β-Glucosidase Activity of Lactiplantibacillus plantarum UNQLp 11 in Different Malolactic Fermentations Conditions: Effect of pH and Ethanol Content

Round 1

Reviewer 1 Report

The manuscript entitled “β-Glucosidase activity of Lactiplantibacillus plantarum UNQLp11 in different malolactic fermentations conditions: effect of pH and ethanol content” is about to characterize the β-glucosidase activity from Lb. plantarum UNQLp11 to get the background data for commercial application.

This manuscript include the enzyme activity at the different condition, HSSE-GC/MS analysis and cell viability to check whether the strain UNQLp11 is good for wine making with good β-glucosidase activity.

Manuscript is written well and experiments were done but this manuscript need modification to improve the quality for publication.

Line 80 and 85: there are underline on the ^oC. Please change that.

Line 77: authors need to provide the information why choose the pH 3.8, 3.5, 3.2. Just because the pH of wine are usually 3.5?

At figure 4, title of X-axis is glycosidase activity but figure title is glucosidase activity. Authors need consistently use the enzyme activity.

At figure 5. Authors need to provide why 5mg/ml of commercial enzyme used in this study because authors compared the activity of glucosidase. If possible authors need additional experiments with higher amount of commercial enzymes to see the difference with low amount of enzyme.

Overall, this manuscript is need some modification to be published.

Author Response

We are now sending you the revised version of the manuscript “Β-GLUCOSIDASE ACTIVITY OF LACTIPLANTIBACILLUS PLANTARUM UNQLP 11 IN DIFFERENT MALOLACTIC FERMENTATIONS CONDITIONS " Manuscript ID: fermentation-1088519, in consonance with reviewers and editor suggestions. Changes made to the manuscript are highlighted in yellow and also answered in this letter.

We are very grateful to the reviewers for their constructive and useful comments. Looking forward to hearing from you,

Yours sincerely,

Dra. E. Elizabeth Tymczyszyn

Reviewer #1:

The manuscript entitled “β-Glucosidase activity of Lactiplantibacillus plantarumUNQLp11 in different malolactic fermentations conditions: effect of pH and ethanol content” is about to characterize the β-glucosidase activity from Lb. plantarumUNQLp11 to get the background data for commercial application. This manuscript include the enzyme activity at the different condition, HSSE-GC/MS analysis and cell viability to check whether the strain UNQLp11 is good for wine making with good β-glucosidase activity. Manuscript is written well and experiments were done but this manuscript need modification to improve the quality for publication.

Line 80 and 85: there are underline on the oC. Please change that.

ANSWER: All symbols were changed

Line 77: authors need to provide the information why choose the pH 3.8, 3.5, 3.2. Just because the pH of wine are usually 3.5?

ANSWER: The pH and ethanol are the main factors that affect the behavior of LAB in wine, and it exerts a selective action on the LAB strains present, determines the viability and growth speed of LAB, the degradation speed of malic acid and its metabolic behavior. The ethanol content could be between 12-14 % v/v and pH is usually 3.5, but it is difficult to predict the pH value in a winery, and pH can be varied due the presence of different concentration of lactic, malic, citric, acetic and tartaric acid, among others. For this reason, several authors modified the pH of wines in the range of 3.2 to 3.8, for research purpose (Davis et al 1986, Gockowiak and Henschke, 2003, Cinquanta et al 2018). In this work, the performance of UNQLp 11 was analyzed at three different pH values: 3.2, 3.8 and at the usual pH value of the wines, 3.5. In order to make clear the use of different pH values, a sentence was added in the Introduction section.

Gockowiak, H., & Henschke, P. A. (2003). Interaction of pH, ethanol concentration and wine matrix on induction of malolactic fermentation with commercial ‘direct inoculation’starter cultures. Australian journal of grape and wine research, 9(3), 200-209.

Cinquanta, L., De Stefano, G., Formato, D., Niro, S., & Panfili, G. (2018). Effect of pH on malolactic fermentation in southern Italian wines. European Food Research and Technology, 244(7), 1261-1268.

Davis, C. R., Wibowo, D. J., Lee, T. H., & Fleet, G. H. (1986). Growth and metabolism of lactic acid bacteria during and after malolactic fermentation of wines at different pH. Applied and environmental microbiology, 51(3), 539-545.

At figure 4, title of X-axis is glycosidase activity but figure title is glucosidase activity. Authors need consistently use the enzyme activity.

ANSWER: The title of the axis was changed and the enzyme activity was consistently change along the manuscript.

At figure 5. Authors need to provide why 5mg/ml of commercial enzyme used in this study because authors compared the activity of glucosidase. If possible authors need additional experiments with higher amount of commercial enzymes to see the difference with low amount of enzyme.

ANSWER: The concentration of the commercial enzyme used was 10 mg/L, according to the supplier recommendations. A paragraph with data of commercial enzyme was added in section “Hydrolysis of octyl β-D-glucopyranoside in sterile Pinot noir wine” for more clarity. At this concentration, excellent results were obtained for hydrolysis of P-NPG in synthetic wine. It is reasonable for the reviewer to recommend testing with more concentration of the enzyme, but due to the Covid-19 pandemic we cannot concur to the laboratory for more experiments. For this reason, a sentence suggesting that concentration of the commercial enzyme must be studied in different wines was added in the text.

Overall, this manuscript is need some modification to be published.

Additionally, the English was rigorously revised along the manuscript, and according reviewers suggestions all sections of the present manuscript was revised and improved.

Reviewer #2: The authors describes the β-glucosidase activity of Lactiplantibacillus plantarum UNQLp11 in different malolactic fermentation conditions. The manuscript contains some interesting points. In addition, the manuscript was well-prepared to understand.

However, the major drawback of this study is the lack of rationale to perform this study. What is the reason of performing experiment using artificial wine? ANSWER: β-glucosidase activity was measured by a colorimetric method (Grimaldi et al 2000 and 2005), the color in the red wine difficults the measurement of the hydrolysis product of p-NPG. For this reason, synthetic wine is a good option for the screening of this enzymatic activity and was used in this work. In order to make clear the use of synthetic wine, a sentence was added in the Introduction section.

What is the reason of choosing Pinot Noir wine? Is it representing many kinds of wine?

ANSWER: North Patagonia is the southernmost wine-producing region of Argentina and one of the most Southern regions in the world. This region has optimal agro-ecological conditions for high quality viticulture, in which Pinot noir have found the optimal conditions to express all its oenological potential. Also, UNQLp 11, strain selected for the fermentation assays was isolated from Patagonian Pinot noir wines (vintages 2012), and according previous studied by our group, is an excellent candidate as autochthonous MLF starter culture (Brizuela et al. 2017, 2018a, b, 2020). Information about the reasons to use Patagonian Pinot noir wine in this study was added in the manuscript (a sentence was added in the Introduction section).

Brizuela, N. S., Bravo-Ferrada, B. M., La Hens, D. V., Hollmann, A., Delfederico, L., Caballero, A., ... & Semorile, L. (2017). Comparative vinification assays with selected Patagonian strains of Oenococcus oeni and Lactobacillus plantarum. LWT, 77, 348-355.

Brizuela, N. S., Bravo-Ferrada, B. M., Pozo-Bayón, M. Á., Semorile, L., & Tymczyszyn, E. E. (2018). Changes in the volatile profile of Pinot noir wines caused by Patagonian Lactobacillus plantarum and Oenococcus oeni strains. Food Research International, 106, 22-28.

Brizuela, N. S., Bravo-Ferrada, B. M., Curilén, Y., Delfederico, L., Caballero, A., Semorile, L., ... & Tymczyszyn, E. E. (2018). Advantages of using blend cultures of native L. plantarum and O. oeni strains to induce malolactic fermentation of Patagonian Malbec wine. Frontiers in microbiology, 9, 2109.

Brizuela, N. S., Franco‐Luesma, E., Bravo‐Ferrada, B. M., Pérez‐Jiménez, M., Semorile, L., Tymczyszyn, E. E., & Pozo‐Bayon, M. A. (2021). Influence of Patagonian Lactiplantibacillus plantarum and Oenococcus oeni strains on sensory perception of Pinot Noir wine after malolactic fermentation. Australian Journal of Grape and Wine Research, 27(1), 118-127.

Is the 'Lactiplantibacillus plantarum' can be used as a starter in real wine production for commercialization? Then, it will greatly affect and differ the taste and flavor of the wine compared to that produced by conventional methods. ANSWER: Oenococcus oeni is the major LAB used in commercial starter cultures for MLF. However, in the last decade, several commercial malolactic starters using Lb. plantarum have been released in the market, such as: Lallemand V22, ML Prime, CHR Hansen Viniflora Nova and the co-inoculant blend Anchor NT 202 (Brizuela et al 2019). Lb. plantarum strains have potential advantages, such as the presence of more flavor-relative genes than O. oeni (Lerm et al 2011), a higher ability to consume malic acid under harsh wine conditions and easy to growth and preserved by drying, with the consequent economic advantages on the production of the indigenous starter cultures (Brizuela et al 2019). In order to make clear the relevance of study Lb. plantarum strains (particularly Patagonian strain UNQLp 11) as autochthonous malolactic starter cultures, a sentence with explanation was added in the introduction section.

Brizuela, N., Tymczyszyn, E. E., Semorile, L. C., La Hens, D. V., Delfederico, L., Hollmann, A., & Bravo-Ferrada, B. (2019). Lactobacillus plantarum as a malolactic starter culture in winemaking: A new (old) player?. Electronic Journal of Biotechnology, 38, 10-18.

Lerm, E., Engelbrecht, L., & Du Toit, M. (2011). Selection and characterisation of Oenococcus oeni and Lactobacillus plantarum South African wine isolates for use as malolactic fermentation starter cultures. South African Journal of Enology and Viticulture, 32(2), 280-295.

Additionally, the English was rigorously revised along the manuscript, and the introduction section was improved. We hope that these clarifications will be able to better explain the experimental design of the present work.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors describes the β-glucosidase activity of Lactiplantibacillus plantarum UNQLp11 in different malolactic fermentation conditions.

The manuscript contains some interesting points.

In addition, the manuscript was well-prepared to understand.

However, the major drawback of this study is the lack of rationale to perform this study.

What is the the reason of performing experiment using artificial wine?

What is the reason of choosing Pinot Noir wine? Is it representing many kinds of wine?

Is the 'Lactiplantibacillus plantarum' can be used as a starter in real wine production for commercialization? Then, it will greatly affect and differ the taste and flavor of the wine compared to that produced by conventional methods. 

Author Response

We are now sending you the revised version of the manuscript “Β-GLUCOSIDASE ACTIVITY OF LACTIPLANTIBACILLUS PLANTARUM UNQLP 11 IN DIFFERENT MALOLACTIC FERMENTATIONS CONDITIONS " Manuscript ID: fermentation-1088519, in consonance with reviewers and editor suggestions. Changes made to the manuscript are highlighted in yellow and also answered in this letter.

We are very grateful to the reviewers for their constructive and useful comments. Looking forward to hearing from you,

Yours sincerely,

Dra. E. Elizabeth Tymczyszyn

Reviewer #2: The authors describes the β-glucosidase activity of Lactiplantibacillus plantarum UNQLp11 in different malolactic fermentation conditions. The manuscript contains some interesting points. In addition, the manuscript was well-prepared to understand.

However, the major drawback of this study is the lack of rationale to perform this study. What is the reason of performing experiment using artificial wine? ANSWER: β-glucosidase activity was measured by a colorimetric method (Grimaldi et al 2000 and 2005), the color in the red wine difficults the measurement of the hydrolysis product of p-NPG. For this reason, synthetic wine is a good option for the screening of this enzymatic activity and was used in this work. In order to make clear the use of synthetic wine, a sentence was added in the Introduction section.

What is the reason of choosing Pinot Noir wine? Is it representing many kinds of wine?

ANSWER: North Patagonia is the southernmost wine-producing region of Argentina and one of the most Southern regions in the world. This region has optimal agro-ecological conditions for high quality viticulture, in which Pinot noir have found the optimal conditions to express all its oenological potential. Also, UNQLp 11, strain selected for the fermentation assays was isolated from Patagonian Pinot noir wines (vintages 2012), and according previous studied by our group, is an excellent candidate as autochthonous MLF starter culture (Brizuela et al. 2017, 2018a, b, 2020). Information about the reasons to use Patagonian Pinot noir wine in this study was added in the manuscript (a sentence was added in the Introduction section).

Brizuela, N. S., Bravo-Ferrada, B. M., La Hens, D. V., Hollmann, A., Delfederico, L., Caballero, A., ... & Semorile, L. (2017). Comparative vinification assays with selected Patagonian strains of Oenococcus oeni and Lactobacillus plantarum. LWT, 77, 348-355.

Brizuela, N. S., Bravo-Ferrada, B. M., Pozo-Bayón, M. Á., Semorile, L., & Tymczyszyn, E. E. (2018). Changes in the volatile profile of Pinot noir wines caused by Patagonian Lactobacillus plantarum and Oenococcus oeni strains. Food Research International, 106, 22-28.

Brizuela, N. S., Bravo-Ferrada, B. M., Curilén, Y., Delfederico, L., Caballero, A., Semorile, L., ... & Tymczyszyn, E. E. (2018). Advantages of using blend cultures of native L. plantarum and O. oeni strains to induce malolactic fermentation of Patagonian Malbec wine. Frontiers in microbiology, 9, 2109.

Brizuela, N. S., Franco‐Luesma, E., Bravo‐Ferrada, B. M., Pérez‐Jiménez, M., Semorile, L., Tymczyszyn, E. E., & Pozo‐Bayon, M. A. (2021). Influence of Patagonian Lactiplantibacillus plantarum and Oenococcus oeni strains on sensory perception of Pinot Noir wine after malolactic fermentation. Australian Journal of Grape and Wine Research, 27(1), 118-127.

Is the 'Lactiplantibacillus plantarum' can be used as a starter in real wine production for commercialization? Then, it will greatly affect and differ the taste and flavor of the wine compared to that produced by conventional methods. ANSWER: Oenococcus oeni is the major LAB used in commercial starter cultures for MLF. However, in the last decade, several commercial malolactic starters using Lb. plantarum have been released in the market, such as: Lallemand V22, ML Prime, CHR Hansen Viniflora Nova and the co-inoculant blend Anchor NT 202 (Brizuela et al 2019). Lb. plantarum strains have potential advantages, such as the presence of more flavor-relative genes than O. oeni (Lerm et al 2011), a higher ability to consume malic acid under harsh wine conditions and easy to growth and preserved by drying, with the consequent economic advantages on the production of the indigenous starter cultures (Brizuela et al 2019). In order to make clear the relevance of study Lb. plantarum strains (particularly Patagonian strain UNQLp 11) as autochthonous malolactic starter cultures, a sentence with explanation was added in the introduction section.

Brizuela, N., Tymczyszyn, E. E., Semorile, L. C., La Hens, D. V., Delfederico, L., Hollmann, A., & Bravo-Ferrada, B. (2019). Lactobacillus plantarum as a malolactic starter culture in winemaking: A new (old) player?. Electronic Journal of Biotechnology, 38, 10-18.

Lerm, E., Engelbrecht, L., & Du Toit, M. (2011). Selection and characterisation of Oenococcus oeni and Lactobacillus plantarum South African wine isolates for use as malolactic fermentation starter cultures. South African Journal of Enology and Viticulture, 32(2), 280-295.

Additionally, the English was rigorously revised along the manuscript, and the introduction section was improved. We hope that these clarifications will be able to better explain the experimental design of the present work.

Reviewer #1:

The manuscript entitled “β-Glucosidase activity of Lactiplantibacillus plantarumUNQLp11 in different malolactic fermentations conditions: effect of pH and ethanol content” is about to characterize the β-glucosidase activity from Lb. plantarumUNQLp11 to get the background data for commercial application. This manuscript include the enzyme activity at the different condition, HSSE-GC/MS analysis and cell viability to check whether the strain UNQLp11 is good for wine making with good β-glucosidase activity. Manuscript is written well and experiments were done but this manuscript need modification to improve the quality for publication.

Line 80 and 85: there are underline on the oC. Please change that.

ANSWER: All symbols were changed

Line 77: authors need to provide the information why choose the pH 3.8, 3.5, 3.2. Just because the pH of wine are usually 3.5?

ANSWER: The pH and ethanol are the main factors that affect the behavior of LAB in wine, and it exerts a selective action on the LAB strains present, determines the viability and growth speed of LAB, the degradation speed of malic acid and its metabolic behavior. The ethanol content could be between 12-14 % v/v and pH is usually 3.5, but it is difficult to predict the pH value in a winery, and pH can be varied due the presence of different concentration of lactic, malic, citric, acetic and tartaric acid, among others. For this reason, several authors modified the pH of wines in the range of 3.2 to 3.8, for research purpose (Davis et al 1986, Gockowiak and Henschke, 2003, Cinquanta et al 2018). In this work, the performance of UNQLp 11 was analyzed at three different pH values: 3.2, 3.8 and at the usual pH value of the wines, 3.5. In order to make clear the use of different pH values, a sentence was added in the Introduction section.

Gockowiak, H., & Henschke, P. A. (2003). Interaction of pH, ethanol concentration and wine matrix on induction of malolactic fermentation with commercial ‘direct inoculation’starter cultures. Australian journal of grape and wine research, 9(3), 200-209.

Cinquanta, L., De Stefano, G., Formato, D., Niro, S., & Panfili, G. (2018). Effect of pH on malolactic fermentation in southern Italian wines. European Food Research and Technology, 244(7), 1261-1268.

Davis, C. R., Wibowo, D. J., Lee, T. H., & Fleet, G. H. (1986). Growth and metabolism of lactic acid bacteria during and after malolactic fermentation of wines at different pH. Applied and environmental microbiology, 51(3), 539-545.

At figure 4, title of X-axis is glycosidase activity but figure title is glucosidase activity. Authors need consistently use the enzyme activity.

ANSWER: The title of the axis was changed and the enzyme activity was consistently change along the manuscript.

At figure 5. Authors need to provide why 5mg/ml of commercial enzyme used in this study because authors compared the activity of glucosidase. If possible authors need additional experiments with higher amount of commercial enzymes to see the difference with low amount of enzyme.

ANSWER: The concentration of the commercial enzyme used was 10 mg/L, according to the supplier recommendations. A paragraph with data of commercial enzyme was added in section “Hydrolysis of octyl β-D-glucopyranoside in sterile Pinot noir wine” for more clarity. At this concentration, excellent results were obtained for hydrolysis of P-NPG in synthetic wine. It is reasonable for the reviewer to recommend testing with more concentration of the enzyme, but due to the Covid-19 pandemic we cannot concur to the laboratory for more experiments. For this reason, a sentence suggesting that concentration of the commercial enzyme must be studied in different wines was added in the text.

Overall, this manuscript is need some modification to be published.

Additionally, the English was rigorously revised along the manuscript, and according reviewers suggestions all sections of the present manuscript was revised and improved.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

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