Oxidation of Terpenoids to Achieve High-Value Flavor and Fragrances—Questioning Microalgae Oxidative Capabilities in the Biotransformation of the Sesquiterpene Valencene and of Selected Natural Apocarotenoids
Round 1
Reviewer 1 Report
Comments
- Simeis and co-workers in this manuscript have very established the protocol for the oxidation of terpenoids leading to high-fragrance and high-flavor and investigated the role of microalgae in such transformation.
- The results achieved in table 2 & 3 suggests the oxidation is initiated or takes place in presence of light and microalgae is not the reason for the transformation. However, can author propose any mechanism for above transformation, this will be very much appreciated or helpful for the readers in understanding the chemistry involved from synthetic chemistry view.
- The soft copies of the GCMS chromatograph should be included in this manuscript for the compound 8, 5, 6, 7 and their respective starting material along with the authentic samples 8, 5, 6, and 7 GCMS chromatograph.
- After biotransformation experiments and after workup process isolation crude 1HNMR and 13 CMR should be taken, which would provide confirmation of the products 8, 5, 6 and 7.
- Authors should elaborate or should mention in detail the calculation for the percentage of compounds 8, 5, 6, 7 from table 2 and table 3.
Comments for author File: 
Comments.pdf
Author Response
- The results achieved in table 2 & 3 suggests the oxidation is initiated or takes place in presence of light and microalgae is not the reason for the transformation. However, can author propose any mechanism for above transformation, this will be very much appreciated or helpful for the readers in understanding the chemistry involved from synthetic chemistry view.
According to the referee suggestion, we added a possible mechanism for the valencene oxidation (Figure 6). It is reasonable that the same mechanism could explain the oxidation of other apocarotenoids.
- The soft copies of the GCMS chromatograph should be included in this manuscript for the compound 8, 5, 6, 7 and their respective starting material along with the authentic samples 8, 5, 6, and 7 GCMS chromatograph.
- After biotransformation experiments and after workup process isolation crude 1HNMR and 13 CMR should be taken, which would provide confirmation of the products 8, 5, 6 and 7.
We have already reported the description of their synthesis (reference 5 and 22 of this work) as well as their full characterization. Therefore, there is no need to report again the 1H and 13C NMR data. The confirmation of the identity of the compounds obtained by biotransformation have been obtained by GC-MS analysis. Moreover, we reported the retention times of all products used in this study.
- Authors should elaborate or should mention in detail the calculation for the percentage of compounds 8, 5, 6, 7 from table 2 and table 3.
We have added a footnote in Table 2 and 3, indicating that the percentage of these compound have been obtained by GC-MS analysis of the crude reaction mixture.
Reviewer 2 Report
TITLE
The title of this research should be restated under the context of the obtained research described in the manuscript. Recognizing that none of the microalgae strains involved, Chlorella sp. (211.8b and 211.8p) and Chlorococcum sp. (JB3), involved in the oxidation of terpanoids, the title of this manuscript should be rethought.
Abstract
The abstract describes as the main approach the use of certain strains of microalgae for the oxidation of terpenoids to obtain natural flavors and flagrances as a novel industrial alternative. But in the same section it is recognized that the oxidation of terpenoids is not related to the presence of microalgae. Therefore, from this point the focus of the article should be rethought. Since the manuscript does not show any evidence of those proposed in the title.
Introduction
The article focuses on the role of microalgae for the oxidation of terpenoids to obtain natural flavors and fragrances by biotransformation. But the experimental evidence shows that there is no biotransformation action in the oxidation of terpenoids, for which it is necessary to rethink the introduction based on the experimental information obtained. A biotransformation action route is proposed, but the experimental evidence does not correspond to what is described in this section. In the manuscript, reference is made to market prices, but in the text there is no economic analysis of the production process of these natural flavors and fragrances.
It is suggested that the introduction section be written from the perspective of the experimental information obtained in this research so that the article is clear in its scientific contribution.
Have a typing error on line 86, need to be corrected.
Materials and Methods
The Materials and Methods section is correctly written about the work presented in this manuscript. It is suggested to correct in section 2.1. Chemicals the information regarding the purity of the products obtained from the epoxidation of α-ionone, theaspirane and β-ionone. The manuscript does not describe the yield and purity of the terpenoids used in the experiment, only the technique used to obtain them is mentioned.
It is suggested that section 2.5. Biotransformation experiments and analysis is more detailed regarding the analysis of the samples obtained that are analyzed to determine the degree of oxidation of the terpenoids. This section contains the information but only generically mentions the operating parameters of the GC-MS analyzes: HP-6890 gas chromatograph. It is important to improve the description of this section to improve the clarity of the information obtained in this investigation.
Results
In the results section, although there is valuable information, it is necessary to improve its presentation and discuss in detail the figures and tables presented. The manuscript in this section should improve the presentation of its figures and tables to give them clarity in the information presented.
Discussion
Section 4. Discussion presents clearly what is presented in the figures and tables of section 3. Results, should be complemented in order to be clear about the real focus of this research. In addition, the discussion continues to focus on the participation of microalgae strains for the biotransformation of terpenoids, but the experimental information presented supports another approach to the investigation.
Conclusion
The conclusions show a scientific fact already described in other investigations, terpenoids are photosensitive and susceptible to oxidation. The new information that is shown in this section is that the microalgae in the studied conditions do not generate any type of biotransformation of the terpenoids. In addition to demonstrating the oxidize valencene to nootkatone. But it is not conclusive with the reaction parameters or with the yields and purities. It is necessary to rethink the approach of the manuscript based on the experimental information that has been obtained. So that the conclusions can provide relevant information for the scientific community.
References
The 28% percent are references from the period 2016-2021, 31% from the period 2010-2015, 41% are references are are lower than the year 2010. In general, 68% of the references it is older than 5 years.
An effort has been made to update the references that support the research. Even so, it is recommended to have a greater number of references no greater than 5 years from the date the manuscript is submitted for review. The updated references allow us to observe the trends in the area of role of microalgae in the oxidation of terpenoids and the novelty of the research described in the manuscript. Currently, the use of microalgae for terpenoid biotransformation is an area of interest in science. Therefore, researchers are encouraged to improve the up-to-dateness of the references presented in this manuscript.
Author Response
-Title:
We modified the title according to the referee comments.
-Abstract:
we rephrased the abstract according to the referee comments.
-Introduction:
We described the reasons of the relevance of the biotechnological processes leading to the natural terpenoids used as flavour ingredients. This discussion is important since helps the readers to understand the relevance of our experimental data, which disprove the microalgae oxidation capabilities.
A complete economic analysis of the production process is out of the study scope.
We modified the introduction section reporting examples of microalgae used as catalyst for the bio-conversion of terpenes. A lot of studies show the potential of the microalgae in the production of secondary metabolites but, according to our knowledge, no other studies were highlighted on the oxidation capabilities of these microorganism on terpenoids recently. If the refery knows something more and wants to share these data, we will be happy to insert the literature him/her suggests.
-Material and methods:
We introduced the purity of the starting materials. For commercial products, we indicated the reported purity whereas for synthesized products we indicated the purity measured by GC-analysis.
The referee states: “This section contains the information but only generically mentions the operating parameters of the GC-MS analyser: HP-6890 gas chromatograph. It is important to improve the description of this section to improve the clarity of the information obtained in this investigation.”
We have described the extraction/derivatization procedure and then the GC-MS equipment, the column and the temperature program. What else is required?
-Results and Discussion:
Results must be clear and concise according to the policy of the journal. We reported the results obtained and we fully explained the data in the Discussion section. What else is required?
In any case, we modified the text adding something more that can help the reader in understanding the data without go ahead in the discussion section.
-Conclusion:
The referee wrote: “ … demonstrating the oxidize valencene to nootkatone. But it is not conclusive with the reaction parameters or with the yields and purities.”
To be honest, we did not understand the referee requirements.
Additionally, at our knowledge, this is the first work reporting the transformation of valencene to nootkatone using water, light and oxygen. The selectivity we obtained is quite unpredictable due the different position that could be oxidised. We don’t think the work proposed does not provide relevant information for the scientific community and we are sure that other experiments are necessary to improve and to go deeper in the mechanism to get the process to the industrial level.
Reviewer 3 Report
Chemistry-1251341
Oxidation of terpenoids to achieve high-value flavor and fragrances. The role of the microalgae and of their growth medium in the biotransformation of the sesquiterpene valencene and of selected apocarotenoids
The authors evaluated the use of three kinds of microalgae to the biotransformation of the different terpenes to get flavor and fragrances of high-relevance natural products.
Other minor requirements are of importance to support the manuscript:
Lines 129, 132, 133, 151, 158, 162, 187, 189, 192, 202, 212, 246: Please insert a space between letters and symbols highlighted in the manuscript PDF version attached.
Lines 87, 91, 103, 225, 263: Please delete the space between symbols highlighted in the manuscript PDF version attached.
Line 173: Please use the bold style on the number highlighted in the manuscript PDF version attached.
Lines 198, 246: Please use capital letter highlighted in the manuscript PDF version attached.
Lines 179, 202: Please use the lowercase letter highlighted in the manuscript PDF version attached.
In Table 2
It said: % of 8 found in
It should say: % of 8 compound found in
Line 112
It said: Tu V.A et al. in 2014 [21]. 4-Acetoxy-β-
It should say: Tu et al. in 2014 [21]. 4-acetoxy-β-
In the section of Reference
Lines 319, 339, 345, 362. The sentences highlighter must be written with lowercase letters
Comments for author File: Comments.pdf
Author Response
Refery 3
Lines 129, 132, 133, 151, 158, 162, 187, 189, 192, 202, 212,246: Please insert a space between letters and symbols highlighted in the manuscript PDF version attached.
Modified Accordingly
Lines 87, 91, 103, 225, 263: Please delete the space between symbols highlighted in the manuscript PDF version attached.
Modified Accordingly
Line 173: Please use the bold style on the number highlighted in the manuscript PDF version attached.
Modified Accordingly
Lines 198, 246: Please use capital letter highlighted in the manuscript PDF version attached.
Modified Accordingly
Lines 179, 202: Please use the lowercase letter highlighted in the manuscript PDF version attached.
Modified Accordingly
In Table 2 It said: % of 8 found in It should say: % of 8 compound found in
Modified Accordingly
Line 112 It said: Tu V.A et al. in 2014 [21]. 4-Acetoxy-β- It should say: Tu et al. in 2014 [21]. 4-acetoxy-β-
Modified Accordingly
In the section of Reference Lines 319, 339, 345, 362. The sentences highlighter must be written with lowercase letters
Modified Accordingly
Reviewer 4 Report
The aim of this study was to investigate the role of three microalgae strains in the oxidation of different terpenoids. It can be seen that in order to ensure the reliability of the experimental results, the author conducted a reasonable grouping test. However, the final results showed that microalgae did not involved in the oxidation of terpenoids, which is contrary to the previous studies. This study proposed the transformation of the four terpenoids is a photochemical reaction that involve the oxygen as oxidant.
Mainly have the following Suggestions:
- It seems that the addition of microalgae was meaningless on the transformation of the reaction. Since the results of this study are inconsistent with previous studies, it is necessary to fully discuss them, including the possible shortcomings of previous studies and the hypothesis mechanism of photochemical reaction.
- The description of the results is too brief and can be extended appropriately.
- Standards synthesized in the laboratory need to provide NMR and mass spectrum data.
- Please mark the name of the compound below the chemical structure in Figure 1 and Figure 2.
- Please consider using a bar chart to show the contents of tables 2 and 3.
- There seems to be a mistake in line 86.
Author Response
1) According to the suggestion of referee 4, we have proposed a mechanism of the photochemical reaction (Figure 6).
Concerning the previous studies, a possible shortcoming could be ascribed to a missing ‘blank experiment’. Anyway, we should not discuss the results previously reported, so we are not willing to include in the work any comment on this point.
2) In any case, we modified the text adding something more that can help the reader in understanding the data without go ahead in the discussion section.
3)1HNMR, 13C NMR and MS spectrum of all the standard compounds have been already reported by us (see the references 5 and 22 of this work).
4) We added the name of the compound in the footnote.
5) We considered this opportunity while preparing the manuscript but the result was not clear and we decided to use a table format. Anyway, we modified the table to describe better the experiments in the text.
6) We corrected the mistake
Round 2
Reviewer 2 Report
TITLE
The title clearly presents the focal point of the manuscript.
Abstract
The abstract adequately describes the main focus of the research developed in this manuscript. The experimental evidence shown in the article coincides with the discussion raised and the conclusions obtained. In addition to raising a review of the information developed in other research on the use of microalgae in the biotransformation of terpenes.
Introduction
The introduction developed in the manuscript adequately describes the experimental evidence obtained in this investigation. It could be improved by including references to current research in the area.
Materials and Methods
The materials and methods section has the appropriate elements to propose the experimental development of the research and guarantee the quality of the experimental results obtained.
Results
In the results section, although there is valuable information. The section can be enriched by making a comparison with the results of other similar investigations. Specifically comparing terpene transporfaction performances and synthesis conditions.
Discussion
The discussion section is clear and concise. Section 4. Discussion presents clearly what is presented in the figures and tables of section 3.
Conclusion
The conclusions raised in this manuscript reinforce the fact that terpenes are photosensitive and susceptible to oxidation. The experimental information obtained in this research shows that the microalgae under the conditions studied do not intervene in the transformation of terpenoids. The investigation could enrich when comparing the results obtained with those of other similar investigations.}
References
The 26% percent are references from the period 2016-2021, 26% from the period 2010-2015, 48% are references are are lower than the year 2010. In general, 74% of the references it is older than 5 years. An effort has been made to update the references that support the research. Even so, it is recommended to have a greater number of references no greater than 5 years from the date the manuscript is submitted for review. The updated references allow us to observe the trends in the area of role of microalgae in the oxidation of terpenoids and the novelty of the research described in the manuscript. Currently, the use of microalgae for terpenoid biotransformation is an area of interest in science. Therefore, researchers are encouraged to improve the up-to-dateness of the references presented in this manuscript.
Author Response
-Introduction:
The introduction developed in the manuscript adequately describes the experimental evidence obtained in this investigation. It could be improved by including references to current research in the area.
As we said in the previous round of comments, we didn’t find any study regarding the oxidation of terpenes using microalgae in the last 5 years and we are still open to add relevant studies suggested by the reviewer. The introduction section must describe what drive the study and what is behind the choices of the authors. This is a full article, not a review in which we have to highlight the current research on the microalgae.
We add in this section a paragraph regarding the biosynthetic capability of microalgae related to terpenes derivatives to stress the enzymatic potential of these microorganisms in transform this class of compounds. To emphasize this point could give more strength to the manuscript because help the reader in understanding the reasoning of the authors.
We want to underline that this study is focused on the oxidation of terpenes and the most important things related to this research area were already summarized in the introduction section.
-Results and Discussion:
In the results section, although there is valuable information. The section can be enriched by making a comparison with the results of other similar investigations. Specifically comparing terpene transporfaction performances and synthesis conditions.
Our policy is to not make any comparison. “Results” are related only to this manuscript. We obtained specific results following a specific strategy in performing the experiments. We can compare results with other researcher’s groups only if:
- We followed the same protocol;
- We used the same strains
- We used the same growth conditions
- We used the same analytical methods
- We used the same substrates
- We used the same instruments
These six points are only examples and we are trying to explain our reasons to the reviewer. The point is we can not compare any result.
Comparing means to make a mistake and could be unfair.
As we said in the previous round of comments, results must be clear and concise even because there is a specific section of the journal (discussion) in which the authors must explore the significance of the results of the work, not repeat them.
-References:
As we mentioned in the answer related to the introduction section, although there is a plethora of new studies on the production of terpenoids (mainly carotenoids) using microalgae, a very limited number of studies described the microalgae-mediated biotransformation of terpenoids. According to referee suggestion, we added few new recent references regarding the biosynthetic potential of microalgae related to terpenes and carotenoids (previously missed). Anyway, the aim of this study is related to the oxidation of terpenes using bio-based and environmental friendly approaches and it is not our fault if the scientific literature lack in this research area. We think we provided sufficient references to the scope of this manuscript.
Reviewer 4 Report
Overall, the manuscript has been modified.
It is correct that the viewpoint of obtaining NOCAKETONE is made by light oxidation.
I hope that the authors can further strengthen reaction and stable the yield in the study.
Author Response
Overall, the manuscript has been modified. It is correct that the viewpoint of obtaining NOCAKETONE is made by light oxidation. I hope that the authors can further strengthen reaction and stable the yield in the study.
We are performing other experiments to improve the potential of this kind of reaction. The optimization will be published in another research paper.
