Analysis of the Genome Architecture of Lacticaseibacillus paracasei UNQLpc 10, a Strain with Oenological Potential as a Malolactic Starter
, Danay Valdés La Hens 2, Liliana Carmen Semorile 2, Emma Elizabeth Tymczyszyn 2,* and Bárbara Mercedes Bravo Ferrada 2
Round 1
Reviewer 1 Report
The manuscript by Iglesias et al. reports about the Lacticaseibacillus paracasei UNQLpc 10 genome and its structure as well as the possible functions of the predicted genes in enological properties, ability to successfully conduct the malolactic fermentation, and potential probiotic properties. Unfortunately, several improvements are needed in terms of clarity. Therefore, the manuscript is not acceptable for publication in its current state.
Specific comments:
Title:
To be more precise, “the malolactic fermentation” must be added to the title:
“Analysis of the genome architecture of Lacticaseibacillus paracasei UNQLpc 10, a strain with potential for malolactic fermentation during winemaking”
or
“Analysis of the genome architecture of Lacticaseibacillus paracasei UNQLpc 10 as potential malolactic starter strain for winemaking”
Abstract:
Line 13- “When incubated in a wine-like medium, it showed good survival capacity and ability to consume L-malic acid.” It is not clear is it the comment of the results obtained in this, or in previous manuscript, because in this manuscript are presented results regarding survival of the Lcb. paracasei UNQLpc 10 strain and malic acid consumption in synthetic wine. These results must be mentioned in the abstract after the sentence “The aim of this work was…” (line 15)
Results:
Table 1 - Instead of “Lpc.” must be “Lcb. paracasei”
Table 2 – the title of this table must be introduced
Figure 4 – introduce word “Lcb. paracasei” before label “UNQLpc 10”
Line 253 – at the end of sentence “Regarding genes with probiotic functionality….” reference regarding data from the literature about exopolysaccharides “extracellularly secreted by many microorganisms” is missing and must be added.
Figure 6 – The title is not correct. Instead of “Evolution of the bacterial population...”, it was shown “Survival of the Lcb. paracasei UNQLpc 10 strain (A) and L-malic acid consumption (MAC) (B) during malolactic fermentation (MLF) in synthetic wine.” Additionally, when you correct the title, the abbreviation on the “y “axis MAC will be explained.
The chapter Discussion starts with the comments about results shown on Figure 6, and experimental results of the manuscript shown before this figure (Tables 1 and 2, and Figures 1-5) are mentioned after, so, rearrangement of the text is necessary according to the order of appearance of the results. Additionally, it would be worth mentioning that the Lacticaseibacillus strains were isolated from a broad range of habitats including not only food (raw or fermented), but also sewage, invertebrates, birds and mammals including human gut of healthy adults as well as breast-fed infants with the probiotic coding potential components of the genome of isolated L. paracasei strains (Florova et al. 2021; Rodrigo-Torres et al. 2022)
Frolova, M., Yudin, S., Makarov, V., Glazunova, O., Alikina, O., Markelova, N., ... & Ozoline, O. Lacticaseibacillus paracasei: Occurrence in the Human Gut Microbiota and K-Mer-Based Assessment of Intraspecies Diversity. Life 11 (2021): 1246.
Rodrigo-Torres, L., Landete, J. M., Huedo, P., Peirotén, Á., Langa, S., Rodríguez-Minguez, E., ... & Arqués, J. L. (2022). Complete genome sequences of Lacticaseibacillus paracasei INIA P272 (CECT 8315) and Lacticaseibacillus rhamnosus INIA P344 (CECT 8316) isolated from breast-fed infants reveal probiotic determinants. Gene, 840, 146743.
Conclusion
Line 412 – Technological properties are wrongly mentioned in the context of potential probiotic properties. Therefore, corrections must be made as follows: “This strain also presents natural tolerance towards a hostile environment as wine (acid, ethanol, and phenolic compounds), a fact that also increases its potential to be used as a live probiotic malolactic starter culture. Finally, UNQLpc 10 has genes that are of technological interest for probiotic use, and more investigation must be carried out to evaluate the potential of the strain in the food industry.”
Author Response
Dear Editor,
We are sending you the revised version of the manuscript “Analysis of the genome architecture of Lacticaseibacillus paracasei UNQLpc10, a strain with oenological potential as malolactic starter "; Manuscript ID: fermentation- 2022575, in consonance with reviewers and editor suggestions. Changes made in 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,
Dr. E. Elizabeth Tymczyszyn
Reviewer #1:
Specific comments:
Title:
To be more precise, “the malolactic fermentation” must be added to the title:
“Analysis of the genome architecture of Lacticaseibacillus paracasei UNQLpc10, a strain with potential for malolactic fermentation during winemaking” or
“Analysis of the genome architecture of Lacticaseibacillus paracasei UNQLpc10 as potential malolactic starter strain for winemaking”.
-ANSWER: The title was changed to “Analysis of the genome architecture of Lacticaseibacillus paracasei UNQLpc10, a strain with oenological potential as a malolactic starter” (Line 2).
Abstract:
Line 13- “When incubated in a wine-like medium, it showed good survival capacity and ability to consume L-malic acid.” It is not clear is it the comment of the results obtained in this, or in previous manuscript, because in this manuscript are presented results regarding survival of the Lcb. paracasei UNQLpc 10 strain and malic acid consumption in synthetic wine. These results must be mentioned in the abstract after the sentence “The aim of this work was…” (line 15)
-ANSWER: Those results correspond to the present study and are mentioned at the final part of the summary (lines 22 – 23).
Results:
Table 1 - Instead of “Lpc.” must be “Lcb. paracasei”
- ANSWER: DONE
Table 2 – the title of this table must be introduced
- ANSWER: A title was introduced: Table 1: In silico analysis of the UNQLpc10 genome looking for genes encoding enzymes of oenological interest
Figure 4 – introduce word “Lcb. paracasei” before label “UNQLpc 10”
- ANSWER: DONE
Line 253 – at the end of sentence “Regarding genes with probiotic functionality….” reference regarding data from the literature about exopolysaccharides “extracellularly secreted by many microorganisms” is missing and must be added.
ANSWER: According to suggestions of reviewer 2, this topic was removed from the manuscript. The probiotic potential of UNQLpc10 will be analyzed in future studies.
Figure 6 – The title is not correct. Instead of “Evolution of the bacterial population...”, it was shown “Survival of the Lcb. paracasei UNQLpc 10 strain (A) and L-malic acid consumption (MAC) (B) during malolactic fermentation (MLF) in synthetic wine.” Additionally, when you correct the title, the abbreviation on the “y “axis MAC will be explained.
- ANSWER: The title of Figure 6 was modified.
The chapter Discussion starts with the comments about results shown on Figure 6, and experimental results of the manuscript shown before this figure (Tables 1 and 2, and Figures 1-5) are mentioned after, so, rearrangement of the text is necessary according to the order of appearance of the results. Additionally, it would be worth mentioning that the Lacticaseibacillus strains were isolated from a broad range of habitats including not only food (raw or fermented), but also sewage, invertebrates, birds and mammals including human gut of healthy adults as well as breast-fed infants with the probiotic coding potential components of the genome of isolated L. paracasei strains (Florova et al. 2021; Rodrigo-Torres et al. 2022)
Frolova, M., Yudin, S., Makarov, V., Glazunova, O., Alikina, O., Markelova, N., ... & Ozoline, O. Lacticaseibacillus paracasei: Occurrence in the Human Gut Microbiota and K-Mer-Based Assessment of Intraspecies Diversity. Life 11 (2021): 1246. Rodrigo-Torres, L., Landete, J. M., Huedo, P., Peirotén, Á., Langa, S., Rodríguez-Minguez, E., ... & Arqués, J. L. (2022). Complete genome sequences of Lacticaseibacillus paracasei INIA P272 (CECT 8315) and Lacticaseibacillus rhamnosus INIA P344 (CECT 8316) isolated from breast-fed infants reveals probiotic determinants. Gene, 840, 146743.
- ANSWER: A rearrangement of the Discussion was done, according to the reviewer suggestions. References were added.
Conclusion
Line 412 – Technological properties are wrongly mentioned in the context of potential probiotic properties. Therefore, corrections must be made as follows: “This strain also presents natural tolerance towards a hostile environment as wine (acid, ethanol, and phenolic compounds), a fact that also increases its potential to be used as a live probiotic malolactic starter culture. Finally, UNQLpc 10 has genes that are of technological interest for probiotic use, and more investigation must be carried out to evaluate the potential of the strain in the food industry.”
- ANSWER: The text of Conclusion was modified according to the reviewer suggestion.
Author Response File: 
Author Response.doc
Reviewer 2 Report
MAIN COMMENTS
This manuscript deals with the study of the potential use of a Lacticaseibacillus paracasei strain for malolactic fermentation (MLF) in winemaking. It seems interesting because this species has never been described in relation with MLF.
Authors' main aim of the work was to analyse the genome structure of this strain and to look for its oenological properties. In addition, cell survival and L-malic acid consumption of this strain were evaluated in wine-like medium at laboratory scale.
Nevertheless, this work has several shortcomings. I summarize below the most concerning aspects.
First, it is not clear why this Lcb. paracasei strain was selected (L63-66 and L87-90). This strain UNQLpc 10 of Lcb. paracasei was isolated in a wine where L. plantarum and O. oeni were dominants. What does "recovered from implantation control assays" mean ? Is there any data or reference of the implantation of this Lcb. paracasei in this wine in comparison with L. plantarum and O. oeni ? Had really this strain a significant role in this MLF ?
If the final goal of this work was to obtain a strain interesting for winemaking, all this analysis of the genome architecture is superfluous. It would be enough analysing the predicted genes with regard the oenological properties. What is the purpose of this detailed description of genome characteristics, relative abundance of clusters and categories and comparison with other Lcb. paracasei strains ? All the information explained in section 3.1 and shown in Figures 1, 2, 3 and 4 has no relevance, and moreover in a strain that is not still used in winemaking or elsewhere.
All the potential probiotic properties (L80, L252-278, L382-402) of this strain have been supposed from its genome characteristics. All this is very speculative and has no sense relating it with the potential use in MLF and winemaking.
In this study the potential use of this strain in MLF has been assayed just in a few assays in wine-like medium, and only bacterial population and L-malic acid consumption have been followed. No other parameters have been studied. It is a poor study of its potentiality, and no comparison of its performing has been made with any other known strain, for instance of O. oeni.
OTHER COMMENTS
In introduction (L27-319) the references on MLF and LAB of grapes and cellars related with it are classical but a bit old. Besides those, I would add some newer ones, such as Sumby et al 2019 App Micro Biot 103:2033; Bartowsky et al 2015 Aust J Grape Wine Res 21:663; Lerm et al 2010 SAfr J Enol Viti 31:186; Franquès et al 2017 LWT Food Sci Tech 81:326; Lorentzen & Lucas 2019 App Micro Biot 103:2937; Cappello et al 2016 Int J Food Micro 243:16.
It would also be interesting mention some other reference related with genomics of LAB and MLF such as Mendoza et al 2017 Front Micro 8:534
L41-47 Lcb. paracasei was isolated or identified in wines in these references, but is there any case where Lcb. paracasei was the dominant in MLF ?
Fig. 5 A comparison of mle gene clusters of this strain of L. paracasei, and other genes, with those of O. oeni would be useful to know the possibilities of UNQLpc 10 in winemaking.
L286 and Fig. 6 A: more than 10^10 CFU/mL reaching 10^12 ? It is impossible, calculations of CFU must be wrong.
L123 and Fig. 6: According Materials & Methods initial inoculated population was 10^7 CFU/mL but in the Figure it is 10^8. As previous note, it must be wrong.
Fig. 6: It seems that is no significant difference between acclimated and non-acclimated cultures. The only apparently significant point is the malic acid consumption at 15 days, but this lower consumption by the non-acclimated culture, due to the decreasing consumption from day 4 is impossible: if the consumption has been more than 80% L-malic acid at day 4, how can it be lower some days after ?
L335-337 This sentence can be removed, it is obvious that you cannot find here eukaryotic genes.
OTHER CORRECTIONS
L117 RPM is not a valid unit of centrifugation because it depends on the radius. Relative centrifugal force (x g) must be used instead.
L277 "bactericins" or bacteriocins ?
L405 paracasei should be in lower case
Fig. 3 "kinchi" should be kimchi
Fig. 6 "Whithout" ?
Author Response
Dear Editor,
We are sending you the revised version of the manuscript “Analysis of the genome architecture of Lacticaseibacillus paracasei UNQLpc10, a strain with oenological potential as malolactic starter"; Manuscript ID: fermentation- 2022575, in consonance with reviewers and editor suggestions. Changes made in 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,
Dr. E. Elizabeth Tymczyszyn
Reviewer #2:
MAIN COMMENTS
This manuscript deals with the study of the potential use of a Lacticaseibacillus paracasei strain for malolactic fermentation (MLF) in winemaking. It seems interesting because this species has never been described in relation with MLF.
Authors' main aim of the work was to analyse the genome structure of this strain and to look for its oenological properties. In addition, cell survival and L-malic acid consumption of this strain were evaluated in wine-like medium at laboratory scale.
Nevertheless, this work has several shortcomings. I summarize below the most concerning aspects.
First, it is not clear why this Lcb. paracasei strain was selected (L63-66 and L87-90). This strain UNQLpc10 of Lcb. paracasei was isolated in a wine where L. plantarum and O. oeni were dominants. What does "recovered from implantation control assays" mean? Is there any data or reference of the implantation of this Lcb.paracasei in this wine in comparison with L. plantarum and O. oeni? Had really this strain a significant role in this MLF?
ANSWER:
In a previous work (Brizuela et al 2018), it was studied the implantation ability, in a Patagonian Malbec wine, of some L. plantarum and O. oeni native strains. The evaluation of the strain cultures implantation was controlled by RAPD PCR with M13 primer, and showed the presence of the inoculated strains after 14 days of incubation. But the percentages of implantation were lower than 100%, suggesting that inoculated strains did not have inhibitory effects on the wine natural microbiota, allowing other LAB species/strains to be involved in fermentation, being the profile corresponding to UNQLp10 the most common of the profiles found. Subsequent sequencing of the 16S rRNA gene confirmed that it belonged to the species Lcb. paracasei.
For this reason, we considered it was important to advance in the knowledge of this strain, expanding the knowledge of the LAB´s oenological diversity and searching new species/strain candidates to design starter cultures.
According to suggestions of reviewer, the text (lines 67-73) was rewritten.
If the final goal of this work was to obtain a strain interesting for winemaking, all this analysis of the genome architecture is superfluous. It would be enough analysing the predicted genes with regard the oenological properties. What is the purpose of this detailed description of genome characteristics, relative abundance of clusters and categories and comparison with other Lcb.paracasei strains? All the information explained in section 3.1 and shown in Figures 1, 2, 3 and 4 has no relevance, and moreover in a strain that is not still used in winemaking or elsewhere.
ANSWER: In accordance with the reviewer's suggestions, Table 1 (characteristics of the Lpc. UNQLpc10 genome) and Figure 4 (Analysis of the COG on the unique genes) were moved to supplementary material, and the Results section was shortened. In addition, in the Discussion section, the text on genomic structure data was reduced and the analysis of genes with oenological potential was emphasized.
All the potential probiotic properties (L80, L252-278, L382-402) of this strain have been supposed from its genome characteristics. All this is very speculative and has no sense relating it with the potential use in MLF and winemaking.
ANSWER: The reviewer is right. Considerations about the probiotic potential of UNQLcp10 were deleted from the manuscript. Experimental analysis of the probiotic potential of this strain will be carried out in the future.
In this study the potential use of this strain in MLF has been assayed just in a few assays in wine-like medium, and only bacterial population and L-malic acid consumption have been followed. No other parameters have been studied. It is a poor study of its potentiality, and no comparison of its performing has been made with any other known strain, for instance of O. oeni.
ANSWER:
The reviewer's comments are correct. But, it is necessary taking into account that for Covid 19 pandemic restriction we did not have access to vintage 2020 and 2021 and also we were restricted to the work on lab. For these reasons, only results in synthetic wine were performed. The aim of this manuscript was to evaluate the possible functions of the predicted genes with regards to their oenological potential as a malolactic starter. The evaluation of implantation in wine and sensory perception of UNQLpc10 should be performed in the future, and the need for more studies was remarked in the discussion and conclusion section of the revised manuscript.
OTHER COMMENTS
In introduction (L27-319) the references on MLF and LAB of grapes and cellars related with it are classical but a bit old. Besides those, I would add some newer ones, such as Sumby et al 2019 App Micro Biot 103:2033; Bartowsky et al 2015 Aust J Grape Wine Res 21:663; Lerm et al 2010 SAfr J Enol Viti 31:186; Franquès et al 2017 LWT Food Sci Tech 81:326; Lorentzen& Lucas 2019 App Micro Biot 103:2937; Cappello et al 2016 Int J Food Micro 243:16.
It would also be interesting mention some other reference related with genomics of LAB and MLF such as Mendoza et al 2017 Front Micro 8:534
ANSWER: the references were added, according the reviewer suggestion
L41-47 Lcb. paracasei was isolated or identified in wines in these references, but is there any case where Lcb. paracasei was the dominant in MLF?
ANSWER: the sentences were re-written for more clarity (Line 42-51 of revised manuscript). In addition, in the paper of López-Sijas et al. 2020, Lcb. paracasei was found as a predominant strain in MLF. This reference was added.
Fig. 5 A comparison of mle gene clusters of this strain of L. paracasei, and other genes, with those of O. oeni would be useful to know the possibilities of UNQLpc 10 in winemaking.
ANSWER: A comparison with O oeni genes was added in the text (Line 218-219) and Figure 2S.
L123 and Fig. 6: According Materials & Methods initial inoculated population was 10^7 CFU/mL but in the Figure it is 10^8. As previous note, it must be wrong.
ANSWER: The reviewer is right. The initial inoculated population was corrected in the section Material and Methods
L286 and Fig. 6 A: more than 10^10 CFU/mL reaching 10^12 ? It is impossible, calculations of CFU must be wrong.
ANSWER: UNQLpc10 was able to reach 1012 CFU/mL in MRS broth, but this value obtained in wine-like medium is curiously elevated. Considering that the condition without ethanol is not relevant for the purpose of the present work, these data were deleted until the impossibility to repeat the experiment before revised submission.
Fig. 6: It seems that is no significant difference between acclimated and non-acclimated cultures. The only apparently significant point is the malic acid consumption at 15 days, but this lower consumption by the non-acclimated culture, due to the decreasing consumption from day 4 is impossible: if the consumption has been more than 80% L-malic acid at day 4, how can it be lower some days after?
ANSWER: In order to avoid misinterpretation, a non-linear regression (one phase-decay) was applied to %MAC in Figure 5 (revised version). The equation was added in the section Material and Methods. Table 2 was added to show the adjusted kinetic parameters, and the new analysis was added in Results.
L335-337 This sentence can be removed, it is obvious that you cannot find here eukaryotic genes.
ANSWER: The sentence was removed
OTHER CORRECTIONS
L117 RPM is not a valid unit of centrifugation because it depends on the radius. Relative centrifugal force (x g) must be used instead. DONE
L277 "bactericins" or bacteriocins? DONE
L405 paracasei should be in lower case DONE
Fig. 3 "kinchi" should be kimchi DONE
Fig. 6 "Whithout" DONE.
Round 2
Reviewer 2 Report
Authors have followed all the suggestions of reviewers and modified the manuscript according them, resulting in a real improvement of quality and significance of this work.
