Molecular Hydrogen Treatment of Sake Yeast and kuratsuki Bacteria Affects Sake Taste

Round 1

Reviewer 1 Report

The manuscript entitled “Molecular hydrogen treatment to sake yeast and kuratsuki bacterium affects taste of sake” deals with investigating the application of molecular hydrogen treatment to two microorganisms used to produce sake before brewing in a co-culture process the evaluation of the sake taste. The authors showed interesting results related to the modification of sake taste with this treatment. However, the statistical analysis of the results should be clearer to evaluate if, in fact, there were differences. Another point is that they didn't prove that the treatment's effect was in the microorganisms' physiology and those affirmatives should be removed.

Introduction

“In contrast, to our knowledge, there are few studies of the effects of H2 on microorganisms.” Which studies are those? Please exemplify in the text.

Methodology

2.1. The name of the species sake yeast Saccharomyces cerevisiae) must be specified here.

Line 27: “as well as PREVENTS…”

Line 36-37: “The sake yeast GENERATES chemical components…”

2.1 Cultivation of microorganisms. This description is confusing.

Line 72: the authors describe the use of water or water containing H2 to treat cells. Are the cultures mixed (both yeast and bacterium) at this stage or are they separated? Which is the concentration of cells at this stage? The use of water without H2 is a control? This water or water containing H2 is removed before inoculation?

Line 74: “As a control, 290 mL water was added along with 60 g of koji...”  Which koji? This needs to be described. Which flasks were used? Are they sterile?

Were the fermentations performed in replicates? The statistical analysis was performed on different samples of the same fermentation or on the results of different fermentations?

Results

Lines 106-107: “Thus, the ethanol fermentation of the sake yeast strain K1401 was not significantly affected by the addition of the kuratsuki Kocuria strain TGY1127_2.” This affirmative cannot be done since ethanol was not monitored during the process.

Line 108: “Additionally, there was no significant variance in the Brix and acidity of sake…” Where is the statistical analysis?

Brix and Acidity: the methodology of determination of those parameters must be specified in the methodology section.

Table 1: statistical analysis of the results must be specified.

Figure 2 is confusing. It is not possible to determine which “bar” correspond to the treatment. Are those replicates? If each treatment has 4 bars from different experiments, there seems to be a huge difference between samples. The ANOVA results must be presented. I believe that these results would be better in a Table.

Conclusions

Lines 174-175: “…H2-induced physiologies of the sake yeast and kuratsuki bacterium continued for a certain period of time after mixture”. It was not proved that the effect of H2 was in the physiology of the microorganisms. This affirmative can’t be done. The differences in sake taste could be from many things. First, the authors must explain better their statistical analysis. These differences could be from different fermentations. Even so, to prove that a modification in the physiology was induced, other metabolism products should be measured.

A minor revision should be performed 

Author Response

Thank you so much for your comments.

The manuscript entitled “Molecular hydrogen treatment to sake yeast and kuratsuki bacterium affects taste of sake” deals with investigating the application of molecular hydrogen treatment to two microorganisms used to produce sake before brewing in a co-culture process the evaluation of the sake taste. The authors showed interesting results related to the modification of sake taste with this treatment. However, the statistical analysis of the results should be clearer to evaluate if, in fact, there were differences. Another point is that they didn't prove that the treatment's effect was in the microorganisms' physiology and those affirmatives should be removed.
Introduction
Q1. “In contrast, to our knowledge, there are few studies of the effects of H2 on microorganisms.” Which studies are those? Please exemplify in the text.
A1. We changed "few" to "no".

Methodology
Q2. 2.1. The name of the species sake yeast Saccharomyces cerevisiae) must be specified here.
A2. We changed "The sake yeast" to "The sake yeast S. cerevisiae".

Q3. Line 27: “as well as PREVENTS…”
A3. We changed "preventing" to "prevents".

Q4. Line 36-37: “The sake yeast GENERATES chemical components…”
A4. We changed "has generated" to "generates".

2.1 Cultivation of microorganisms. This description is confusing.
Q5. Line 72: the authors describe the use of water or water containing H2 to treat cells. Are the cultures mixed (both yeast and bacterium) at this stage or are they separated? Which is the concentration of cells at this stage? The use of water without H2 is a control? This water or water containing H2 is removed before inoculation?
A5. We changed "Solutions" to "These four solutions, sake yeasts suspended in water, sake yeasts suspended in H2-water, kuratsuki Kocuria suspended in water, and kuratsuki Kocuria suspended in H2-water,".

Q6. Line 74: “As a control, 290 mL water was added along with 60 g of koji...”  Which koji? This needs to be described. Which flasks were used? Are they sterile?
A6. We added "(Isenou, Tokyo)". This koji has been already used in our previous study (reference [13]).

Q7. Were the fermentations performed in replicates? The statistical analysis was performed on different samples of the same fermentation or on the results of different fermentations?
A7. We have no biological replicate in this experiment. However, we performed different experiments and confirmed the effects of H2 treatment.

Results
Q8. Lines 106-107: “Thus, the ethanol fermentation of the sake yeast strain K1401 was not significantly affected by the addition of the kuratsuki Kocuria strain TGY1127_2.” This affirmative cannot be done since ethanol was not monitored during the process.
A8. We deleted the sentence, "Thus, the ethanol fermentation of the sake yeast strain K1401 was not significantly affected by the addition of the kuratsuki Kocuria strain TGY1127_2."
 
Q9. Line 108: “Additionally, there was no significant variance in the Brix and acidity of sake…” Where is the statistical analysis?
A9. We added the following in Materials and Methods, "The Kolmogorov–Smirnov test was performed to compare the Brix and acidity change patterns."

Q10. Brix and Acidity: the methodology of determination of those parameters must be specified in the methodology section.
A10. We added the following in Materials and Methods, "For the Brix test, 0.3 mL of the sample solution was used; for acidity, 0.6 mL of the sample solution was diluted 20 times with water."
 
Q11. Table 1: statistical analysis of the results must be specified.
A11. We have no technical replicates.
 
Q12. Figure 2 is confusing. It is not possible to determine which “bar” correspond to the treatment. Are those replicates? If each treatment has 4 bars from different experiments, there seems to be a huge difference between samples. The ANOVA results must be presented. I believe that these results would be better in a Table.
A12. We added the following in Figure legend, "Each measurement was repeated four times. Each treatment has four bars from different experiments." We changed "p < 0.05" to "p < 0.05, ANOVA", and added the following, "(in pairwise t test)".

Conclusions
Q13. Lines 174-175: “…H2-induced physiologies of the sake yeast and kuratsuki bacterium continued for a certain period of time after mixture”. It was not proved that the effect of H2 was in the physiology of the microorganisms. This affirmative can’t be done. The differences in sake taste could be from many things. First, the authors must explain better their statistical analysis. These differences could be from different fermentations. Even so, to prove that a modification in the physiology was induced, other metabolism products should be measured.
A13. We deleted the following, "Thus, H2-induced physiologies of the sake yeast and kuratsuki bacterium continued for a certain period of time after mixture. Furthermore, ".

Reviewer 2 Report

Paper with the title "Molecular hydrogen treatment to sake yeast and kuratsuki bacterium affects taste of sake" is a manuscript that addresses the effects of molecular hydrogen (H2) on microorganisms, the study of sake yeast strain K1401 and Kocuria strain TGY1127_2 of kuratsuki bacteria. in brewing. Sake was required to undergo H2 treatment microorganisms involved in the preparation of sake to increase the variety and satisfy the market. It is an interesting study, which approaches a less treated subject. This paper can be accepted after some improvements:

You stated "The Brewing Company of Japan (Jozo-Kyokai) manages and sells sake yeast strains (Kyokai yeast strains) that have been established in selected sake breweries in Japan" for what purpose is it marketed?

Please explain why the kuratsuki strain Kocuria TGY1127_2 does not convert rice starch to sugar and does not convert sugar to ethanol.

Please detail for what purpose you used molecular hydrogen both in the introduction part and in the results section.

Please justify the choice for the chosen strains.

Author Response

Thank you so much for your comments.

Paper with the title "Molecular hydrogen treatment to sake yeast and kuratsuki bacterium affects taste of sake" is a manuscript that addresses the effects of molecular hydrogen (H2) on microorganisms, the study of sake yeast strain K1401 and Kocuria strain TGY1127_2 of kuratsuki bacteria. in brewing. Sake was required to undergo H2 treatment microorganisms involved in the preparation of sake to increase the variety and satisfy the market. It is an interesting study, which approaches a less treated subject. This paper can be accepted after some improvements:
Q1. You stated "The Brewing Company of Japan (Jozo-Kyokai) manages and sells sake yeast strains (Kyokai yeast strains) that have been established in selected sake breweries in Japan" for what purpose is it marketed?
A1. We changed “The Brewing Company of Japan (Jozo-Kyokai) manages and sells sake yeast strains (Kyokai yeast strains) that have been established in selected sake breweries in Japan [10]” to the following, “The Brewery Society of Japan (Jozo-Kyokai) manages and sells sake yeast strains (Kyokai yeast strains), which were established in selected sake breweries in Japan because the flavor and taste of sake produced using naturally occurring yeasts are unstable and not always satisfactory [10].”

Q2. Please explain why the kuratsuki strain Kocuria TGY1127_2 does not convert rice starch to sugar and does not convert sugar to ethanol.
A2. We added the following, “Kocuria strain TGY1127_2 lacks amylase, and no significant difference in Brix change was detected between the solutions of koji with and without TGY1127_2 [13].”

Q3. Please detail for what purpose you used molecular hydrogen both in the introduction part and in the results section.
A3. We added the following in Introduction, “Generally, environmental conditions, such as culture conditions, affect bacterial properties. If the properties of kuratsuki bacteria are altered by H2 treatment, the effects of kuratsuki Kocuria with and without such treatment on the taste of sake may differ.” In addition, we added the following in Results and Discussion, “These results indicated that H2 treatment affected the properties of kuratsuku Kocuria and sake yeast.”

Q4. Please justify the choice for the chosen strains.
A4. We added the following in Materials and Methods, “K1401 has been frequently used by sake breweries in Japan and was used in our experiments [13, 18]. TGY1127_2 was isolated, classified, and used in our experiments [13, 18].”

Round 2

Reviewer 1 Report

Several questions weren't answered:

Q5. Line 72: the authors describe the use of water or water containing H2 to treat cells. Are the cultures mixed (both yeast and bacterium) at this stage or are they separated? Which is the concentration of cells at this stage? The use of water without H2 is a control? This water or water containing H2 is removed before inoculation?
A5. We changed "Solutions" to "These four solutions, sake yeasts suspended in water, sake yeasts suspended in H2-water, kuratsuki Kocuria suspended in water, and kuratsuki Kocuria suspended in H2-water,".

Q9. Line 108: “Additionally, there was no significant variance in the Brix and acidity of sake…” Where is the statistical analysis?
A9. We added the following in Materials and Methods, "The Kolmogorov–Smirnov test was performed to compare the Brix and acidity change patterns." I am still looking for the statistical analysis!

Q10. Brix and Acidity: the methodology of determination of those parameters must be specified in the methodology section.
A10. We added the following in Materials and Methods, "For the Brix test, 0.3 mL of the sample solution was used; for acidity, 0.6 mL of the sample solution was diluted 20 times with water." This is not methodology! Which equipment was used? How can you measure acidity just by diluting the sample? 

Q11. Table 1: statistical analysis of the results must be specified.
A11. We have no technical replicates. How can you state that something really happened without confirming it? For me, if there are no replicates, there is no way to publish these results!

Minor revision of English language

Author Response

Thank you so much for helpful your comments.

Q5. Line 72: the authors describe the use of water or water containing H2 to treat cells. Are the cultures mixed (both yeast and bacterium) at this stage or are they separated? Which is the concentration of cells at this stage? The use of water without H2 is a control? This water or water containing H2 is removed before inoculation?
A5. We changed "Solutions" to "These four solutions, sake yeasts suspended in water, sake yeasts suspended in H2-water, kuratsuki Kocuria suspended in water, and kuratsuki Kocuria suspended in H2-water,".
We showed separated four solutions, which is NOT mixed! We added each concentrations. We have shown the control which is not treated with molecular hydrogen. We have already shown that 10 mL of each solution was added! Molecular hydrogen quickly diffuses into the air and is out of solution.

Q9. Line 108: “Additionally, there was no significant variance in the Brix and acidity of sake…” Where is the statistical analysis?
A9. We added the following in Materials and Methods, "The Kolmogorov–Smirnov test was performed to compare the Brix and acidity change patterns." I am still looking for the statistical analysis!
We performed the KS test using R software, used command "ks.test()". As the result, p-values were > 0.05. We do not need to show the process in this text, because we declared "Data are available upon request" in Data Availability Statement.

Q10. Brix and Acidity: the methodology of determination of those parameters must be specified in the methodology section.
A10. We added the following in Materials and Methods, "For the Brix test, 0.3 mL of the sample solution was used; for acidity, 0.6 mL of the sample solution was diluted 20 times with water." This is not methodology! Which equipment was used? How can you measure acidity just by diluting the sample? 
We have already shown that we used PAL-BX/ACID (ATAGO, Tokyo, Japan). Probably the reviewer do not know and do not use it. It does not need any parameters. It needs only sample.

Q11. Table 1: statistical analysis of the results must be specified.
A11. We have no technical replicates. How can you state that something really happened without confirming it? For me, if there are no replicates, there is no way to publish these results!
We deleted Table 1, chapter 3.2, and the related references.

Round 3

Reviewer 1 Report

Q10. Brix and Acidity: the methodology of determination of those parameters must be specified in the methodology section.
A10. We added the following in Materials and Methods, "For the Brix test, 0.3 mL of the sample solution was used; for acidity, 0.6 mL of the sample solution was diluted 20 times with water." This is not methodology! Which equipment was used? How can you measure acidity just by diluting the sample? 
We have already shown that we used PAL-BX/ACID (ATAGO, Tokyo, Japan). Probably the reviewer do not know and do not use it. It does not need any parameters. It needs only sample.

AUTHORS MUST UNDERSTAND THAT THE METHODOLOGY NEEDS TO BE DESCRIBED IN DETAIL TO MAKE IT POSSIBLE TO REPRODUCE DATA AND FOR THE READER TO UNDERSTAND THE RESULTS AND THEIR UNCERTAINTIES. IT IS NOT INTENDED JUST FOR THE REVIEWERS. "PAL-BX/ACID" IS THE MODEL OF THE EQUIPMENT. the equipment must be specified.

It is ok

Author Response

Thank you so much. We added "digital refractometer".