Utilization of Quinoa Post-Fermentation Waste as a Medium for Carotenoid Production by Yeast

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study researched the application of fermentation wastes as medium in carotenoid production.

Although the results indicated the feasibility of some candidate wastes, the whole study lacked in-depth research. e.g. (1)what's the major bottlenecks in applying fermentation wastes? (2) the major components in carotenoids can be affected by different mediums?

In addition, routine methods in fermentation studies are lacked in this manuscript, any process  data in fermentation techniques (any checking points during 5 day phase)??? other than FTIR spectrum, any quantitative and qualitative assays have been ferformed by this study?

Pay afftention to reduce the utilization of AI tool in preparation of the manuscript. e.g. table head of Table 2. seems to be done by AI.

Author Response

Dear Reviewer 1.,

Thank you for a helpful review of our manuscript. All comments helped us to clarify our results and to improve the readability of the article. Your suggestions have been incorporated as appropriate into the revised version of the manuscript. The manuscript has been modified, reorganized, and completed. Please find below our answers. We appreciate your contribution that helps improve the manuscript.

Although the results indicated the feasibility of some candidate wastes, the whole study lacked in-depth research. e.g. (1)what's the major bottlenecks in applying fermentation wastes? (2) the major components in carotenoids can be affected by different mediums?

Added the information in line 79-81.

In addition, routine methods in fermentation studies are lacked in this manuscript, any process  data in fermentation techniques (any checking points during 5 day phase)??? other than FTIR spectrum, any quantitative and qualitative assays have been ferformed by this study?

The main objective of our research was to determine the possibility of using plant digestate as a substrate for carotenoid synthesis by yeast. Therefore, in preliminary studies, tests were carried out to confirm the ability of yeast to reproduce in digestate, and their growth over time was analysed. The aim was to determine the cultivation time required to obtain the highest carotenoid effect from the tested strains. We decided that publishing these results was not essential to the objective of the study.  During this time, checks were also carried out on changes in basic parameters, such as pH.

Quantitative analyses were performed using a spectrophotometer, measuring absorbance at wavelengths corresponding to carotenoids. Using the molar extinction coefficient for a specific wavelength corresponding to specific carotenoid fractions, their presence can be confirmed.  This method is used by researchers, and the results obtained are the basis for drawing conclusions. However, we considered the results due to the similar wavelengths corresponding to specific fractions, the results were considered questionable and were not presented in the manuscript. A methodology for determining carotenoid fractions using HPLC is currently being developed.

Pay afftention to reduce the utilization of AI tool in preparation of the manuscript. e.g. table head of Table 2. seems to be done by AI.

The table title has been corrected. AI was not used.

Reviewer 2 Report

Comments and Suggestions for Authors

The topic of this study is of great interest. I suggest adding some parts to the introduction and checking the other sections carefully, as reported in the detailed comments.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer 2.,

Thank you for a helpful review of our manuscript. All comments helped us to clarify our results and to improve the readability of the article. Your suggestions have been incorporated as appropriate into the revised version of the manuscript. The manuscript has been modified, reorganized, and completed. Please find below our answers. We appreciate your contribution that helps improve the manuscript.

Lines 55–56: Please add references.

Reference added.

Lines 62–73: Implement this section, which provides more generic information. After describing the various types of research, focus this section on the type of plant-based waste you want to apply to your study.

Information about quinoa has been added to the line 75-79.

Lines 74–75: The aim of the study is too generic. Please rephrase the sentence.

The aim of the research was clarified.

Table 1: Check the names of the strains.

Strain names have been corrected.

Paragraphs 2.2 and 2.3: These two paragraphs are the same.

The title of the paragraph has been corrected.

Table 2: Check the title.

The table title has been corrected.

Table 4: The title is incorrect, and the author is reported in both column variants II.

The table title and description of variants have been corrected.

Tables 5–6: Eliminate the standard deviation.

The standard deviation has been removed from the description.

Line 347: Why was quinoa fermentation waste chosen as a culture medium?

Mentioned in line 75-79 and 212-218. Furthermore, in the case of the presented studies, fermented quinoa grains were used as animal feed in other studies conducted by our team. We wanted to use the resulting waste as a valuable nutrient for yeast growth and carotenoid synthesis. This allows us to reduce the costs of microbial carotenoid production and eliminates the generation of post-production waste from fermentation processes.

Line 371: (TF): Add the name in full.

Full name added.

Conclusion: The research presented demonstrated the possibility of synthesizing bioactive carotenoids on a waste substrate, in this case plant post-ferment. What could the main limitations of this approach be?

Added this information in line 216-218.

 

Reviewer 3 Report

Comments and Suggestions for Authors

see attached file

Comments for author File: Comments.pdf

Author Response

Dear Reviewer 3.,

Thank you for a helpful review of our manuscript. All comments helped us to clarify our results and to improve the readability of the article. Your suggestions have been incorporated as appropriate into the revised version of the manuscript. The manuscript has been modified, reorganized, and completed. Please find below our answers. We appreciate your contribution that helps improve the manuscript.

Use of acronyms (TF, VC, CB) The manuscript currently employs the abbreviations TF, VC, and CB to describe carotenoid-related parameters. Although the authors have used this acronyms before (ref 12) in non-English journals, these acronyms are non-standard in the carotenoid and microbial biotechnology literature in English. In addition, although they are defined in table footers and in section 4.4 (page 10), they are widely used throughout the text, even in the abstract. I think they should be defined the first time they are mentioned. In addition, to improve transparency and align the manuscript with common conventions, I strongly recommend replacing these acronyms with widely accepted terminology. The manuscript says that total carotenoid content was calculated according to Chen et al. 2006 [34]. I think it would be good to use the same nomenclature used in reference 34: instead of “VC”, “TC” for total carotenoid content — carotenoids expressed per volume of culture (mg/L) should be used.

Abbreviations are explained when first used. Abbreviations have been changed from TF to TFC and VC to VCC in accordance with the designations adopted by:

Rodríguez, N. A. T., Quiñones-Cerna, C. E., Castillo, H. M. R., Cruz-Monzon, J. A., Butrón, F. J. H., & Soto, J. C. R. (2023). Optimization of total carotenoid production by Rhodotorula mucilaginosa from artichoke agroindustrial waste using response surface methodology. Environmental Research, Engineering and Management, 79(2), 111-121.

Reference 34 does not indicate how the “TF” — carotenoid content expressed in μg/g d.w can be calculated. Therefore, it should be described in section 4.4.

The formula for calculating TFC is provided in line 394-400.

CB is used for “dry cell weight”. I think it would be more reasonable to use DCW. This nomenclature is more intuitive, corresponds to common usage in microbial carotenoid production studies and avoids ambiguity

A change has been made.

Corrections have been made in accordance with the guidelines in the tables.

Heading “2.3. Culture media” Repeated. Possibly the authors mean “Statistical analysis”.

Corrected.

Table 2, Heading. Please use a proper heading, not the manuscript template.

Corrected.

entry SR-20 inconsistent decimal notation 1,05 ± 0.02 433,90 ± 7.81

Corrected.

2.4. FTIR analysis

Figure 1

Hard copy of measured FTIR spectra is always welcomed. However, from figure 1 it is obvious that the carotenoid content was extremely low, as absorbance bands have really small intensities (lower than 2%). One of the greatest advantages of FTIR is that it allows accumulation of scans. Setting the parameters to a higher number of scans should allow to increase the intensity of the signals.

Due to low absorbance, scans are presented as % of transmittance (%T). This mode is typically used in the literature on the subject which cytate in this manuscript. The numbers of scans of the sample was 20. According to the instruction manual, 10 scans are enough and we have doubled this number. In the literature cited in manuscript [24, 25, 26, 27, 28] Authors did not provide the number of scans.

Although all QATR FTIR instruments have a CO2 signal cancellation procedure, it is obvious that for extremely weak signals, the CO2 absorption band can not be completely suppressed. This is the case of this spectra. The absorption bands around 2370 cm-1 are due to residual CO2, and not to carotenoids.

Thank you for a valuable comment. We have added this information to the discussion.

If possible, I would recommend the authors to measure the IR again with a higher number of o scans. In any case, the number of scans used should be mentioned in section 4.7.

The numbers of scans of the sample was 20. According to the instruction manual, 10 scans are enough and we have doubled this number. We have added this information to section 4.7. Thank you for the comment.

Please be aware that IR spectra show “absorption bands”, not peaks. Therefore, that should be replaced in lines 160, 168 and 170.

Thank you for suggestion. We have replaced this term.

Table 3 and line 336 Change mucialginosa for mucilaginosa

Changed.

Line 172. Heading 2.4 should be 2.4, and not FTIR analysis

Changed.

Table 4 Includes an erroneous header (“Variant II Variant II”), which should be corrected to reflect the appropriate experimental variants.

Corrected.

Tables 5 and 6 A format suggestion. First column can be wider, in such a way that the third entry can occupy just one line. It is a bit confusing in the present format.

Tables 5 and 6 have been formatted in accordance with the guidelines.

Section 3.2. FTIR analysis: should be renamed 3.3

Corrected.

• I really do not think that maxima at 1508 cm-1 and 1560 cm-1 are due to N-O bounds. First, N-O bounds are hardly found in nature. Second, the absorption bands around 1550–1600 cm−1 correspond to C=C double bond stretching vibrations of β-carotene. Sentence in 298 should be rephrased accordingly.

Thank you for the commend. We have revised the text according to your suggestion and reference [25]. In addition we have added the sentence regarding absorption band 1008, which may be associated with rocking vibrations of the (RH) C=C (RH) groups of carotenoids [29].

• In addition, aromatic rings are very rare in carotenoids. The absorption bands around 1650 cm-1 are more likely due to C=O groups conjugated with C=C. Although β-carotene does not have oxygen functional groups, C=O groups are not that rare (see reference: Lóránd, T., Deli, J., Molnár, P. and Tóth, G. (2002), FT-IR Study of Some Carotenoids. HCA, 85: 1691-1697.

We have changed the explanation of 1654 cm-1 absorption band according to your comment and suggested literature. Thank you.

• Line 300. The band a 2920 cm-1 is due to aliphatic C-H (sp3 carbons, not sp2). I do not see the relevance of reference 22 in this context, as it does not discuss the IR absorption bands regarding the stereochemistry of double bonds (cis/trans). In any case, the comment “a peak at 2924 cm-1 was observed, indicating the presence of the trans-CH=CH group found in β-carotene” is incorrect and should be suppressed.

Answer: We have changed the discussion regarding the band 2924 cm-1 according to your suggestion and reference. Thank you for the comment.

Table in review

We have added the detailed information (bending or stretching) according to your comment. However in the reference [26] band 2920 cm-1 is attributed to C-H bending.

References Journal names should be abbreviated following journal recommendations.

Corrected.

Reviewer 4 Report

Comments and Suggestions for Authors

Title & Abstract

• Line 2: “plant-based post-fermentation waste” — consider specifying “quinoa post-fermentation waste” to improve precision.

• Lines 16–33: Abstract is informative, but the flow is slightly fragmented. Suggest combining lines 21–25 into one coherent sentence describing the methodological steps.

• Line 31: “formulat” → should be “formulate” or “formulation”.

Introduction

• Lines 43–44: “carotenoids stimulates the immune system” → should be “carotenoids stimulate” (subject–verb agreement).

• Lines 48–50: Consider briefly quantifying the economic disadvantages of synthetic carotenoid production (one sentence) to strengthen rationale.

• Lines 62–73: Good discussion of plant waste types. However, the novelty gap (limited studies on post-ferments) could be more strongly emphasized—suggest adding a sentence about why fermented quinoa is unique.

• Line 70: “under-researched area.” → consider citing at least one supporting reference if available.

Methods (Clarity & Terminology)

• Line 102: Section titled “Culture media” appears twice (2.2 and 2.3). Rename one to avoid confusion.

• Lines 133–142: Clarify why YPG medium produced lower TF compared with post-fermentation waste. A brief biochemical explanation would help readers.

• Line 348: “Fermentation was performed at 5% v/v using L. plantarum” — specify inoculum concentration (cfu/mL) if available.

• Line 382: Powder drying at 50 °C for one hour may degrade carotenoids; consider noting this limitation in Methods or Discussion.

Results

• Lines 103–111: Statistical significance is mentioned but the test used is not specified here (ANOVA described later). Suggest adding “(ANOVA, p < 0.05)” for clarity.

• Lines 117–118: Provide a brief explanation why lentil-origin strains R-1 and R-2 perform better (mentioned later in Discussion but could be summarized here).

• Line 162: FTIR results mention “no effect” — consider briefly describing what would be expected if there was an effect (for context).

• Lines 167–171: FTIR peak assignments should cite references directly in-text (e.g., β-carotene functional groups).

• Line 179: Table 4 caption incorrectly says “Total carotenoid content in powder” — should be “DPPH reduction activity”.

Figures & Tables

• Figure on Page 5: axes labels are small; recommend improving readability.

• Table 2 (Line 128):

  • Some decimal commas (e.g., “1,05”) use comma instead of period. Ensure consistent formatting (MDPI style uses periods).

  • Ensure consistent significant figures across rows.

• Table 3 (Line 156): TF values expressed in mg/g d.w. but earlier TF was μg/g d.w. Clarify unit conversion or adjust terminology.

Discussion

• Lines 225–227: Statement “possibility of using post-fermentation waste as nutrient source” — suggest quantifying its performance relative to literature.

• Lines 243–246: Very interesting ecological explanation for strain performance; consider expanding by adding one supporting reference.

• Lines 250–270: Co-culture discussion could benefit from briefly explaining why L. plantarum enhances or decreases TF depending on strain.

• Lines 282–288: Add a sentence discussing possible carotenoid oxidation pathways during drying.

• Line 291: Section heading misnumbered (“3.2. FTIR analysis”). Should be “3.3” based on previous headings.

Conclusion

• Lines 430–433: Good practical implications. Consider adding one sentence on potential scalability or industrial applicability.

Minor Language & Formatting Corrections

• Replace all comma decimals with period decimals (e.g., “1,05” → “1.05”).

• Correct spacing before units (e.g., “mg/g d.w.”).

• Ensure consistent italics for species names (Rhodotorula mucilaginosa, Lactobacillus plantarum, etc.).

• Standardize “carotenogenic yeasts” vs “carotenoid-producing yeasts” usage.These two references mighe be helpful to improve the quality of this manuscript: A comprehensive review of the use of sensors for food intake detection (https://www.sciencedirect.com/science/article/pii/S0924424720306464);

• Co-delivery of curcumin and quercetin in the bilayer structure based on complex coacervation (https://www.sciencedirect.com/science/article/pii/S0268005X23004538)

Author Response

 

Dear Reviewer 4.,

Thank you for a helpful review of our manuscript. All comments helped us to clarify our results and to improve the readability of the article. Your suggestions have been incorporated as appropriate into the revised version of the manuscript. The manuscript has been modified, reorganized, and completed. Please find below our answers. We appreciate your contribution that helps improve the manuscript.

Line 2: “plant-based post-fermentation waste” — consider specifying “quinoa post-fermentation waste” to improve precision.

Corrected.

Lines 16–33: Abstract is informative, but the flow is slightly fragmented. Suggest combining lines 21–25 into one coherent sentence describing the methodological steps.

Corrected.

Line 31: “formulat” → should be “formulate” or “formulation”.

Corrected.

Lines 43–44: “carotenoids stimulates the immune system” → should be “carotenoids stimulate” (subject–verb agreement).

Corrected.

Lines 48–50: Consider briefly quantifying the economic disadvantages of synthetic carotenoid production (one sentence) to strengthen rationale.

Information added in line 47-50.

Lines 62–73: Good discussion of plant waste types. However, the novelty gap (limited studies on post-ferments) could be more strongly emphasized—suggest adding a sentence about why fermented quinoa is unique.

Added information in line 75-79.

Line 70: “under-researched area.” → consider citing at least one supporting reference if available.

Thank you for your opinion, corrected.

Line 102: Section titled “Culture media” appears twice (2.2 and 2.3). Rename one to avoid confusion.

Corrected.

Lines 133–142: Clarify why YPG medium produced lower TF compared with post-fermentation waste. A brief biochemical explanation would help readers.

Added explanation in line  136 and 238-242.

Line 348: “Fermentation was performed at 5% v/v using L. plantarum” — specify inoculum concentration (cfu/mL) if available.

Added information about inoculum - 6.0 x 10⁵ cfu/ml in line 369.

Line 382: Powder drying at 50 °C for one hour may degrade carotenoids; consider noting this limitation in Methods or Discussion.

Added the information in line 291-293.

Lines 103–111: Statistical significance is mentioned but the test used is not specified here (ANOVA described later). Suggest adding “(ANOVA, p < 0.05)” for clarity.

Added.

Lines 117–118: Provide a brief explanation why lentil-origin strains R-1 and R-2 perform better (mentioned later in Discussion but could be summarized here).

Added.

Line 162: FTIR results mention “no effect” — consider briefly describing what would be expected if there was an effect (for context).

Added explanation in line 165-167.

Lines 167–171: FTIR peak assignments should cite references directly in-text (e.g., β-carotene functional groups).

Added the information in line 164.

Line 179: Table 4 caption incorrectly says “Total carotenoid content in powder” — should be “DPPH reduction activity”.

Corrected.

Figure on Page 5: axes labels are small; recommend improving readability.

Corrected.

Table 2 (Line 128):

• Some decimal commas (e.g., “1,05”) use comma instead of period. Ensure consistent formatting (MDPI style uses periods).
• Ensure consistent significant figures across rows.

Checked and corrected.

Table 3 (Line 156): TF values expressed in mg/g d.w. but earlier TF was μg/g d.w. Clarify unit conversion or adjust terminology.

The unit mg/g d.w. was used for the obtained powder to make the results more transparent. Furthermore, this allowed for a comparison of the total carotenoid content in the powder with the results of other authors who also used this unit.

Lines 225–227: Statement “possibility of using post-fermentation waste as nutrient source” — suggest quantifying its performance relative to literature.

Corrected.

Lines 243–246: Very interesting ecological explanation for strain performance; consider expanding by adding one supporting reference.

Thank you for your opinion. Added reference.

Lines 250–270: Co-culture discussion could benefit from briefly explaining why L. plantarum enhances or decreases TF depending on strain.

Wydaje mi się, że zostało to wytłumaczone 274-276.

Lines 282–288: Add a sentence discussing possible carotenoid oxidation pathways during drying.

Added the information in line 298-302.

Line 291: Section heading misnumbered (“3.2. FTIR analysis”). Should be “3.3” based on previous headings.

Corrected.

Lines 430–433: Good practical implications. Consider adding one sentence on potential scalability or industrial applicability.

Thank you for your suggestion.

Minor Language & Formatting Corrections

• Replace all comma decimals with period decimals (e.g., “1,05” → “1.05”).

Corrected.

• Correct spacing before units (e.g., “mg/g d.w.”)

Corrected.

• Ensure consistent italics for species names (Rhodotorula mucilaginosa, Lactobacillus plantarum, etc.).

Checked.

• Standardize “carotenogenic yeasts” vs “carotenoid-producing yeasts” usage.These two references mighe be helpful to improve the quality of this manuscript: A comprehensive review of the use of sensors for food intake detection (https://www.sciencedirect.com/science/article/pii/S0924424720306464);
• Co-delivery of curcumin and quercetin in the bilayer structure based on complex coacervation

Thank you very much for pointing out very interesting scientific articles

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Although the obvious format mistakes were corrected in the revised manuscript. However, additional experimental improvement was not seen in the revised manuscript. 

as seen in previous comment (AS SEEN THE FOLLOWING PARAGRAPH), only spectrum assay cannot guarantee the final production of objective products. THIS IS THE MAJOR BOTTLENECK FOR THE MANUSCRIPT.

In addition, routine methods in fermentation studies are lacked in this manuscript, any process  data in fermentation techniques (any checking points during 5 day phase)??? other than FTIR spectrum, any quantitative and qualitative assays have been ferformed by this study?

 

Author Response

Dear Reviewer 1.,

Thank you for a helpful review of our manuscript. All comments helped us to clarify our results and to improve the readability of the article. Your suggestions have been incorporated as appropriate into the revised version of the manuscript. The manuscript has been modified, reorganized, and completed. Please find below our answers. We appreciate your contribution that helps improve the manuscript.

Although the obvious format mistakes were corrected in the revised manuscript. However, additional experimental improvement was not seen in the revised manuscript. As seen in previous comment (AS SEEN THE FOLLOWING PARAGRAPH), only spectrum assay cannot guarantee the final production of objective products. THIS IS THE MAJOR BOTTLENECK FOR THE MANUSCRIPT.

In addition, routine methods in fermentation studies are lacked in this manuscript, any process  data in fermentation techniques (any checking points during 5 day phase)??? other than FTIR spectrum, any quantitative and qualitative assays have been ferformed by this study?

The publication presents the first in a planned series of studies.  The aim of the research was to determine the ability of yeast strains to synthesise carotenoids on waste substrate in order to select the most optimal strains for further studies, in which a detailed analysis of the obtained carotenoids is planned. In addition, FTIR analysis provided insight into the structure of the carotenoids obtained. However, the amount of β-carotene obtained, determined by spectrophotometry, was added. In subsequent stages of the research, HPLC chromatography is expected to be used – the methodology is currently being established.

Diagrams showing biomass growth depending on the cultivation variant used, in which waste was used as the substrate, have also been added.

We appreciate your valuable comments.

Yours sincerely,

Ewa Kulczyk-Małysa.