuscript.
Answer: Dear referee, thank you for the observation. We checked and corrected the references format.
- Results and Discussion: Some chemical names (ie (NH4)2SO4) are not formatted correctly (subscripts). This needs to be corrected throughout the manuscript.
Answer: Corrected.
Figure 4: “distilled” is spelled incorrectly in A) and B)
Answer: Dear referee, thank you for the observation. We checked and corrected the figure.
Results and Discussion, Line 390: The terminology “slightly close” is unclear and should be reworded.
Answer: Dear referee, we have rewritten the sentence. Thank you.
It is unclear from the conclusion section whether the optimal conditions identified in this study represent a viable means of commercially producing the compounds of interest. The authors should provide some comment as to the practical utility of this method, particularly if it is necessary for glucose to be added to the agricultural waste product for optimal production. Although some general comment is provided, it is not supported by facts that convince the reader that this method has practical utility.
Answer: Dear referee, thank you for your question. In fact, we based this study on a preliminary comparison of different forms of supplementation, from nitrogen and trace element sources to carbon sources such as glucose, a simpler carbohydrate, and sucrose, a slightly more complex carbohydrate. In this study, we compared systems without supplementation, and it was clear that the system needs the addition of nutrient solutions to increase the productivity of aroma compounds. However, the addition of sucrose was just as efficient as glucose, proving to be a lower-cost indication. However, other carbon sources are being studied, including hydrolyzed starch and others. As this is a preliminary study, we are working to optimize the system, as well as investigating other parameters, such as the apparatus for SSF, optimizing humidity control, as well as other microorganisms from our collection. Although it is a simple method, SSF presents a high risk of contamination, does not allow efficient temperature control and can present mass transfer problems. However, it is a practical tool for recovering agro-industrial waste. The methodology for extracting aroma constituents was very successful, and is one of the great advantages of this work, with the combination of SSF with this technique being a simple procedure that can be scaled up. However, in order to affirm its viability precisely, we need an economic study, which was not carried out at this stage.
We have modified the text of the conclusion section.
Reviewer 2 Report
Comments and Suggestions for AuthorsI am very grateful to you for the invitation to review the manuscript microbiolres-3112377 by Anjos and coauthors "Valorization of Residual Babassu Mesocarp Biomass to Obtain Aroma Compounds by Solid-State Fermentation”. In this work, solid-state fermentation (SSF) was applied to babassu mesocarp (MB) for the low-cost bioproduction of natural aroma compounds having Trichoderma harzianum (IOC 4042) and Geotrichum candidum (CCT 1205) as microbial agents. The work is interesting but needs adjustments to increase the quality of the material.
Comments:
- Lines 14-15: Specify the difference between “using defatted (MB-ENG) and defatted (MB-DES) babassu mesocarp.” The description in line 107 is “using greased (MB-ENG) and defatted (MB-DES) babassu mesocarp as support.” Please check throughout the text.
- Lines 15-16: Specify the term “hydration solution of different compositions better.” What composition?
- Line 28: Change the repeated keywords to different terms not included in the title.
- Line 37: Review the correct way of presenting references. Change “[1, 2, 3, 4]” to “[1-4]”. Review throughout the text.
- Line 39: Describe the abbreviation “GDP” on its first occurrence.
- Line 46: Highlight the global production of waste (tons or % of what is processed/produced) and more clearly specify the associated risks of accumulation.
- Lines 48-50: Please update the data related to waste. There are several reports on the enormous amount of by-products generated.
- Lines 59-64: Better describe the characteristics and advantages of using the indicated microorganisms.
- Lines 66-68: Rewrite the sentence in a more continuous form (In this process/ In this type of Fermentation).
- Line 72: Better describe the problems associated with improper disposal.
- Introduction: Briefly describe the problems or limitations associated with solid-state fermentation and the indicated microorganisms.
- Lines 74-75: Use italics appropriately. Review the use throughout the text.
- Line 77 and throughout the text: Standardize the use of the terminology "waste" or "by-products."
- Lines 74-79: The authors should indicate the amount of babassu produced and the proportions between the different plant fractions, mainly indicating the amount of by-products generated along the chain.
- Lines 80-86: Please use the most recent data regarding the aroma and fragrance market.
- Line 89: Indicate the main applications and usage sectors.
- Lines 93-96: Indicate possible waste generated in each case to compare the compound's acquisition more effectively.
- Lines 97-101: Please include more information about the compound's recovery.
- Figure 1: Include the image of the whole fruit.
- Lines 118-120: Please remove the sentence about recent studies and indicate only the study reference.
- Line 126: greased babassu mesocarp? Review throughout the text. Refer to it as a "whole" or other specification to avoid misunderstandings.
- Lines 148-149; 174 and throughout the text: Observe the correct use of subscripts in chemical formulas.
- Lines 184-188: Better explain this sentence. If no analytical medium was adopted, describe only the identification from the GC.
- Line 233: Verify the correct citation format.
- Lines 228-231: Indicate the babassu's composition, specifying the material's starch concentration.
- Line 239-240: The description in line 107 is "using greased (MB-ENG) and defatted (MB-DES) babassu mesocarp as support." Please check throughout the text.
- Line 241: Check if "surface topography" is more appropriate than "textural characteristics."
- Lines 170-172; 262-265: Use the corresponding acronym in English. Check throughout the text.
- Lines 267-277: The role of the hydration mechanism and the different solutions should be better explored. Highlight the role of the components in microbial metabolism.
- Lines 293-294: Highlight how the components favor microbial growth.
- Line 303: Verify the correct citation format.
- Figures 4 and 5: Please apply statistical analysis to facilitate the presentation of the results, allowing better means for aroma production.
- Line 441: Verify the correct citation format.
- Line 475: Verify the correct citation format.
- Line 488: Change “FES” to “SSF”. Check throughout the text.
Author Response
Referee 2
I am very grateful to you for the invitation to review the manuscript microbiolres-3112377 by Anjos and coauthors "Valorization of Residual Babassu Mesocarp Biomass to Obtain Aroma Compounds by Solid-State Fermentation”. In this work, solid-state fermentation (SSF) was applied to babassu mesocarp (MB) for the low-cost bioproduction of natural aroma compounds having Trichoderma harzianum (IOC 4042) and Geotrichum candidum (CCT 1205) as microbial agents. The work is interesting but needs adjustments to increase the quality of the material.
Comments:
- Lines 14-15: Specify the difference between “using defatted (MB-ENG) and defatted (MB-DES) babassu mesocarp.” The description in line 107 is “using greased (MB-ENG) and defatted (MB-DES) babassu mesocarp as support.” Please check throughout the text.
Answer: Dear referee, thank you for your note. We have added a better description of fattened and defatted babassu mesocarp in the abstract, as well as in the introduction and material and methods sections. The difference is that MB-ENG is the in-natura babassu mesocarp, bringing with it the oleaginous residues after the industrial extraction process. MB-DES, on the other hand, is the residual babassu mesocarp that has undergone a degreasing process using a Soxhlet extractor. We have also updated these acronyms to IN-BM (in-natura babassu mesocarp) e DEF-BM (defatted babassu mesocarp).
- Lines 15-16: Specify the term “hydration solution of different compositions better.” What composition?
Answer: Done.
- Line 28: Change the repeated keywords to different terms not included in the title.
Answer: Done.
- Line 37: Review the correct way of presenting references. Change “[1, 2, 3, 4]” to “[1-4]”. Review throughout the text.
Answer: Done.
- Line 39: Describe the abbreviation “GDP” on its first occurrence.
Answer: Done.
- Line 46: Highlight the global production of waste (tons or % of what is processed/produced) and more clearly specify the associated risks of accumulation.
Answer: Done. Thank you for the suggestion. We have included the information in the text and specified some of the associated risks of waste accumulating in the environment. Some examples of these risks are: increased greenhouse gas (GHG) emissions, river and soil pollution and the proliferation of disease-transmitting vectors.
- Lines 48-50: Please update the data related to waste. There are several reports on the enormous amount of by-products generated.
Answer: Dear referee, thank you for your note. We've updated the information in the text.
- Lines 59-64: Better describe the characteristics and advantages of using the indicated microorganisms.
Answer: Dear referee, thank you for the observation. The microorganisms Trichoderma harzianum and Geotrichum candidum were selected for this work because of their ability to metabolize lignocellulosic biomass and produce high-value bioactive compounds. Among the various bioactive compounds that can be produced by these microorganisms are aroma compounds, which can also be produced using a lignocellulosic source rich in available nutritional constituents. In addition, these microorganisms are GRAS (generally recognized as safe) and their use in this process allows aroma compounds to be used in the cosmetics and food industry and gives the product a natural connotation. The bioproduction of flavors has already been explored by our group using other types of lignocellulosic waste and the efficiency of this waste as biomass has been demonstrated, also using T. harzianum and G. candidum for fermentation. We therefore decided to investigate the productive potential of these microorganisms for the production of aromas using this waste. We've improved the explanation in the text.
- Lines 66-68: Rewrite the sentence in a more continuous form (In this process/ In this type of Fermentation).
Answer: Done.
- Line 72: Better describe the problems associated with improper disposal.
Answer: Done. Thank you for the observation. We have included some examples in the text.
- Introduction: Briefly describe the problems or limitations associated with solid-state fermentation and the indicated microorganisms.
Answer: Done.
- Lines 74-75: Use italics appropriately. Review the use throughout the text.
Answer: Done. Thank you for the observation.
- Line 77 and throughout the text: Standardize the use of the terminology "waste" or "by-products."
Answer: Done
- Lines 74-79: The authors should indicate the amount of babassu produced and the proportions between the different plant fractions, mainly indicating the amount of by-products generated along the chain.
Answer: Done. Thank you for the suggestion. We have added the information requested in the text.
- Lines 80-86: Please use the most recent data regarding the aroma and fragrance market.
Answer: Done. Thank you for the suggestion. The data included in the text on this subject is from 2023 and forecasts this scenario for 2027 (2022-2027). The report on this market is published annually and the data for 2024 is not yet available. As a result, we were unable to insert more up-to-date data.
- Line 89: Indicate the main applications and usage sectors.
Answer: Done. Thank you for the suggestion. Flavour compounds are used in industry to characterize, improve, standardize, reconstruct the aroma/flavour or mask unwanted aromas/flavours of: cakes, candies, cookies, chewing gum, yoghurts, oral medicines and soaps, for example. Thus, the food, pharmaceutical, cosmetics and fragrance industries have the greatest potential for the application of these compounds.
- Lines 93-96: Indicate possible waste generated in each case to compare the compound's acquisition more effectively.
Answer: Dear referee, we better discussed the both pathways for the aroma compounds obtaining.
- Lines 97-101: Please include more information about the compound's recovery.
Answer: Done. Thank you for the suggestion. After 7 days of fermentation, the vials were quickly closed with a screw cap with a silicone septum, in order to trap the aroma compounds in these containers until the extraction stage. The extraction or recovery of the compounds for analysis was carried out using the solid phase microextraction (SPME) technique, using 100 μm thick PDMS (polydimethylsiloxane) coated fibers to capture the compounds. This step took place in the headspace of the vials and to carry out this method, 5.0 mL of saline solution (25% m/v) was added to the fermentations. The vials were subjected to constant magnetic stirring in a water bath at 79ºC for 30 minutes.
- Figure 1: Include the image of the whole fruit.
Answer: Done. Thank you for the suggestion. We've added a more illustrative figure.
- Lines 118-120: Please remove the sentence about recent studies and indicate only the study reference.
Answer: Done. Thank you for the suggestion. We've removed the sentence.
- Line 126: greased babassu mesocarp? Review throughout the text. Refer to it as a "whole" or other specification to avoid misunderstandings.
Answer: Done. Thank you for the observation. We have revised and corrected the term.
- Lines 148-149; 174 and throughout the text: Observe the correct use of subscripts in chemical formulas.
Answer: Done. Thank you for the observation.
- Lines 184-188: Better explain this sentence. If no analytical medium was adopted, describe only the identification from the GC.
Answer: Done. Thank you for the observation.
- Line 233: Verify the correct citation format.
Answer: Done.
- Lines 228-231: Indicate the babassu's composition, specifying the material's starch concentration.
Answer: Done.
- Line 239-240: The description in line 107 is "using greased (MB-ENG) and defatted (MB-DES) babassu mesocarp as support." Please check throughout the text.
Answer: Dear referee, thank you for your question. Indeed it wasn't clear about the difference between the two biomasses. The purpose of this pre-treatment was to study the influence of the residual lipid and extractive components on obtaining aroma compounds and assimilating the residual BM during the fermentation process, identifying some type of metabolic inhibition or activation in the strains studied. To do this, part of the raw MB was subjected to a lipid extraction process using soxhlet, and the fraction obtained was called (BM-DEF). The in natura fraction was called (BM-IN). We have changed the nomenclatures throughout the text, and explain this procedure further in section 2.2 as well as in the introduction section.
- Line 241: Check if "surface topography" is more appropriate than "textural characteristics."
Answer: Dear referee, thank you for the correction. We have changed in the text.
- Lines 170-172; 262-265: Use the corresponding acronym in English. Check throughout the text.
Answer: Done.
- Lines 267-277: The role of the hydration mechanism and the different solutions should be better explored. Highlight the role of the components in microbial metabolism.
Answer: Dear referee, thank you for the suggestion. The purpose of fermentation hydration solutions is to provide a humid environment for the development of microorganisms, simulating their natural habitat. These solutions also contain micronutrients such as zinc, sulphur, magnesium, manganese and yeast extract, for example, which play an extremely important role in the metabolism of microorganisms. Mainly because they are required by the cellular machinery as cofactors for various processes.
- Lines 293-294: Highlight how the components favor microbial growth.
Answer: Dear referee, thank you for the suggestion. The components of the solution with 3 salts favor microbial growth because they have a positive effect on the metabolism of these microorganisms. Some components of the solution containing the 7 salts may have had a negative effect on growth during fermentation, which may have had an impact on the production of aroma compounds.
- Line 303: Verify the correct citation format.
Answer: Done.
- Figures 4 and 5: Please apply statistical analysis to facilitate the presentation of the results, allowing better means for aroma production.
Answer: Dear referee, thank you for your suggestion. We have applied a statistical analysis to better understand and discuss the results.
- Line 441: Verify the correct citation format.
Answer: Done.
- Line 475: Verify the correct citation format.
Answer: Done.
- Line 488: Change “FES” to “SSF”. Check throughout the text.
Answer: Done. Thank you for the observation.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have satisfactorily responded to all my questions and made the necessary changes to the manuscript.
