Ultrasound-Assisted Kinetics of Alcoholic Fermentation at Varying Power Levels for the Production of Isaño Wine (Tropaeolum tuberosum)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates the effects of ultrasound-assisted fermentation on isaño wine production at different power levels, exploring how ultrasound technology can optimise the alcoholic fermentation of isaño wine, particularly on yeast growth kinetics, ethanol yield and substrate consumption. The application of ultrasound to isaño wine fermentation is novel and has the potential to impact the production of functional beverages in the study.

However, some problems must be major revised in the manuscript, particularly the depth of discussion in the introduction and conclusion. Of note, authors have to refine the novelty and significance of the study, which is very important for getting it published.

 

1. Introduction

The introduction provides a comprehensive background on isaño (Tropaeolum tuberosum), its nutritional and medicinal properties, and its potential use in fermented beverages. The introduction also effectively introduces the concept of using ultrasound technology to enhance fermentation processes, citing previous studies that have explored similar applications in other fermentation systems. However, there are still some issues that need to be revised by the author.

Line 38-52 Both of these paragraphs introduce isaño, so they can be elaborated as one paragraph.

Line 38-39 Three isaño genotypes were used in the study, and it should be detailed here why these three were used as study subjects?

Line 55-58 More detailed discussion on the specific challenges faced in fermenting isaño wine, particularly in relation to its starch content.

Line 58-60 Pulsed electric fields, gamma radiation, high pressure and ultrasound are listed here as reducing production time, so why choose ultrasonic instead of the remaining three processes? Detail the advantages and disadvantages of the remaining three methods.

Line 67-80 Re-summarizing the passage to avoid literature listing.

Line 80 Restatement of the novelty of applying ultrasound to isaño fermentation.

Line 83-85 Why these specific power levels were chosen and how they relate to the potential industrial application of ultrasound in the wine production? Additionally, the study design could have included a broader range of ultrasound frequencies to explore their effects on fermentation kinetics.

2. Materials and methods

The study have chosen to investigate the effect of ultrasound at different power levels (0 W, 100 W, 200 W, and 300 W) on the fermentation kinetics of three isaño genotypes. The use of a completely randomized design with triplicates ensures statistical robustness. The inclusion of a control group (0 W) allows for a clear comparison of the effects of ultrasound. 

Line 90-91 Detailed description of the sampling method. How are samples stored and at what temperature?

Line 91-92 Does rinsing with tap water before processing affect the wine fermentation?

2.2. The section on ultrasound application could include more details on the equipment used, such as the type of ultrasonic bath and how the power levels were calibrated.

Line 109-110 Provide more information on the specific strain of Saccharomyces cerevisiae used and its relevance to the study.

2.3.2 Give the standard curve of the ethanol concentration.

2.3.3 The authors describe the use of the DNS method for the assessment of residual sugars. DNS assay is susceptible to interferences and therefore results in inaccurate analyses.

2.3.4 The detail messages of digital refractometer.

2.3.5 DoseResp, SGompertz and Logistic models are also often used to evaluate wine fermentation kinetics, so why were they not used in this study?

3. Results and discussion

The results section could benefit from a more detailed statistical analysis, particularly in comparing the effects of different ultrasound power levels.

Providing more detailed mechanistic insights into how ultrasound affects yeast metabolism.

3.2. Expanded discussion of the mechanism of ethanol production in ultrasound-assisted fermentation and how it is affected, rather than a simple comparison of the literature.

3.3. and 3.4.  Why the 100 W treatment was the most effective, possibly linking it to the specific physiological responses of the yeast to ultrasound.

4. Conclusions

The conclusion could be strengthened by discussing the broader implications of the findings for the food and beverage industry, particularly in terms of scalability and potential challenges in industrial application.

 

Line 68, 73, 110, etc. Check the full text, latin genus names of micro-organisms all in italics.

Line 62, 104, etc. Check the full text, only one space character can exist between words.

Line 195-197 Delete this paragraph.

Some references are formatted incorrectly, proofread carefully and make corrections. For example 37, 41...

There are minor grammatical errors, such as inconsistent verb tenses and occasional awkward phrasing.

Comments on the Quality of English Language

The language of this manuscript is simple and clear. However, the entire text needs to be checked and corrected for inconsistent verb tenses and occasional misuse of words.

Author Response

Comments 1: Line 38-52 Both of these paragraphs introduce isaño, so they can be elaborated as one paragraph.

Response 1: Both paragraphs introduce Isaño, so they have been merged into a single paragraph to improve coherence and avoid redundancy.

Comments 2: Line 38-39 Three isaño genotypes were used in the study, and it should be detailed here why these three were used as study subjects?

Response 2: The following text has been added: “There are several varieties, the most common being yellow, purple, and yellow with purple eyes”.

Comments 3: Line 55-58 More detailed discussion on the specific challenges faced in fermenting isaño wine, particularly in relation to its starch content.

Response 3: The following text has been added to address the specific challenges of Isaño wine fermentation related to its starch content: The fermentation of Isaño presents several challenges, particularly the conversion of starch into fermentable sugars and yeast inhibition due to the presence of glucosinolates and phenolic compounds. To overcome these obstacles, a physical, chemical, or enzymatic pretreatment, or a combination of these, must be applied to hydrolyse the starch and release simple sugars, such as glucose and maltose, which can be efficiently utilised by yeasts in winemaking [21].

Comments 4: Line 58-60 Pulsed electric fields, gamma radiation, high pressure and ultrasound are listed here as reducing production time, so why choose ultrasonic instead of the remaining three processes? Detail the advantages and disadvantages of the remaining three methods.

Response 4: Based on the literature review, we found that ultrasound is the most suitable technique for enhancing fermentation, particularly due to its ability to improve yeast cell permeability and its lower risk compared to other methods such as gamma radiation, high hydrostatic pressure, and pulsed electric fields. These alternative techniques involve significant operational challenges, including higher costs, safety concerns, and potential negative effects on yeast viability.

Given that our research facilities are equipped with ultrasound technology, we chose to focus on its advantages for our study. We have now included a general overview of the alternative techniques, highlighting their potential benefits and limitations based on existing literature ( Galván-D’Alessandro , & Carciochi, R. A. (2018) and Zhang, W., Wang, L., & Li, D. (2022)).

Galván-D’Alessandro, L., & Carciochi, R. A. (2018). Fermentation Assisted by Pulsed Electric Field and Ultrasound: A Review. Fermentation, 4(1), 1. https://doi.org/10.3390/fermentation4010001

Zhang, W., Wang, L., & Li, D. (2022). Potential applications of pulsed electric field in the fermented wine industry. Frontiers in Microbiology, 13, 966825. https://doi.org/10.3389/fmicb.2022.966825

Comments 5: Line 67-80 Re-summarizing the passage to avoid literature listing.

Response 5: It has been summarised: Several studies have reported on the use of ultrasound in fermentation processes, highlighting that its effect depends on the growth phase of the yeast, as well as the frequency and power of the ultrasound applied. It has been reported that the application of ultrasound in the fermentation of cider with Hansenia sp., at a frequency of 40 kHz and a power of 100 W accelerated the growth of the yeast, reaching values ​​up to 10 times higher than those of the control group [26]. Similarly, in beer fermentation with Saccharomyces cerevisiae, ultrasonic treatment at 40 kHz and 120 W increased the glucose fermentation rate by 2.3 and 2.5 times at temperatures of 30 °C and 20 °C, respectively [27]. Other studies reported that the use of 40 kHz and 160 W improved ethanol production by 13.18% [28], while the use of 23 kHz and 120 W increased it by 19.33% [29].

Comments 6: Line 80 Restatement of the novelty of applying ultrasound to isaño fermentation.

Response 6: The following text has been added: Taken together, these results demonstrate that the use of ultrasound in fermentation reduces process time and improves product quality characteristics [24]. Furthermore, it is regarded as a green, sustainable, innovative, cost-effective, rapid, portable, and easy-to-use technology, and its non-destructive nature makes it highly suitable for fermentation processes [31]. However, its effectiveness depends on the type and frequency of ultrasound, the type of antinutrient, the duration of exposure and the composition of the food.

Comments 7: Line 83-85 Why these specific power levels were chosen and how they relate to the potential industrial application of ultrasound in the wine production? Additionally, the study design could have included a broader range of ultrasound frequencies to explore their effects on fermentation kinetics.

Response 7: The selection of specific ultrasound power levels in the aforementioned study responded to the need to evaluate how different intensities influence the alcoholic fermentation of isaño (Tropaeolum tuberosum). By applying power levels ranging from 100 to 300 W, the goal was to identify an optimal balance that maximizes benefits such as yeast growth and ethanol production, without incurring the adverse effects that can arise with higher powers. The selected powers are related to potential industrial uses. The frequency used in the present study was 50 kHz; a broader range of frequencies will be studied in future research.

 

1. Materials and methods

The study have chosen to investigate the effect of ultrasound at different power levels (0 W, 100 W, 200 W, and 300 W) on the fermentation kinetics of three isaño genotypes. The use of a completely randomized design with triplicates ensures statistical robustness. The inclusion of a control group (0 W) allows for a clear comparison of the effects of ultrasound. 

Comments 8: Line 90-91 Detailed description of the sampling method. How are samples stored and at what temperature?

Response 8:

• The following text was included: "Samples of each genotype were collected using a random sampling method."
• They were stored in a dark location at 25 ± 2°C with a relative humidity of 68.1 ± 1.5%.

Comments 9: Line 91-92 Does rinsing with tap water before processing affect the wine fermentation?

Response 9: All samples were washed with water to remove any soil residue, which could otherwise affect product quality.

Comments 10: 2.2. The section on ultrasound application could include more details on the equipment used, such as the type of ultrasonic bath and how the power levels were calibrated.

Response 10: The following text has been added: "equipped with a multi-frequency ultrasound generator (QX Ultrasonic, Beijing, China)."

Comments 11: Line 109-110 Provide more information on the specific strain of Saccharomyces cerevisiae used and its relevance to the study.

Response 11: Saccharomyces cerevisiae EC-1118 was used.

Comments 12: 2.3.2 Give the standard curve of the ethanol concentration.

Response 12: The standard curve equation is as follows:

y = 0.1503x + 0.0153

R² = 0.9998

Comments 13: 2.3.3 The authors describe the use of the DNS method for the assessment of residual sugars. DNS assay is susceptible to interferences and therefore results in inaccurate analyses.

Response 13: The 3,5-dinitrosalicylic acid (DNS) method is a widely used colorimetric technique for determining reducing sugars in biological and industrial samples. However, it has certain limitations that may affect result accuracy, such as susceptibility to interference from other compounds, variability in the reaction, and sugar degradation at high temperatures. To enhance accuracy and minimise interference, alternative methods such as high-performance liquid chromatography (HPLC) and enzymatic assays can be considered. Nevertheless, in this study, the DNS method demonstrated adequate performance, as evidenced by the observed trends in biomass consumption kinetics during fermentation.

 

Comments 14: 2.3.4 The detail messages of digital refractometer.

Response 14: Soluble solids were measured using a tabletop Milwaukee MA871-BOX digital refractometer (Milwaukee Instruments, Inc., NC, USA).

Comments 15: 2.3.5 DoseResp, SGompertz and Logistic models are also often used to evaluate wine fermentation kinetics, so why were they not used in this study?

Response 15: The aim was to model microbial growth as a function of nutrient availability. The Monod model was chosen as the most appropriate option, as it is based on real biochemical mechanisms. In contrast, models such as DoseResp, SGompertz, and Logistic are more suited to other types of processes, such as pharmacological, ecological, or population growth studies.

1. Results and discussion

The results section could benefit from a more detailed statistical analysis, particularly in comparing the effects of different ultrasound power levels.

Providing more detailed mechanistic insights into how ultrasound affects yeast metabolism.

Comments 16: 3.2. Expanded discussion of the mechanism of ethanol production in ultrasound-assisted fermentation and how it is affected, rather than a simple comparison of the literature.

Response 16: The following text has been added: Ultrasound treatment increased ethanol production in S. cerevisiae because it promotes yeast growth by increasing membrane permeability and enzymatic activities [43]. Ultra-sound intensity affects cellular ultrastructure. Previous studies have shown that, in sam-ples without ultrasonic treatment, cells remain intact and maintain a uniform morpholo-gy, with the cytoplasm and organelles homogeneously distributed, along with a clearly visible nuclear region. However, when ultrasonic treatment is applied at 102.8 W for 10 minutes, the cellular structure becomes irregular, with no visible organelles in the cyto-plasm, the presence of vacuoles, and signs of plasmolysis in some cells. At a higher inten-sity of 288 W for the same duration, significant damage is observed, with evidence of plasmolysis [44].

Comments 17: 3.3. and 3.4.  Why the 100 W treatment was the most effective, possibly linking it to the specific physiological responses of the yeast to ultrasound.

Response 17: Ultrasound intensity has a significant impact on cellular ultrastructure, which likely explains why the 100 W treatment was the most effective. Previous studies have shown that, in samples without ultrasonic treatment, cells maintain an intact, uniform morphology, with the cytoplasm and organelles homogeneously distributed and a clearly visible nuclear region. However, when ultrasonic treatment is applied at 102.8 W for 10 minutes, the cellular structure becomes irregular, with no visible organelles in the cytoplasm, the appearance of vacuoles, and signs of plasmolysis in some cells. At higher intensities, such as 288 W for the same duration, more severe damage is observed, with evident plasmolysis [44]. These results suggest that the 100 W treatment might be optimal for stimulating yeast growth and metabolic activity, as it promotes beneficial physiological responses without causing excessive damage. At lower intensities, the ultrasound might not provide enough energy to induce significant changes, while at higher intensities, the damage to cellular structures may outweigh the benefits, leading to a decrease in yeast viability.

1. Conclusions

The conclusion could be strengthened by discussing the broader implications of the findings for the food and beverage industry, particularly in terms of scalability and potential challenges in industrial application.

Comments 18: Line 68, 73, 110, etc. Check the full text, latin genus names of micro-organisms all in italics.

Response 18: This has been corrected in the article.

Comments 19: Line 62, 104, etc. Check the full text, only one space character can exist between words.

Response 19: This has been corrected in the article.

 

Comments 20: Line 195-197 Delete this paragraph.

Response 20: This paragraph has been deleted as requested.

 

Comments 21: Some references are formatted incorrectly, proofread carefully and make corrections. For example 37, 41...

Response 21: This has been corrected in the article.

There are minor grammatical errors, such as inconsistent verb tenses and occasional awkward phrasing.

Comments on the Quality of English Language

The language of this manuscript is simple and clear. However, the entire text needs to be checked and corrected for inconsistent verb tenses and occasional misuse of words.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript describes research that investigated the effects of ultrasound application and the isano genotypes on growth kinetics and ethanol fermentation of S. cerevisiae cultures, using hydrolyzed isano must as the substrate. The results indicated that an ultrasonic power applied at 100 W positively affected fermentation parameters and increased the ethanol yield. Higher ultrasonic powers, however, had less positive effects. In addition, among three different isano genotypes tested, the purple isano had the highest ethanol yield. The research described in the manuscript is original. The results could be interesting for potential biotechnological applications regarding isano wine production. However, there are some major and minor points that need to be considered and/or improved:

- Section 2.3.1, line 130: why are the plates incubated at 36-38 C, and not at 30 C or 25 C (the fermentation temperature used in this study – line 116)? The authors should clarify these temperature differences in their experimental procedures.

- The authors indicated in the Methods section (lines 103-108) that the isano must with a high starch content was treated with alpha-amylase and amyloglucosidase, before the yeast fermentation. It is known that the yeast S. cerevisiae cannot utilize starch. The use of isano must as a substrate for S. cerevisiae fermentation therefore requires these enzymatic treatment steps which may be costly at large scale. The authors should discuss this issue and the potential use of alternative strategies such as development /use of starch-degrading, recombinant S. cerevisiae strains etc. by checking the related literature.

- Results, Section 3.1.: Is the S. cerevisiae strain used in this study a flocculating strain? Did the authors observe differences in cell flocculation/aggregation under the microscope etc.? They should clarify these points in Section 3.1.

- Regarding Fig 1A,B and C, the authors should comment on or suggest a possible explanation for the decrease in biomass concentrations of the cultures treated with ultrasound after the 60th hour of fermentation, particularly as there is no decrease in ethanol production of these cultures after the 60th hour.

- The authors should verify/comment on the umax value (0.015) observed for the purple genotype at 100 W. This value seems to be too low. All their other results generally indicate the best performance with the purple genotype and at 100 W. Thus, a higher mmax would be expected at 100 W than any of the other conditions ( control, 200 and 300 W).

- line 41, please correct as: ‘… Andean populations, mashua (isano) is also recognized for….’

- line 287, please correct as: ‘… with the increase in biomass…’

- line 294, please correct as ‘The must provides a favorable….’

- In Table 1, line 143: please indicate the concentration values in g/L instead of mg/dL.

- Section 2.3.3., line 163: please indicate the centrifugation rate in g, not in rpm.

- Section 2.3.5., lines 171-172: please use superscripts for h-1 and  subscripts for Ks and umax. Please check and correct such issues throughout the whole manuscript (e.g. in lines 181-182, lines 330-339, 344-352 and 358.

- Results and Discussion, lines 195-197: please remove these instructions.

- Please write the genus and species names in italic: (Saccharomyces cerevisiae, Tropaeolum tuberosum, S. cerevisiae, Lactobacillus sakei etc.)

- line 222, please write species names in lowercase letters (e.g. cerevisiae, not Cerevisiae).

- line 328, please correct as Monod.

 

- line 334-335, please correct as: ‘Under control conditions, the max values were 0.0007……..’ (the authors should indicate at the beginning of that sentence that those data belong to the control condition.)

-line 340, Table 3, please correct as Y  x/s, and Y p/s. (They are currently written as And x/s , And p/s).

- line 341, Table 3, please indicate what each of these different superscripts (a, b,c,d) stand for, e.g. are they different significance levels, if so which letter belongs to which.

- lines 345-346, please check Table 3 and correct that sentence accordingly. It should be: ‘For yellow isano, umax increased from 0.006…..to 0.029……. as the power decreased from 300 to 100 W.’

- lines 347-348, please correct as: ‘……to 0.211…..h-1, as the power decreased from 300 to 100 W.’  

- lines 348-349, please correct as: ‘..., as the power increased from 100 W to 300 W. Specifically,it was…’  

 

 

 

 

 

Comments on the Quality of English Language

Quality of English language is good. However, English proofreading is  recommended for some minor issues. 

Author Response

Reviewer 2

Comments and Suggestions for Authors

The manuscript describes research that investigated the effects of ultrasound application and the Isaño genotypes on growth kinetics and ethanol fermentation of S. cerevisiae cultures, using hydrolyzed Isaño must as the substrate. The results indicated that an ultrasonic power applied at 100 W positively affected fermentation parameters and increased the ethanol yield. Higher ultrasonic powers, however, had less positive effects. In addition, among three different Isaño genotypes tested, the purple Isaño had the highest ethanol yield. The research described in the manuscript is original. The results could be interesting for potential biotechnological applications regarding Isaño wine production. However, there are some major and minor points that need to be considered and/or improved:

Comments 1: - Section 2.3.1, line 130: why are the plates incubated at 36-38 C, and not at 30 C or 25 C (the fermentation temperature used in this study – line 116)? The authors should clarify these temperature differences in their experimental procedures.

Response 1: There was a typo in the manuscript. The incubation temperature for OGYE agar is 20–25 °C for 48 h to 5 days. The confusion arose from the mention of different temperatures in different sections of the study. We clarify that this incubation temperature is used to ensure adequate yeast growth on the plates, while the fermentation conditions (25 °C) are related to the active phase of the fermentation process in the Isaño must.

Comments 2: - The authors indicated in the Methods section (lines 103-108) that the isano must with a high starch content was treated with alpha-amylase and amyloglucosidase, before the yeast fermentation. It is known that the yeast S. cerevisiae cannot utilize starch. The use of isano must as a substrate for S. cerevisiae fermentation therefore requires these enzymatic treatment steps which may be costly at large scale. The authors should discuss this issue and the potential use of alternative strategies such as development /use of starch-degrading, recombinant S. cerevisiae strains etc. by checking the related literature.

Response 2: It is true that the yeast Saccharomyces cerevisiae cannot naturally degrade starch due to the lack of the enzymes necessary to hydrolyze it into fermentable sugars. In the present study, enzymatic treatments with alpha-amylase and amyloglucosidase were necessary to release fermentable sugars from the starch in the Isaño must, making the must suitable for fermentation with S. cerevisiae. Regarding large-scale cost, this is a factor to be taken into account in industrial applications. However, a viable alternative to reduce costs could be the development of recombinant S. cerevisiae strains that possess the ability to degrade starch, as has been explored in previous studies. The use of genetically modified strains could lead to improved fermentation efficiency and reduce the need for external enzymatic treatments.

Comments 3: - Results, Section 3.1.: Is the S. cerevisiae strain used in this study a flocculating strain? Did the authors observe differences in cell flocculation/aggregation under the microscope etc.? They should clarify these points in Section 3.1.

Response 3: The S. cerevisiae strain used in this study was EC-1118 LALVIN®, a commercial strain commonly used for wine fermentation. This strain exhibits high fermentative vigor, low nitrogen demand, and tolerance to a wide range of temperatures. Furthermore, it is known for its ability to flocculate well with compact lees, making it suitable for the production of sparkling wines and late-harvest wines. Regarding microscopic observation of cell flocculation/aggregation, no significant differences were found during the fermentation process in the studied cultures. This aspect will be considered for analysis in future research.

 

 

Comments 4: - Regarding Fig 1A, B and C, the authors should comment on or suggest a possible explanation for the decrease in biomass concentrations of the cultures treated with ultrasound after the 60th hour of fermentation, particularly as there is no decrease in ethanol production of these cultures after the 60th hour.

Response 4: The decrease in biomass concentrations observed in ultrasound-treated cultures after 60 hours of fermentation could be related to cell death induced by the ultrasound treatment. In ultrasound-assisted fermentation, the stationary phase is shortened compared to conventional fermentation due to the physical and chemical effects generated by ultrasound, which favor the growth and metabolic activity of microorganisms. Despite the decrease in biomass, ethanol production continues to increase slowly, as the yeasts continue to ferment and produce ethanol as they progress through the process. This phenomenon is due to the fact that the yeast cells remain active in terms of metabolic production, although their physical growth is impaired.

Comments 5: - The authors should verify/comment on the umax value (0.015) observed for the purple genotype at 100 W. This value seems to be too low. All their other results generally indicate the best performance with the purple genotype and at 100 W. Thus, a higher mmax would be expected at 100 W than any of the other conditions (control, 200 and 300 W).

Response 5: The μ_max value observed for the purple genotype at 100 W (0.015) may seem low, but it is important to consider that the maximum specific growth rate (μ_max) can be influenced by several factors, including the effects of antinutritional compounds present in Isaño. Although the purple genotype showed the best overall performance in terms of ethanol production at 100 W, the low μ_max could be related to the release of inhibitory compounds during fermentation. These compounds could interfere with cell growth, reflected in the low μ_max value. This aspect will be addressed in future research to better understand the relationship between the antinutrient content in Isaño must and yeast growth rate.

Comments 6: - line 41, please correct as: ‘… Andean populations, mashua (Isaño) is also recognized for….’

Response 6: It has been corrected in the article

Comments 7: - line 287, please correct as: ‘… with the increase in biomass…’

Response 7: It has been corrected in the article

Comments 8: - line 294, please correct as ‘The must provides a favorable….’

Response 8: It has been corrected in the article

 

Comments 9: - In Table 1, line 143: please indicate the concentration values in g/L instead of mg/dL.

Response 9:

Amount of ethanol	Concentration (g/L)
0 mL/50 mL	0
0.5 mL/50 mL	0.5518
1.0 mL/50 mL	1.1034
1.5 mL/50 mL	1.6552
2.0 mL/50 mL	2.2069
2.5 mL/50 mL	2.7587
3.0 mL/50 mL	3.3104

 

Comments 10: - Section 2.3.3., line 163: please indicate the centrifugation rate in g, not in rpm.

Response 10: It has been corrected in the article

Comments 11: - Section 2.3.5., lines 171-172: please use superscripts for h-1 and  subscripts for Ks and umax. Please check and correct such issues throughout the whole manuscript (e.g. in lines 181-182, lines 330-339, 344-352 and 358.

Response 11: It has been corrected in the article

Comments 12: - Results and Discussion, lines 195-197: please remove these instructions.

Response 12: It has been corrected in the article

Comments 13: - Please write the genus and species names in italic: (Saccharomyces cerevisiae, Tropaeolum tuberosum, S. cerevisiae, Lactobacillus sakei etc.)

Response 13: It has been corrected in the article

Comments 14: - line 222, please write species names in lowercase letters (e.g. cerevisiae, not Cerevisiae).

Response 14: It has been corrected in the article

Comments 15: - line 328, please correct as Monod.

Response 15:  It has been corrected in the article

Comments 16: - line 334-335, please correct as: ‘Under control conditions, the max values were 0.0007……..’ (the authors should indicate at the beginning of that sentence that those data belong to the control condition.)

Response 16: It has been corrected in the article

Comments 17: -line 340, Table 3, please correct as Y  x/s, and Y p/s. (They are currently written as And x/s , And p/s).

Response 17: It has been corrected in the article

Comments 18: - line 341, Table 3, please indicate what each of these different superscripts (a, b,c,d) stand for, e.g. are they different significance levels, if so which letter belongs to which.

 

Response 18: It has been corrected in the article

Comments 19: - lines 345-346, please check Table 3 and correct that sentence accordingly. It should be: ‘For yellow isano, umax increased from 0.006…..to 0.029……. as the power decreased from 300 to 100 W.’

Response 19: It has been corrected in the article

Comments 20: - lines 347-348, please correct as: ‘……to 0.211…..h-1, as the power decreased from 300 to 100 W.’  

Response 20: It has been corrected in the article

Comments 21: - lines 348-349, please correct as: ‘..., as the power increased from 100 W to 300 W. Specifically,it was…’  

Response 21: It has been corrected in the article

Comments on the Quality of English Language

Quality of English language is good. However, English proofreading is recommended for some minor issues. 

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The reviewed manuscript is satisfactorily written. The experimental design is quite good. The results obtained are statistically processed and the presentation of the results is appropriate. The fermentation kinetics is related to three very related parameters (alcohol, reducing sugars and ^oBrix). The growth kinetics (yeast biomass concentration) and all kinetic model calculations are well presented. The discussion of the results needs to be extended, and some additional relevant references should be included.

Comments for author File: Comments.pdf

Author Response

Comment 1: The reviewed manuscript is satisfactorily written. The experimental design is quite good. The results obtained are statistically processed and the presentation of the results is appropriate. The fermentation kinetics is related to three very related parameters (alcohol, reducing sugars and ^oBrix). The growth kinetics (yeast biomass concentration) and all kinetic model calculations are well presented. The discussion of the results needs to be extended, and some additional relevant references should be included.

 

Response 1: The discussions have been expanded, and some references have been included to better support the results. In addition, all the observations highlighted in the manuscript have been addressed.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

In this revised version of the manuscript, the authors have addressed all points raised by the reviewer. The revised manuscript has been significantly improved.