New Malolactic Bacteria Strains Isolated from Wine Microbiota: Characterization and Technological Properties
, Claudiu-Ioan Bunea 2,†, Ancuța Nechita 1, Florin Dumitru Bora 3, Simona Isabela Dunca 4, Andrei Mocan 5,6 and Roxana Mihaela Filimon 1,*Round 1
Reviewer 1 Report
The manuscript “New malolactic bacteria strains isolated from wine microbiota. Characterisation and technological properties” approaches the isolation and characterization of indigenous bacterial strains with potential use as starter cultures for the fermentation process.
However, I believe that some points in the manuscript need to be better elucidated by the authors.
- Regarding the methodologies for identifying the isolates, I believe that the use of molecular techniques associated with physiological identification tests would be essential.
-In the screening for the production of enzymes, were the tests performed in replicates? It would be interesting to quantify the size of the halos found for a less subjective assessment. I believe that it is not necessary to place figures elucidating these results. The results of the tables would be enough to understand the text.
- In the specialized literature there are several tests for the production of more specific biogenic amines with quantitative results.
- In my opinion, the manuscript does not present any significant news, other than the characterization of bacterial strains isolated from the fermentation process. To suggest the use of these isolates as initiators of the fermentation process for wine production, it would be interesting to carry out tests on a laboratory scale and then on a pilot scale.
Author Response
Editor,
Fermentation
January, 2022
Dear Editor,
I am pleased to submit the first revision of the original manuscript entitled: “New malolactic bacteria strains isolated from wine microbiota. Characterisation and technological properties” authors: Răzvan Vasile Filimon, Claudiu-Ioan Bunea, Ancuța Nechita, Florin Dumitru Bora, Simona Isabela Dunca, Andrei Mocan and Roxana Mihaela Filimon, for publication in Fermentation MDPI journal.
We would like to thank the reputable reviewers for their time and suggestions, which will definitely improve the manuscript. With great respect and gratitude, we will respond to each reviewer according to the suggestions made.
Reviewer 1.
- Regarding the methodologies for identifying the isolates, I believe that the use of molecular techniques associated with physiological identification tests would be essential.
In our labs, we do not have the opportunity to perform genetic tests at this time, but we plan to perform these molecular tests. But, as we mentioned in the manuscript, the API 50 CH (BioMérieux, France) is a standardized system used to differentiate LAB isolates. API 50 CH tests were extensively used for phenotypic characterization of bacterial species isolated from grape must, wine and dairy product, previous results obtained being highly correlated with genetic tests.
- In the screening for the production of enzymes, were the tests performed in replicates? It would be interesting to quantify the size of the halos found for a less subjective assessment. I believe that it is not necessary to place figures elucidating these results. The results of the tables would be enough to understand the text.
As we mentioned in 2.7. Statistical Procedure subsection, all determinations were carried out in triplicate from independent cultures of each bacterial strain.
As you very well suggested, we measured the halos for each analysis, but being a qualitative determination, we preferred to highlight the phenomenon without having divergences in terms of standardization of the working method.
As a different reviewer suggested, „data were illustrated by figures, photo captures or tables/graphic which make the paper friendly reader either for people generally interested in the subject, not only for the specialists”. If necessary, we can remove the images if the editor considers that they are not conclusive.
- In the specialized literature there are several tests for the production of more specific biogenic amines with quantitative results.
The aim was not to determine the concentrations of biogenic amines (by HPLC), but to eliminate from the study any bacterial strain capable of producing such compounds by decarboxylation of amino acids.
- In my opinion, the manuscript does not present any significant news, other than the characterization of bacterial strains isolated from the fermentation process. To suggest the use of these isolates as initiators of the fermentation process for wine production, it would be interesting to carry out tests on a laboratory scale and then on a pilot scale.
Respectfully, thank you for all your suggestions. We considered it appropriate to share our results, especially after evaluating the new valuable straines of lactic acid bacteria in synthetic media. Also, we continued the experiments in wine (both at laboratory and micropilot level – data in progress), and the partial results confirmed the results obtained in this first study. Data regarding the LAB testing in wine will be the subject of a new paper, as soon as possible.
All authors thank you for the favorable evaluation and for all the competent sugestions.
We appreciate your time and look forward to your positive response.
Sincerely,
Filimon R.V., PhD
Senior researcher, Department of Winemaking and Wine microbiology
Research Development Station for Viticulture and Winemaking Iasi, Romania
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The paper entitled: New malolactic bacteria strains isolated from wine microbiota. Characterization and technological properties, is related to putative novel bacteria isolated from wine industry. The paper is dived into three section and close with conclusion and literature data.
The first part is dedicated to Introduction which is very well documented and argued for the subject of the paper. This part is close with the aim of the work which was the characterization and assessment of technological properties of new malolactic bacteria strains isolated from red wine microbiota, in order to determine their potential to be used as starter cultures to induce the malolactic fermentation of wine.
The second part, Materials and methods, is subdivided in other seven subchapters and described in details the protocols and methodology used accordingly with the aim of the paper. Each method is very well described for further reproduction or is very well argued with the literature data.
The chapter dedicated to Results and Discussion is also subdivided in six subchapters and showed in a very well way they obtained results which are discussed accordingly. Some subchapters, six for example, is also subdivided, in order to explain in a very good order the data and argued for their high scientific values. All the data are illustrated by figures, photo captures or tables/graphic which make the paper friendly reader either for people generally interested in the subject not only for the specialists.
The main conclusions of the work were that 67 indigenous catalase-negative and Gram-positive lactic acid bacteria strains were isolated from red wines undergoing spontaneous malolactic fermentation and two of them showing high potential for future use in the winemaking process as malolactic bacterial starter cultures, in order to obtain high quality wines with balanced acidity and increased typicity.
The papaer have 46 literature titles strongly correlated with the subject of the work.
Author Response
Editor,
Fermentation
January, 2022
Dear Editor,
I am pleased to submit the first revision of the original manuscript entitled: “New malolactic bacteria strains isolated from wine microbiota. Characterisation and technological properties” authors: Răzvan Vasile Filimon, Claudiu-Ioan Bunea, Ancuța Nechita, Florin Dumitru Bora, Simona Isabela Dunca, Andrei Mocan and Roxana Mihaela Filimon, for publication in Fermentation MDPI journal.
We would like to thank the reputable reviewers for their time and suggestions, which will definitely improve the manuscript. With great respect and gratitude, we will respond to each reviewer according to the suggestions made.
Reviewer 2.
Thank you for the favorable evaluation and for all the competent comments.
All authors thank you for the favorable evaluation and for all the competent sugestions.
We appreciate your time and look forward to your positive response.
Sincerely,
Filimon R.V., PhD
Senior researcher, Department of Winemaking and Wine microbiology
Research Development Station for Viticulture and Winemaking Iasi, Romania
Author Response File: Author Response.pdf
Reviewer 3 Report
Provide methods for wine analysis: pH, SO2, volatile acidity, total acidity, alcohol content, reducing sugars/substances as mentioned at pag. 5 lines 230-233 and pH device used.
At pag. 5 line 214, specify device model used.
At pag. 5 line 237-238, please provide reference for statement.
At pag. 8 line 294, please revise the number of strains showing high potential for malolactic bioconversion.
Author Response
Editor,
Fermentation
January, 2022
Dear Editor,
I am pleased to submit the first revision of the original manuscript entitled: “New malolactic bacteria strains isolated from wine microbiota. Characterisation and technological properties” authors: Răzvan Vasile Filimon, Claudiu-Ioan Bunea, Ancuța Nechita, Florin Dumitru Bora, Simona Isabela Dunca, Andrei Mocan and Roxana Mihaela Filimon, for publication in Fermentation MDPI journal.
We would like to thank the reputable reviewers for their time and suggestions, which will definitely improve the manuscript. With great respect and gratitude, we will respond to each reviewer according to the suggestions made.
Reviewer 3.
Provide methods for wine analysis: pH, SO2, volatile acidity, total acidity, alcohol content, reducing sugars/substances as mentioned at pag. 5 lines 230-233 and pH device used.
We completed at the 2.2. Isolation and Purification of Indigenous LAB Strains subsection: The main physico-chemical parameters of wines were determined acording to the Compendium of international methods of wine and must analysis of OIV (with the appropriate citation).
At pag. 5 line 214, specify device model used.
We specified at the 3.1. Isolation of Indigenous LAB Strains subsection: (pH meter InoLab Level 1, with SenTix 81 electrode; WTW, Germany).
At pag. 5 line 237-238, please provide reference for statement.
„...diversity and density of LAB are influenced by both the grape cultivar and microclimate conditions” is our conclusion, as a result of the studies undertaken, it is not quoted from the literature.
In order to clarify the situation, we have added text with some mentions from the literature: „Similar findings were previously reported by Muñoz et al. (27) and Dicks and Endo (28), which showed that the density of LAB in the initial phases of winemaking (the must phase and onset of alcoholic fermentation) ranges from approximately 103 to 104 CFU/mL and the numbers and proportions of species vary according to the ripeness and condition of the grapes at the time of harvesting. Moreover, De Orduna mentioned that climate conditions are exerting a profound influence on wine microbiology (29), while Bokulich et al. introduced the term “microbial terroir”, as determining factor in regional variation among wine grapes (30).
At pag. 8 line 294, please revise the number of strains showing high potential for malolactic bioconversion.
No need to revise, of the 67 initial malolactic strains, only 12 strains showing high potential for malolactic bioconversion were retained and further characterized.
All authors thank you for the favorable evaluation and for all the competent sugestions.
We appreciate your time and look forward to your positive response.
Sincerely,
Filimon R.V., PhD
Senior researcher, Department of Winemaking and Wine microbiology
Research Development Station for Viticulture and Winemaking Iasi, Romania
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Dear authors and editor. I believe that as requested revisions were answered. The manuscript has been sufficiently improved for publication in this journal.
