Review Reports - Oenological Capabilities of Yeasts Isolated from High-Sugar Matrices (Manna and Honey) as Potential Starters and Co-Starters for Winemaking

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article deals with the evaluation of the enological potential of yeasts isolated from manna and honey, including Saccharomyces and non-Saccharomyces species. The experiments follow the usual approach to select wine strains and are well described and the results are interesting for the scientific and technical communities.

Authors should stress, at the conclusions, that fermentations were performed with pasteurized grape juice which is understandable as an initial screening but may not be representative of real ferments, particularly when skin maceration is used.

Minor corrections

Line 29, remove )

Line 51, correct to Interestingly

Lines 204, 282, remove paragraph

Table 1, insert string origin, manna or honey

Line 276, correct to demonstrated

Line 372, insert reference

Author Response

R.1- The article deals with the evaluation of the enological potential of yeasts isolated from manna and honey, including Saccharomyces and non-Saccharomyces species. The experiments follow the usual approach to select wine strains and are well described and the results are interesting for the scientific and technical communities.

AU. Thank you for your comments. We modified the manuscript following your suggestions and all the changes in the text were highlighted in yellow.

R.1- Authors should stress, at the conclusions, that fermentations were performed with pasteurized grape juice which is understandable as an initial screening but may not be representative of real ferments, particularly when skin maceration is used.

AU. Thank you for this suggestion, the section regarding the conclusions was modified according to your comment (L.447-450; L.459-461).

R.1- Line 29, remove )

AU. Done (L.27).

R.1- Line 51, correct to Interestingly

AU. Done (L.48).

R.1- Lines 204, 282, remove paragraph

AU. Done (L.201-202; L.278-280).

R.1- Table 1, insert string origin, manna or honey

AU. Done (L.241).

R.1- Line 276, correct to demonstrated

AU. Done (L.272).

R.1- Line 372, insert reference

AU. Done (L.366).The new reference [45] was added in the references section (L. 624).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript of the original scientific article has a very well-defined problem and review of publications in the introductory part. The Materials and Methods chapter is described in detail and no corrections are necessary. The experimental part is appropriately designed, and the obtained results are statistically analysed.

In the research, appropriate molecular techniques were used to investigate yeast diversity and extensive technological characterization was carried out. The most oenologically interesting strains with limited H₂S production and increased tolerance to ethanol, osmotic stress and copper were selected.

Many results are presented in detail in a total of five tables and four figures. The commentary and discussion of the results is excellent, and includes a total of 47 contemporary literature references. The authors appropriately conclude that further studies are needed to determine how these selected strains affect VOCs when exposed to sequential inoculation with S. cerevisiae.

Comments for author File: Comments.pdf

Author Response

R.3- The manuscript of the original scientific article has a very well-defined problem and review of publications in the introductory part. The Materials and Methods chapter is described in detail and no corrections are necessary. The experimental part is appropriately designed, and the obtained results are statistically analysed. In the research, appropriate molecular techniques were used to investigate yeast diversity and extensive technological characterization was carried out. The most oenologically interesting strains with limited H₂S production and increased tolerance to ethanol, osmotic stress and copper were selected. Many results are presented in detail in a total of five tables and four figures. The commentary and discussion of the results is excellent, and includes a total of 47 contemporary literature references. The authors appropriately conclude that further studies are needed to determine how these selected strains affect VOCs when exposed to sequential inoculation with S. cerevisiae.

AU. Dear author, thank you for your kind comments. We greatly appreciate your comments. We modified the manuscript following your suggestions and all the changes in the text were highlighted in green.

R.3- Line 23 change “enhancing” with “the improvement of”

AU. Done (L.21).

R.3- Line 24 change “perspective” with “point of view”

AU. Done (L.22).

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

However, relying solely on S. cerevisiae has led to a 42

decrease in microbial diversity within the final product, resulting in a flattening of sensory 43

characteristics.

This is a statement not supported by research data. Need citations for this statement or delete this statement.

diverse array of other yeast species and genera that can persist during fermentation. 46

These non-Saccharomyces yeasts, while of secondary importance in must fermentation, are 47

sometimes considered spoilage microorganisms

Not only non-saccharomyces yeast persist through fermentation. Also diverse strains of S. cerevisiae can be found in inoculated and in spontaneous fermentations.

Additionally, unwanted volatile phenols, such as those produced 50

by Brettanomyces spp. [3],

Need citation for Brettanomyces here. Ref (3) is not correct here.

Specifically, they contribute to improving wine complexity, texture, and flavour 53

integration in spontaneous fermentations since the 1980s [4].

Specifically, they can contribute to improving wine complexity, texture, and flavour 53

integration in spontaneous fermentations since the 1980s [4].

It is an overstatement to say that wine fermentations conducted with S. cerevisiae starter cultures are lacking complexity, texture, flavor. Show evidence.

non-Saccharomyces yeasts 84

generally exhibit lower fermentation performance and do not numerically dominate the 85

entire fermentation process due to their limited tolerance to ethanol. 86

Lines 84-86 change to: non-Saccharomyces yeasts generally exhibit lower fermentation performance and do not numerically dominate the entire fermentation process due to their limited tolerance to ethanol and SO2.

Line 89 change to: This makes them suitable candidates for co-inoculation or sequential inoculation alongside S. cerevisiae.

Lines 139-156

Question the usefulness of the glucosidase activity test used. Because this test only uses on glycoside substrate seems to very much underestimate the glycosidic activity by yeast on glycosides found in grape must and wine. Thus, characterization by this test is very limited. In my view this test employed for screening these yeast isolates is meaningless and should be omitted from the paper. The test only says that only one isolate was able to hydrolyze this substrate but it can not tell you how active these yeast isolates might be against diverse glycosides found in grape must and wine.

Line 321: In terms of weight loss percentage, both the TR1 and TR4 trials exhibited greater losses compared to 322

the TRC control.

This sentence is unclear. Fig 3 shows that all yeasts tested here reach the same percent weight loss by around day 25-30. The rate of weight loss – fermentation rate - was different. Fermentation rate was highest for TR4, followed by TR2, TR1, and finally TR3.

Line 398: In terms of glycerol content, the highest values were observed in trials inoculated with L. thermotolerans (7.4 g/L) compared to other treatments involving non-Saccharomyces yeasts.

Be specific. List the final glycerol content for all strains tested. Simply listing one value and saying this was higher is not clear.

Minor editing of the English language use is needed to correct grammatical mistakes and limit redundancy of words to help make the writing more concise.

Example:

The yeasts employed in this research work belongs to the collection of the 111

Department of Agricultural, Food and Forest Sciences (SAAF; University of Palermo, 112

Italy),

The yeasts studied here in this research work belongs to the collection of the 111

Department of Agricultural, Food and Forest Sciences (SAAF; University of Palermo, 112

Italy), they were isolated from manna and honey by-products they were isolated from manna and honey by-products

Define ‘manna’. Do the authors mean ‘honeydew’ the insect secretion?

Also, what ‘honey by-products’ are the authors referring to?

To make an initial discrimination of yeast, 38 isolates were analysed

For initial discrimination, 38 yeast isolates were analyzed...

Comments on the Quality of English Language

Minor editing of the English language use is needed to correct grammatical mistakes and limit redundancy of words to help make the writing more concise.

Author Response

R.4- However, relying solely on S. cerevisiae has led to a decrease in microbial diversity within the final product, resulting in a flattening of sensory characteristics.

This is a statement not supported by research data. Need citations for this statement or delete this statement.

AU. Thanks for this comment, the statement has been removed from the text.

R.4- diverse array of other yeast species and genera that can persist during fermentation. These non-Saccharomyces yeasts, while of secondary importance in must fermentation, are sometimes considered spoilage microorganisms

Not only non-Saccharomyces yeast persist through fermentation. Also diverse strains of S. cerevisiae can be found in inoculated and in spontaneous fermentations.

AU. Thanks for your suggestion, we modified the sentence, including a new reference, which was also added in the list. All the changes in the text were highlighted in turquoise (L.41-43; L.501-502).

R.4- Additionally, unwanted volatile phenols, such as those produced by Brettanomyces spp. [3],

Need citation for Brettanomyces here. Ref (3) is not correct here.

AU. Done, the reference has been changed (L509-511).

R.4- Specifically, they contribute to improving wine complexity, texture, and flavour integration in spontaneous fermentations since the 1980s [4].

It is an overstatement to say that wine fermentations conducted with S. cerevisiae starter cultures are lacking complexity, texture, flavor. Show evidence.

AU. We totally agree with your comment, thus we have removed the previous statement that fermentation solely with S. cerevisiae might result in a wine that lacks complexity.

R.4- non-Saccharomyces yeasts generally exhibit lower fermentation performance and do not numerically dominate the entire fermentation process due to their limited tolerance to ethanol.

Lines 84-86 change to: non-Saccharomyces yeasts generally exhibit lower fermentation performance and do not numerically dominate the entire fermentation process due to their limited tolerance to ethanol and SO₂.

AU. Done. Additionally, two new references have been included to the list (L.80-82; L.561-565).

R.4- Line 89 change to: This makes them suitable candidates for co-inoculation or sequential inoculation alongside S. cerevisiae.

AU. Done (L.85-86).

R.4- Lines 139-156: Question the usefulness of the glucosidase activity test used. Because this test only uses on glycoside substrate seems to very much underestimate the glycosidic activity by yeast on glycosides found in grape must and wine. Thus, characterization by this test is very limited. In my view this test employed for screening these yeast isolates is meaningless and should be omitted from the paper. The test only says that only one isolate was able to hydrolyze this substrate but it can not tell you how active these yeast isolates might be against diverse glycosides found in grape must and wine.

AU. We agree with your comment, thus we have removed the test regarding glucosidase activity from MM section 2.3 “Technological screening”, from RD section 3.2 “Technological characteristics of yeast strains” and 3.3 “Microfermentation”, from table 2 and from References list.

R.4- Line 321-322: In terms of weight loss percentage, both the TR1 and TR4 trials exhibited greater losses compared to the TRC control.

AU. Thanks for your observation. We have modified the entire paragraph following your suggestion (L.330-334).

R.4- Line 398: In terms of glycerol content, the highest values were observed in trials inoculated with L. thermotolerans (7.4 g/L) compared to other treatments involving non-Saccharomyces yeasts.

Be specific. List the final glycerol content for all strains tested. Simply listing one value and saying this was higher is not clear.

AU. We agree with your comment, therefore we have changed the sentence and listed glycerol values for each strain tested (L.409-412).

R.4- Minor editing of the English language use is needed to correct grammatical mistakes and limit redundancy of words to help make the writing more concise.

Example:

The yeasts employed in this research work belongs to the collection of the Department of Agricultural, Food and Forest Sciences (SAAF; University of Palermo, Italy),

The yeasts studied here in this research work belongs to the collection of the Department of Agricultural, Food and Forest Sciences (SAAF; University of Palermo, Italy), they were isolated from manna and honey by-products they were isolated from manna and honey by-products

Define ‘manna’. Do the authors mean ‘honeydew’ the insect secretion?

Also, what ‘honey by-products’ are the authors referring to?

To make an initial discrimination of yeast, 38 isolates were analysed For initial discrimination, 38 yeast isolates were analyzed...

AU. English has been revised throughout the manuscript. A brief description of manna and honey by-products has been included in the introduction section (L.89-90; L.96-98; L.111; L.117).

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript "Oenological capabilities of yeasts isolated from high sugar matrices (manna and honey) as potential starters and co-starters
for winemaking" present very interesting results on potential of non-Saccharomyces yeast for winemaking. After reading the article carefully, I confirm that it can be published without any corrections.

Author Response

R.5- The manuscript "Oenological capabilities of yeasts isolated from high sugar matrices (manna and honey) as potential starters and co-starters
for winemaking" present very interesting results on potential of non-Saccharomyces yeast for winemaking. After reading the article carefully, I confirm that it can be published without any corrections.

AU. Dear author, we appreciate your warm words. We are very grateful for your feedback.

Author Response File: Author Response.pdf