Increased Varietal Aroma Diversity of Marselan Wine by Mixed Fermentation with Indigenous Non-Saccharomyces Yeasts
Round 1
Reviewer 1 Report
Reviewer Recommendation and Comments for Manuscript Number: fermentation-1291730
Title: Increased varietal aroma diversity of Marselan wine by mixed fermentation with indigenous Non-Saccharomyces yeasts
Comments to the Author
The work seems to have been carried out competently and properly. I would recommend that the authors pay particular attention to improving the grammar of their manuscript by cooperating with a native English language professional. Also, please verify the terminology of all volatile aroma compounds. Discussions should be rewritten as this section tended to sound more as a repetition of the results. Generally, in the discussion section one needs to address the results scientifically in relation to cited literature. Please add recent literature references.
Line 95: Please add more information about the winemaking process.
Line 97: Please detail the methods used for determining the physicochemical parameters.
Line 114: For section “GC-MS Analysis”, cite a valid literature reference.
Line 199: In table 1, correct the term ethyl octanoateinstead of “ethyl octoate”
Lines 257: Please rephrase this sentence: “Ethyl laurate was significantly different among all groups, increasing as fermentation process.”
Line 261,464, 466: Please replace “easters” with esters.
Line 273: The “cozier” is a term that is not used for wine smell. Please add a reference or remove it.
Line 299: This is hard to follow. Please rephrase the sentence “Figure 2 showed that the relative content of phenyl ethanol, ethyl caproate in wine 299 was 50-110 and 10-20 times than must, respectively.”
Line 326: make a new section for the PCA analysis because it used as variables the aroma compounds and has nothing to do with sensory analysis of wines.
The "discussion" section is poor particularly in terms of literature review. Compare obtained results with similar/contrasting literature findings.
Author Response
We would like to thank the reviewers for their insightful comments. Our responses are detailed below. The changes are highlighted in yellow in the manuscript file uploaded as supporting information for review.
Point 1:
Line 95: Please add more information about the winemaking process.
Response 1: We truly thank you for reading carefully and patiently and sorry for our careless.
We have added the detailed information about the winemaking progress in the 2.5 Fermentation Methods cited as: “The 2017 HL Marselan grape must was used as material for all trails. The frozen grape juice was placed to 4℃, centrifuged at high speed in a 50 mL tubes. The supernatant was filtered with 0.65 then 0.45 µm filters to obtain the sterile grape juice. Yeasts were inoculated at 28°C for 48 hours until the amount was 107 CFU/mL. 300 mL sterile grape juice was measured. For single-strain fermentation groups, the pre-cultured Saccharomyce yeasts (FS36, YT28 and HL12) were inoculated. For mixed-strain fermentation groups, non-Saccharomyce yeasts (FS31, YT2 and HL9) were inoculated (with the ratio of 1%).Three parallels for each group were set. After the inoculation, all samples were placed in the incubator at 20°C. On the 4th day, the pre-cultured Saccharomyce yeasts (FS36, YT28 and HL12) were added to the corresponding mixed groups (with the ratio of 1%). On the 0th, 2, 4, 6, 10, 14, 19 days, the wine bottles were fully shaken, samples were taken to detect the yeast number, Brix, pH, residual sugar, acid, ethanol, glycerol, and volatile aroma components. When the yeast began to decline and the Brix value kept stable, the fermentation was terminated. The supernatant of wine was taken and stored at 4°C. So the total six trials were: (1) single fermentation with FS36 (short for FS36); (2) mixed fer-mentation of FS36 and FS31 ( short for FS Mixed); (3) single fermentation with YT28( short for YT28); (4) mixed fermentation of YT28 and YT2 (short for YT Mixed); (5) s single fermentation with HL12( short for HL12); (6) mixed fermentation of HL12 and HL9 (short for HL Mixed). The specific details were shown below”(Line152-170)
And we added the Table 2 to make it clear.
Point 2:
Line 97: Please detail the methods used for determining the physicochemical parameters.
Response 2: Thank you for advice.
We have added detailed methods as follows: “Glucose, fructose, ethanol, glycerol were all detected by high performance liquid chromatography (HPLC)[24].The chromatographic conditions : sugar analysis column Aminex HPX-87H (300 mm×7.8 mm), column temperature 55°C, differential detector (RID, Waters-2414), internal temperature 40°C, 0.005 mol/L H2SO4, injection volume 10 μL, flow rate 0.5 mL/min isocratic elution, qualitative by retention time, and quantitative peak area. pH and Brix were detected by pH meter and Brix meter. Based on our previous method, the flow rate was modified to 0.8 mL/min, RP-HPLC was used to detect eight organic acids. The chromatographic conditions are: TechMate ST-C (4.6 mm×250 mm, 5 μm), column temperature 25°C, ultraviolet detector (PAD, Waters-2996), detection wave length 210 nm, mobile phase: ammonium dihydrogen phosphate (NH4H2PO4) 40 mmol/L (4.6 g/L), H3PO4 adjusted pH to 2.5, injection volume 10 μL, flow rate 0.8 mL/min, isocratic elution, qualitative retention time, and peak area quantification.” (Line 192-203)
Point 3:
Line 114: For section “GC-MS Analysis”, cite a valid literature reference.
Response 3: Sorry for our careless.
We have cited the vaild literature reference “Suzzi, G.; Arfelli, G.; Schirone, M.; Corsetti, A.; Perpetuini, G.; Tofalo, R. Effect of grape indigenous Saccharomyces cerevisiae strains on Montepulciano d’Abruzzo red wine quality. Food Res. Int. 2012, 46, 22–29.
- Sun, Y.; Li, E.; Qi, X.; Liu, Y. Changes of diversity and population of yeasts during the fermentations by pure and mixed inoculation of Saccharomyces cerevisiae strains. Ann. Microbiol. 2015, 65, 911–919” on the section “GC-MS Analysis” (Line 123,133)
Point 4:
Line 199: In table 1, correct the term ethyl octanoate instead of “ethyl octoate”
Response 4: Thank you for your careful check. We have revised the sentences accordingly.
We have corrected all the term “ethyl octanoate” instead of “ethyl octoate”.
Point 5:
Lines 257: Please rephrase this sentence: “Ethyl laurate was significantly different among all groups, increasing as fermentation process.”
Response 5: Thank you for advice. We have revised the sentences accordingly.
“Ethyl laurate was significantly different among all groups, increasing as fermentation process.” Is revised as “Compared with must, the content of ethyl laurate was significantly increased as fermentation process” (Line 331)
Point 6:
Line 261,464, 466: Please replace “easters” with esters.
Response 6: So sorry for our careless and thank you for your careful check.
We have corrected all the misspellings.
Point 7:
Line 273: The “cozier” is a term that is not used for wine smell. Please add a reference or remove it.
Response 6: Thank you for your professional advice.
We have deleted the word “cozier”.
Point 8:
Line 299: This is hard to follow. Please rephrase the sentence “Figure 2 showed that the relative content of phenyl ethanol, ethyl caproate in wine 299 was 50-110 and 10-20 times than must, respectively.”
Response 8: We are so sorry to confuse you and thank you for your careful advice. We have revised the sentences accordingly.
“Figure 2 showed that the relative content of phenyl ethanol, ethyl caproate in wine 299 was 50-110 and 10-20 times than must, respectively” is revised as “Compared with must, the relative content of phenethyl alcohol (50-100 fold change), ethyl caprylate (480-670 fold change) and ethyl caproate (10-20 fold change) was much higher in wine, these aromas were produced by yeasts during wine fermentation.” (Line 412-414)
Point 9:
Line 326: make a new section for the PCA analysis because it used as variables the aroma compounds and has nothing to do with sensory analysis of wines.
Response 8: Thank you for your professional advice.
We have made a new section for PCA analysis and the new subtitle is “The PCA analysis of Marselan wine” (Line 450)
Point 10:
The "discussion" section is poor particularly in terms of literature review. Compare obtained results with similar/contrasting literature findings.
Response 10: Thank you for the helpful comments.
We have polished the discussion and added the recommended comments.
Author Response File: 
Author Response.docx
Reviewer 2 Report
This manuscript aims to identify characteristic aromas of Marselan must and wine with various yeast fermentation parameters, however, the method used for volatiles analysis does not provide sufficient analytical rigor. The analysis of the samples is fairly unusual – 2 hour incubation followed by 50 min of fiber exposure. But regardless of these parameters, the analysis appears to be reporting chemical compounds strictly based on the NIST library identification. For that reason, all compounds are only ‘tentatively’ identified. This would require retention indices and match factors to be included to better understand how reliable the data is. Some compounds reported (e.g. calamine, 2,4-di-terbutyl phenol, etc.) are quite uncommon or unreported in wine at meaningful (aroma-relevant) levels. Furthermore, there are some very unusual results for a Vitis Vinifera spp. For example, it would be expected that all grape musts would contain fairly high levels of C6-alcohols, yet many of the musts had no detectable levels according to table 1. For the wines, it was reported that ethyl decanoate and ethyl 9-decanoate were the most abundant ethyl esters, but that is highly unusual as they should be found at lower levels than esters such as ethyl hexanoate and ethyl octanoate. This might suggest that the method of analysis and matrix effects aren’t properly analyzed and normalized for. Without proper quantitation (or more appropriate methods for semi-quantitative analysis), I’m not certain that any conclusions could be drawn from the results.
Author Response
Point 1: This manuscript aims to identify characteristic aromas of Marselan must and wine with various yeast fermentation parameters, however, the method used for volatiles analysis does not provide sufficient analytical rigor.
Response 1: Thank you for your professional comments.
I totally agree with your comments on this method. The semi-quantitative method for chemicals cannot provide sufficient analytical rigor, but it still reflects a kind of tendency to help us find more possibility on exploration. In our study, the comparison between different fermentation groups is the key point instead of finding new compounds, so semi- quantitative analysis is appropriate. Besides, in the field of aroma analysis in grape must and wine, GC-MS method has been widely used in many studies. A specific internal standard like 2-octanol needs to be added to obtain the semi-quantitative content of chemicals through calculating the peak area. Just as what we adopted, some studies also applied headspace solid phase microextraction or rotary evaporation concentration coupled with GC-MS to amplify the signals of aroma concentration when obtaining the initial aroma extract, which is conducive to the precision and accuracy of GC-MS. In this way, numerous compounds can be easily detected and relatively quantified. However, the disadvantage that it cannot get the absolute content of a certain compound is obvious, which is unfriendly for a deeper understanding of the relationship between the detected material and compounds. We have read some paper related with wine aroma analysis and found many of them chose to use this method, and the references were listed below.
[1] Tufariello M, Fragasso M, Pico J, et al. Influence of non-Saccharomyces on wine chemistry: A focus on aroma-related compounds[J]. Molecules, 2021, 26(3): 644.
[2] Zhang B Q , Yu L , Duan C Q , et al. Use of Torulaspora delbrueckii Co-fermentation With Two Saccharomyces cerevisiae Strains With Different Aromatic Characteristic to Improve the Diversity of Red Wine Aroma Profile[J]. Frontiers in Microbiology, 2018, 9:606.
[3] Escribano-Viana R, González-Arenzana L, Portu J, et al. Wine aroma evolution throughout alcoholic fermentation sequentially inoculated with non-Saccharomyces/Saccharomyces yeasts[J]. Food Research International, 2018, 112: 17-24.
[4] Padilla B, Zulian L, Ferreres À, et al. Sequential inoculation of native non-Saccharomyces and Saccharomyces cerevisiae strains for wine making[J]. Frontiers in microbiology, 2017, 8: 1293.
[5] Escribano-Viana R, González-Arenzana L, Portu J, et al. Wine aroma evolution throughout alcoholic fermentation sequentially inoculated with non-Saccharomyces/Saccharomyces yeasts[J]. Food Research International, 2018, 112: 17-24.
[6] Binati R L, Junior W J F L, Luzzini G, et al. Contribution of non-Saccharomyces yeasts to wine volatile and sensory diversity: A study on Lachancea thermotolerans, Metschnikowia spp. and Starmerella bacillaris strains isolated in Italy[J]. International journal of food microbiology, 2020, 318: 108470.
Point 2: The analysis of the samples is fairly unusual –2 hour incubation followed by 50 min of fiber exposure.
Response 2: Thank you for your advice.
We have checked the experiment records again and added some details for the method as follows: “The volatile compounds of Marselan wine samples were extracted using the Head Space Solid Phase Microextraction (HS-SPME) method as described[22]. For each analysis, 2.5 g of sodium chloride (NaCl) and 10 μL of 2-octanol (internal standard), were added to 5 mL of must sample into a 20 mL headspace screw vial. Helium (He) was the carrier gas at a constant flow rate of 1 mL/min. The SPME fiber was inserted through the needle and exposed into the headspace of the vial to adsorb volatile compounds at 45 ℃ for 50 min and then immediately injected into the gas chromatography injection port at 250 ℃ for 2.5 min to desorb volatile compounds; afterwards, GC-MS separation, analysis and identification was performed.
The volatile compounds from the samples were analyzed by Gas Chromatography and Mass Spectrometry (GC–MS) according to a previous description with some modi-fication[23]. GCMS-QP2010Ultra (SHIMADZU, Japan) was used with a DB-Wax capillary column (30 mm length × 0.25 mm i.d. × 0.25 µm film thickness) and splitless injection mode was adopted. High purity helium, as a carrier gas, was used at a constant flow rate of 1 mL/min. The gas chromatographic oven was set at 50 °C for 5 min, increased until 230 °C at a rate of 5 °C/min, and finally maintained for 30 min. Other conditions included an interface temperature of 250 °C, emission current of 100 µA, and mass spectra were obtained at 70 eV in the electron ionization+ (EI +) mode.The mass spectral identification of volatile compounds in the samples was carried out by comparing to the National In-stitute of Standards and Technology (NIST) 2014 and Wiley 8.0 database. Qualitative analysis of mass spectral data was verified by comparing the retention indices and mass spectra of identified compounds. The qualitative aroma components were qualitatively determined by NIST2011 and Demo library. Volatile compounds extraction (µg/L) = (peak area of volatile compounds / peak area of internal standard × mass of internal standard) / mass of sample.”(Line 122-149)
Point 3: But regardless of these parameters, the analysis appears to be reporting chemical compounds strictly based on the NIST library identification. For that reason, all compounds are only ‘tentatively’ identified. This would require retention indices and match factors to be included to better understand how reliable the data is. Some compounds reported (e.g. calamine, 2,4-di-terbutyl phenol, etc.) are quite uncommon or unreported in wine at meaningful (aroma-relevant) levels.
Response 3: Thank you for your helpful advice.
Your comments are totally right about this point. It is indeed insufficient to just rely on NIST library for qualitative identification, in consideration of 1D Gas-Phase substances may not be separated, which means there could be several substances below one peak. NIST library only selects the most likely one, possibly the substance is not right.
Point 4: Furthermore, there are some very unusual results for a Vitis Vinifera spp. For example, it would be expected that all grape musts would contain fairly high levels of C6-alcohols, yet many of the musts had no detectable levels according to table 1.
Response 4: Thank you for your professional advice.
As we mentioned above, we adopted the semi-qualitative GC-MS analysis so the actual content of compounds cannot be reflected in Marselan must or wine. From the results, C6 alcohols were not detected, but this did not mean that C6 alcohols don’t exist in our samples. The possibility for empty detection could be the detection method not optimized to the maximum or this kind of substance has a low response on the column that we used.
Point 5: For the wines, it was reported that ethyl decanoate and ethyl 9-decanoate were the most abundant ethyl esters, but that is highly unusual as they should be found at lower levels than esters such as ethyl hexanoate and ethyl octanoate. This might suggest that the method of analysis and matrix effects aren’t properly analyzed and normalized for. Without proper quantitation (or more appropriate methods for semi-quantitative analysis), I’m not certain that any conclusions could be drawn from the results.
Response 5: Thank you for your advice.
In this research, we applied the semi-qualitative GC-MS so the actual content of compounds cannot be reflected, no matter in must or wine. In addition, the response of substances through GC-MS detection was also verified and fluctuant in different experiments. We think it is meaningful to compare a specific compound among different fermentation groups rather than try to figure out the content in one sample. Ethyl decanoate and ethyl 9-decanoate were much higher than ethyl hexanoate and ethyl octanoate, probably due to the higher sensitivity to the detection, other than the actual content from the sample.
Author Response File: Author Response.docx
Reviewer 3 Report
The article titled „Increased varietal aroma diversity of Marselan wine by mixed fermentation with indigenous Non-Saccharomyces yeasts“ represents interesiting work covering the aromatic profile assesment of Marselan musts and as well as wines obtained with indogenous Saccharomycess and non- Saccharomycess yeast. Although GC-MS results seems interesting there are several major drawbacks listed below:
Major issues
Please explain add how panel tranning was performed. Adequate selection and later traning of panellist is important factor in every sensory analysis. And add more information about wine tasting conditions.
Please explain why you didn’t use a commercial Saccharomyces yeast as control? I personally think that direct comparison with control is monumental in studies like presented.
Some parts of manuscript have low quality of English, ms should be checked by native English speaker.
Minor issues
Page 3 Line 101 – please give full name for YPD medium
Page 3 Line 123 – please insert the temperature gradient (ºC/per min until 230 ºC)
It is not possible to understand which must was used in vinification process. Was mixture of 4 musts or one specific? Please add information.
Please add all methods (at least the reference) which you used to obtain results from Table S2.
Table S2 – How do you explain that trials with mixed yeasts produced more glycerol and higher alcohol (not statistically significant but trend is obvious) respective to pure-yeast trials? It is well know that higher production of glycerol could result in lower level of alcohol in wines, which is one of the strategies to mitigate reduce alcohol in wines.
Goold, Hugh D., et al. "Yeast's balancing act between ethanol and glycerol production in low‐alcohol wines." Microbial biotechnology 10.2 (2017): 264-278.
English and typos
Page 1 Line 29 and Page 3 Line 100 S. cerevisiae, please check the entire manuscript.
It should be non-Saccharomyces, please correct thought entire manuscript.
Page 3 Line 116 – term “aged” might not be appropriate, please rephrase it same for “aging” 2 rows below.
Page 3 Line 129 – The frozen grape juice was placed to 4C…
Page 3 Line 130 – centrifuged at high in a 50 mL, the entire sentence should be rephrased.
Page 3 131-132 – Please rephrase sentence.
Page 4 Line 164 – barely research was found – is not correct formulation of the sentence
Page 4 lines 186-187 – I don’t understand the sentence, please rephrase it.
Table 2 - Esters
Author Response
Point 1: Please explain add how panel training was performed. Adequate selection and later training of panelist is important factor in every sensory analysis. And add more information about wine tasting conditions.
Response 1: We truly thank you for reading carefully and patiently and sorry for our careless.
We have added the more detailed information about the training process and wine tasting conditions as follows: “Wine aroma was evaluated in triplicates by a tasting panel consisting of four females and four males trained with a 54-aroma kit (Le Nez du Vin ®, France) for three weeks. During the training, the performances were evaluated by aroma sense test every five days until their identification accuracy for each aroma reached above 95%. The analysis was conducted in a tasting room at 23 °C. Approximately 30 mL wine (15 °C) was held in a black wine glass. Throughout the wine sensory analysis, the samples were presented in a random order. The interval between two samples was 5 min. The panelists evaluated wine aroma according to the following procedure: they smelled the aroma of wine sample for approximately 5-8 s, then shook the wine to smell the aroma for 5-10 s, defined aromas and scored. The sensory descriptors were alcohol, floral, citrus, stone fruits, berries, dry fruits, herbs and fermentative aromas. The samples were quantitatively measured on a 5-point interval scale to grade the intensity (1-very weak; 2-weak; 3-medium; 4-intense; 5-very intense)” (Line 177-189)
Point 2: Please explain why you didn’t use a commercial Saccharomyces yeast as control? I personally think that direct comparison with control is monumental in studies like presented.
Response 2: Thank you for your professional comments.
Like you mentioned, it is indeed that commercial Saccharomyces yeasts are often used as control in many studies. But in this study, our purpose was to explore the characteristic of indigenous yeasts and how local non-Saccharomyces effect the Saccharomyces, especially on wine aromas. We are curious about the performance of these local yeasts. These strains come from different wine making areas in China, with promising ability on practice in the future. We did not consider the commercial yeast because this was not the point of this work.
Point 3: Some parts of manuscript have low quality of English, ms should be checked by native English speaker.
Response 3: Thank you for your professional comments.
We have polished the paper.
Point 4: Page 3 Line 101 – please give full name for YPD medium
Response 4: Thank you for your professional advice.
We have added the full name “yeast extract peptone dextrose medium (YPD, yeast extract 10 g/L, peptone 20 g/L and glucose 20 g/L)” (Line 106)
Point 5: Page 3 Line 123 – please insert the temperature gradient (ºC/per min until 230 ºC)
Response 5: Thank you for your professional advice. We have revised the sentences accordingly.
“DB-5 MS column temperature program conditions: initial temperature 50℃, hold for 5 min, then increased to 230°C and kept for 30 minutes” is revised as “The gas chromatographic oven was set at 50 °C for 5 min, increased until 230 °C at a rate of 5 °C/min, and finally maintained for 30 min” (Line 140)
Point 6: It is not possible to understand which must was used in vinification process. Was mixture of 4 musts or one specific? Please add information.
Response 6: Sorry for my careless and thank you for your kind suggestions.
We have added more detailed clarification about the vinification process in the Fermentation methods like this “The 2017 HL Marselan grape must was used as material for all trails” (Line 152).
Point 7: Please add all methods (at least the reference) which you used to obtain results from Table S2.
Response 7: Sorry for my careless and thank you for your helpful advice.
We have added more detailed information about the methods as follows: “Glucose, fructose, ethanol, glycerol were all detected by high performance liquid chromatography (HPLC)[22].The chromatographic conditions : sugar analysis column Aminex HPX-87H (300 mm×7.8 mm), column temperature 55°C, differential detector (RID, Waters-2414), internal temperature 40°C, 0.005 mol/L H2SO4, injection volume 10 μL, flow rate 0.5 mL/min isocratic elution, qualitative by retention time, and quantitative peak area. pH and Brix were detected by pH meter and Brix meter. Based on our previous method, the flow rate was modified to 0.8 mL/min, RP-HPLC was used to detect eight organic acids. The chromatographic conditions are: TechMate ST-C (4.6 mm×250 mm, 5 μm), column temperature 25°C, ultraviolet detector (PAD, Waters-2996), detection wave length 210 nm, mobile phase: ammonium dihydrogen phosphate (NH4H2PO4) 40 mmol/L (4.6 g/L), H3PO4 adjusted pH to 2.5, injection volume 10 μL, flow rate 0.8 mL/min, isocratic elution, qualitative retention time, and peak area quantification” (Line192-203).
Point 8: Table S2 –How do you explain that trials with mixed yeasts produced more glycerol and higher alcohol (not statistically significant but trend is obvious) respective to pure-yeast trials? It is well known that higher production of glycerol could result in lower level of alcohol in wines, which is one of the strategies to mitigate reduce alcohol in wines.
Goold, Hugh D., et al. "Yeast's balancing act between ethanol and glycerol production in low‐alcohol wines." Microbial biotechnology 10.2 (2017): 264-278.
Response 8: Thank you for your professional advice.
I carefully read the paper that you cited above and searched some papers, and here is my explanation for this question:
(1) It is indeed that the mixed fermentation with non-Saccharomyces increased the glycerol and decreased alcohol in many studies, and many wine markers select these typical yeasts to make wine with lower alcohol and higher glycerol on purpose. However, there is no absolute negative relationship between both factors, which means high glycerol production cannot guarantee low alcohol. A previous study showed that hybrids from S. cerevisiae × S. uvarum increased glycerol production compared to the S. cerevisiae parent; however, no reduction of ethanol concentration was observed (Coloretti et al., 2006). Glycerol production in Saccharomyces cerevisiae can also be induced by the addition of sulfite to the growth medium, which cannot be used for ethanol production due to the combination of sulfite and acetaldehyde. Thus, another study used sulfite to select yeasts with good adaption of high osmotic pressure, and this strategy was very successful, whereby exposure to sulfite over 300 generations resulted in a 46% increase in glycerol yield. But the ethanol production was just a minor decrease (Kutyna et al., 2012). For a single yeast, it is inconclusive to define the balance between them, not to mention the complicated mixed-strain fermentation. Therefore, it is possibly not reasonable to deduce the content of any one according to the other.
(2) Even though glycerol and ethanol are the main metabolites of carbon flux, there still remains a lot of other carbonic metabolites like organic acids, which could disturb the balance by changing the redox system within yeast cells, especially on NAD+/NADH cycle. In Table S2, the mixed fermentation (red square, HL and YT, except for FS) had lower content of organic acids (acetic acid, citric acid and tartaric acid) than single-strain groups (blue square), which could partly explain why the alcohol was not reduced in non-Saccharomyces groups, because the reduced carbon for forming organic acids possibly transformed to the alcohol partly.
Ref:
Coloretti F, Zambonelli C, Tini V. Characterization of flocculent Saccharomyces interspecific hybrids for the production of sparkling wines[J]. Food microbiology, 2006, 23(7): 672-676.
Kutyna, D.R., Varela, C., Stanley, G.A., Borneman, A.R., Henschke, P.A., and Chambers, P.J. (2012) Adaptive evolution of Saccharomyces cerevisiae to generate strains with enhanced glycerol production. Appl Microbiol Biotechnol 93: 1175–1184.
Point 9: Page 1 Line 29 and Page 3 Line 100 S. cerevisiae, please check the entire manuscript. It should be non-Saccharomyces, please correct thought entire manuscript.
Response 9: Thank you for your kind remind.
Line 29: “The current set of commercial S. cerevisiae strains and its derived hybrids is insufficient to provide novel properties to wine, stressing the need for new and improved strains for the industry”.
This sentence is to emphasize the limitation of commercial Saccharomyces, so here should be S. cerevisiae.
Line 100: “Six autochthonous strains, including three S. cerevisiae strains (FS36, YT28 and HL12) and three non-Saccharomyces strains (FS31, YT2 and HL9)”
Six indigenous yeast strains were used in this study( to be clear, you could see the new added Table 1 in the revised manuscript), three of them (FS36, YT28 and HL12) belong to Saccharomyces, and the other three (FS31, YT2 and HL9) were non-Saccharomyces. These yeasts were selected and identified in our lab previously. So here should be S. cerevisiae.
Point 10: Page 3 Line 116 – term “aged” might not be appropriate, please rephrase it same for “aging” 2 rows below.
Response 10: Thank you for your professional advice. We have revised the sentences accordingly.
We rewrote the 2.4 HS-SPME–GC–MS analysis and added more details into the method. (Line 131-149)
Point 11: Page 3 Line 129 – The frozen grape juice was placed to 4C…
Response 11: Thank you for your professional advice. We have revised the sentences accordingly.
“Place the frozen grape juice at 4°C to slowly thaw. After the thawing is complete, centrifuge at high speed with a 50 mL centrifuge tube, take the supernatant and remove the precipitate” is revised as “The frozen grape juice was placed to 4℃, centrifuged at high speed in a 50 mL tubes. The supernatant was filtered with 0.65 then 0.45 µm filters to obtain the sterile grape juice.” (Line 152-154).
Point 12: Page 3 Line 130 – centrifuged at high in a 50 mL, the entire sentence should be rephrased.
Response 12: Thank you very much for your advice. We have revised the sentences accordingly.
“Place the frozen grape …” is revied as “The frozen grape juice was placed to 4℃, centrifuged at high speed in a 50 mL tubes. The supernatant was filtered with 0.65 then 0.45 µm filters to obtain the sterile grape juice. Yeasts were inoculated at 28°C for 48 hours until the amount was 107 CFU/mL. 300 mL sterile grape juice was measured. For single-strain fermentation groups, the pre-cultured Saccharomyce yeasts (FS36, YT28 and HL12) were inoculated. For mixed-strain fermentation groups, non-Saccharomyce yeasts (FS31, YT2 and HL9) were inoculated (with the ratio of 1%). Three parallels for each group were set. After the inoculation, all samples were placed in the incubator at 20°C. On the 4th day, the pre-cultured Saccharomyce yeasts (FS36, YT28 and HL12) were added to the corresponding mixed groups (with the ratio of 1%). On the 0th, 2, 4, 6, 10, 14, 19 days, the wine bottles were fully shaken, samples were taken to detect the yeast number, Brix, pH, residual sugar, acid, ethanol, glycerol, and volatile aroma components. When the yeast began to decline and the Brix value kept stable, the fermentation was terminated. The supernatant of wine was taken and stored at 4°C. So the total six trials were: (1) single fermentation with FS36 (short for FS36); (2) mixed fermentation of FS36 and FS31 ( short for FS Mixed); (3) single fermentation with YT28( short for YT28); (4) mixed fermentation of YT28 and YT2 (short for YT Mixed); (5) s single fermentation with HL12( short for HL12); (6) mixed fermentation of HL12 and HL9 (short for HL Mixed). The specific details were shown below” (Line 152-170)
Point 13: Page 3 131-132 – Please rephrase sentence.
Response 13:
Thank you for your advice. We have revised the sentences accordingly.
“Filter the clarified grape …is revised as “The frozen grape juice was placed to 4℃, centrifuged at high speed in a 50 mL tubes. The supernatant was filtered with 0.65 then 0.45 µm filters to obtain the sterile grape juice. Yeasts were inoculated at 28°C for 48 hours until the amount was 107 CFU/mL. 300 mL sterile grape juice was measured.” (Line 152-155)
Point 14: Page 4 Line 164 – barely research was found – is not correct formulation of the sentence.
Response 14: Thank you for your advice. We have revised the sentences accordingly.
“However, barely research was found to investigate the characteristic aromas in Marselan must” is revised as “However, few research was found to investigate the characteristic aromas of Marselan must.” (Line 216)
Point 15: Page 4 lines 186-187 – I don’t understand the sentence, please rephrase it.
Response 15: Thank you for your advice. We have revised the sentences accordingly.
“For a clearer comparison among different samples, Figure S1 demonstrated the characteristic aroma compounds expressed as the percentage of the total concentration”is revised as “To compare all the samples, the relative content of characteristic aroma compounds was showed in Figure S1. ”(Line 238)
Point 16: Table 2 - Esters
Response 16: Thank you for your detailed advice.
All “Ester” spellings were checked.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Accept in present form.
Reviewer 2 Report
I think the authors put a lot of effort into revising which is much appreciated. Both on expanding on methodology and results/conclusions. However, I don't think the concerns regarding the way the data is shared from the GC-MS results have been fully addressed.
There are still many uncommon compounds that are not typically reported in grapes and wine that are heavily referred to in conclusions. For example, in line 353-354 the authors state that "Calamenene as one of the most typical aromas in Marselan grape". This could potentially be true, but it is unclear if the analytical method gave accurate identification of the compound for such a definitive statement. I have not seen this compound previously described as an important key aroma compounds in grapes or wine. I would strongly recommend tables 3 and 4 to include the NIST match factor and retention index (and CAS number). The authors stated they chose the "top hit", but it is not unusual for the top hit to be wrong. Is the match factor at over 900 or is it 600? It is misleading to annotate the compound without confirmation with authentic standards. If the authors prefer, they may choose to move some of the uncommon compounds (e.g. violine, calamenene, samenene) to supplementary material.
I think section 3.3 and 3.4 (line 303) as a whole needs to be written more carefully as none of the results are definitive. I would suggest taking more careful consideration for any definitive statements since the aroma data is not quantitative. For example, in line 313-314 it states that ethyl decanoate is the most abundant ester in all samples. As mentioned in previous comments this doesn't make sense as almost all standard wines would have higher concentration of ethyl octanoate and ethyl hexanoate. The authors mentioned that it could be that ethyl decanoate simply had a higher response (presumably from matrix effects), but that does not mean it is present in higher concentrations. If the absolute peak area is not directly related to concentration then the language needs to be more casual not to mislead readers as to the relative importance of tentatively identified compounds. Furthermore, just because a compound is volatile does not mean that it is aroma-active, so careful consideration is needed when referring to characteristic aroma of Marselan wine. For example, 2,4-di-tert-butylphenol is an odorless compound and should have no aroma relevance to the grapes and wine.
It is unclear if the authors are referring to β-damascenone or β-damascone throughout the manuscript, which are different compounds. β-damascenone is generally considered as important to wines.
The Discussion goes into depth into much of the aroma chemistry but it is important it supports any findings, there are references to pathways and compounds that were not measured in the study, and these portions should be omitted or have the relevance of why they were included explained.
Comment for Figure 1: how can the measured brix be at ~8 at the end of fermentation if glucose and fructose both dropped to 0, shouldn't that be 0 as well?
Reviewer 3 Report
The authors significantly improved the manuscript. Therefore I suggest acceptance in its current form.
