Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The MS by Quach et al. Presented functional genomic and Phenotypic Analysis of Lactiplantibacillus pentosus strain P7 which was Isolated from Pickled Mustard Greens and Revealed its Capacity for EPS, B-Vitamin, and Lactic Acid Production. Generally, the MS is well-prepared. But there are still some minor issues need to be corrected.

1. For the antioxidant potential assay, it suggest to add a positive control when the measurement was performed.
2. Fig. 1, C, for L. planetarium ATCC 14917T and DSM 20174T, these two type strains are the same strain which are deposited in two different culture collection. please recheck and correct it.
3. References section, please recheck the formats of some references, for consistent format. Especially, some names of the journals. 

Author Response

Responses (#) to the Reviewers' Comments are given in red:

The MS by Quach et al. presented functional genomic and phenotypic analysis of Lactiplantibacillus pentosus strain P7 which was isolated from pickled mustard greens and revealed its capacity for EPS, B-vitamin, and lactic acid production. Generally, the MS is well-prepared. But there are still some minor issues need to be corrected.

1. For the antioxidant potential assay, it suggests to add a positive control when the measurement was performed.

# We thank the reviewer for the suggestion. The growth of S. cerevisiae is not affected negatively or positively by ascorbic acid under normal condition as our tests in this manuscript and other related papers (Quach et al. 2024; Vu et al. 2024). Therefore, in the spot, 0.2 mg ascorbic acid mixed to 2 mM H₂O₂ was used as the positive control and single ascorbic acid was not performed.

References

Quach NT, Nguyen TTA, Vu THN, Nguyen TTN, Tran XK, Chu NH, Ta TTT, Chu HH, Phi QT. New insight into protective effect against oxidative stress and biosynthesis of exopolysaccharides produced by Lacticaseibacillus paracasei NC4 from fermented eggplant. Curr Genet. 2024 14;70(1):7. doi: 10.1007/s00294-024-01292-8.

Vu THN, Pham NS, Quach NT, Le PC, Pham QA, Ngo CC, Nguyen VT, Anh DH, Quang TH, Chu HH, et al. Fusarium foetens AQF6 Isolated from Amentotaxus ynnanensis H.L.Li as a Prolific Source of Antioxidant Compounds. Applied Sciences. 2024; 14(5):2048. doi.org/10.3390/app14052048

2. 1C, for L. plantarum ATCC 14917^T and DSM 20174^T, these two type strains are the same strain which are deposited in two different culture collection. Please recheck and correct it.

# We appreciate the comment made by the reviewer and agree that two type strains are the same strain. L. plantarum ATCC 14917^T was sequenced by the 454 platform under accession number ACGZ00000000, while the genome L. plantarum DSM 20174^T sequenced by both PacBio and Illumina MiSeq was deposited in GenBank database under accession number CP039121. As a result, 2 type strain genomes are automatically available in the The Type (Strain) Genome Server (TYGS) database. Since the query genome is specified by default in the TYGS, we could not exclude neither ATCC 14917^T or DSM 20174^T. Our result in Fig. 1C is similar to Huidrom et al. 2024 and Kingkaew et al. 2024.

References

Huidrom S, Ngashangva N, Khumlianlal J, Sharma KC, Mukherjee PK, Devi SI. Genomic insights from Lactiplantibacillus plantarum BRD3A isolated from Atingba, a traditional fermented rice-based beverage and analysis of its potential for probiotic and antimicrobial activity against Methicillin-resistant Staphylococcus aureus. Front Microbiol. 2024 Mar 27;15:1357818. doi: 10.3389/fmicb.2024.1357818. 

Kingkaew E, Tanaka N, Shiwa Y, Sitdhipol J, Nuhwa R, Tanasupawat S. Genomic Assessment of Potential Probiotic Lactiplantibacillus plantarumCRM56-2 Isolated from Fermented Tea Leaves. Trop Life Sci Res. 2024 Jul;35(2):249-269. doi: 10.21315/tlsr2024.35.2.12.

3. References section, please recheck the formats of some references, for consistent format. Especially, some names of the journals.

# We would like to thank the reviewer for the valuable comments and have taken it on board.

Reviewer 2 Report

Comments and Suggestions for Authors

1- While the authors state novelty in B-vitamin production by L. pentosus, the discussion should better position P7’s yields relative to other Lactobacillus species and to industrial benchmarks. For example, riboflavin and pyridoxine levels are low compared to some L. plantarum and L. paracasei strains this needs a deeper comparative evaluation. Expand the discussion to include a quantitative comparison table of vitamin production across related species/strains. Clearly indicate whether the yields are industrially significant or whether strain improvement would be essential.

2- While EPS yield and antioxidant capacity are reported, there is no structural characterization (monosaccharide composition, molecular weight, rheological properties) which is crucial for industrial applicability. If available, provide preliminary EPS composition data, or at minimum, discuss the expected composition based on genetic and literature evidence.

3- Some methods lack critical detail (e.g., number of biological replicates for vitamin assays, statistical tests used for comparing yields, criteria for “significant difference” in antioxidant assays). The number of independent experiments and technical replicates. Statistical tests applied, p-value thresholds, and software used. Error bars’ meaning in figures (SD vs SEM).

4- The identification of biosynthetic genes is based on in silico annotation without transcriptomic or proteomic validation. While full omics validation may not be feasible for this submission, acknowledge this limitation and suggest RNA-seq or qPCR confirmation in future work to strengthen genotype–phenotype links.

5- The lactic acid production rate (91.2 ± 12.3 g/L in 24 h) is high, but no discussion is given on scalability, tolerance to high substrate concentration, or downstream processing challenges. Add a short industrial applicability section discussing: How P7’s yields compare with commercial lactic acid producers. Whether it can ferment inexpensive substrates (e.g., agro-waste). Potential for co-production strategies.

6- Some figures (e.g., phylogenomic tree in Fig. 1C, EPS activity in Fig. 3B) are small or lack scale bars and detailed legends, reducing their interpretability.

7- The phrase “remains underexplored” could be replaced with “has been underexplored” for grammatical accuracy. Include quantitative highlights (EPS, lactic acid, vitamin yields) directly in the abstract for stronger impact.

8- The rationale for selecting pickled mustard greens as the source should be better justified mention microbial diversity or prior reports of high-EPS-producing strains from this niche.

9- Clarify the reason for selecting sucrose concentration (60 g/L) in EPS production. Specify brand/model for spectrophotometer and centrifuge when relevant to ensure reproducibility.

10- Some sentences are overly descriptive of results; more critical analysis is needed on why P7 shows these traits and how genetic elements may confer advantages.

Comments on the Quality of English Language

Seek help from English language professional 

Author Response

1- While the authors state novelty in B-vitamin production by L. pentosus, the discussion should better position P7’s yields relative to other Lactobacillus species and to industrial benchmarks. For example, riboflavin and pyridoxine levels are low compared to some L. plantarum and L. paracasei strains this needs a deeper comparative evaluation. Expand the discussion to include a quantitative comparison table of vitamin production across related species/strains. Clearly indicate whether the yields are industrially significant or whether strain improvement would be essential.

# We would like to thank the valuable comments made by the reviewer. We seriously improved the discussion and added new Table 2 for comparison of riboflavin, pyridoxine, and pyridoxine production by lactobacilli.

2- While EPS yield and antioxidant capacity are reported, there is no structural characterization (monosaccharide composition, molecular weight, rheological properties) which is crucial for industrial applicability. If available, provide preliminary EPS composition data, or at minimum, discuss the expected composition based on genetic and literature evidence.

# We agreed to the reviewer that structural characteristics are needed to highlight the potential of EPS for industrial applicability. Due to the labor-intensive and time-consuming experiments, we intend to investigate the structure of the EPS, along with its potential bioactivities, in the future study. In the revised manuscript, the discussion related the EPS’s composition is improved. We kindly hope the reviewer will consider our situation and allow us to omit the additional characterization of the EPS in this manuscript.

3- Some methods lack critical detail (e.g., number of biological replicates for vitamin assays, statistical tests used for comparing yields, criteria for “significant difference” in antioxidant assays). The number of independent experiments and technical replicates. Statistical tests applied, p-value thresholds, and software used. Error bars’ meaning in figures (SD vs SEM).

# We appreciate the thoughtful comments made by the reviewer and have taken them on board. For each representative result, the experiment was performed in triplicate. The missing information was added in the current version.

In addition, the use of “significant difference” was wrongly mentioned in the antioxidant assays. We would like to correct them throughout the manuscript.

4- The identification of biosynthetic genes is based on in silico annotation without transcriptomic or proteomic validation. While full omics validation may not be feasible for this submission, acknowledge this limitation and suggest RNA-seq or qPCR confirmation in future work to strengthen genotype–phenotype links.

# Many thanks! We have improved the discussion and conclusions following the reviewer’s insightful comments.

5- The lactic acid production rate (91.2 ± 12.3 g/L in 24 h) is high, but no discussion is given on scalability, tolerance to high substrate concentration, or downstream processing challenges. Add a short industrial applicability section discussing: How P7’s yields compare with commercial lactic acid producers. Whether it can ferment inexpensive substrates (e.g., agro-waste). Potential for co-production strategies.

# The authors fully agreed with the reviewer’s valuable suggestions and have addressed missing information related to lactic acid production in the final paragraph of the Discussion section.

6- Some figures (e.g., phylogenomic tree in Fig. 1C, EPS activity in Fig. 3B) are small or lack scale bars and detailed legends, reducing their interpretability.

# Following the reviewer’s comments, Fig. 1, Fig. 3 and their captions were improved.

7- The phrase “remains underexplored” could be replaced with “has been underexplored” for grammatical accuracy. Include quantitative highlights (EPS, lactic acid, vitamin yields) directly in the abstract for stronger impact.

# We would like to thank the reviewer for the valuable correction and have taken it on board. As the result, the Abstract was revised.

8- The rationale for selecting pickled mustard greens as the source should be better justified mention microbial diversity or prior reports of high-EPS-producing strains from this niche.

# We appreciate the helpful suggestion made by the reviewer and added it in the last paragraph of Introduction section.

9- Clarify the reason for selecting sucrose concentration (60 g/L) in EPS production. Specify brand/model for spectrophotometer and centrifuge when relevant to ensure reproducibility.

# We thank the reviewer for the suggestion. As shown in our previous study (Quach et al., 2024), the maximum EPS production by Lacticaseibacillus paracasei NC4 was achieved by culturing in MRS medium containing 60 g/L sucrose at 37°C for 48 h. Therefore, 60 g/L sucrose was used for the EPS experiments. Also, we realized our mistakes and corrected the sub-sections 2.1 and 2.5.

References

Quach NT, Nguyen TTA, Vu THN, Nguyen TTN, Tran XK, Chu NH, Ta TTT, Chu HH, Phi QT. New insight into protective effect against oxidative stress and biosynthesis of exopolysaccharides produced by Lacticaseibacillus paracasei NC4 from fermented eggplant. Curr Genet. 2024 May 14;70(1):7. doi: 10.1007/s00294-024-01292-8

10- Some sentences are overly descriptive of results; more critical analysis is needed on why P7 shows these traits and how genetic elements may confer advantages.

# We would like to thank the reviewer for the comments. Indeed, to know better genetic elements underlying biotechnological traits, the genomic diversity, evolutionary history, and the potential ecological differentiation of the species might be performed by extensive comparative genomic analysis of at least 30 L. pentosus strains. It is very challenging for us to perform this analysis and integrate it with the current results. We sincerely hope the reviewer understands our current limitations and see our efforts undertaken in this study."

Reviewer 3 Report

Comments and Suggestions for Authors

In the manuscript named “Functional Genomic and Phenotypic Analysis of Lactiplantibacillus pentosus P7 Isolated from Pickled Mustard Greens Reveals Capacity for Exopolysaccharide, B-Vitamin, and Lactic Acid Production”, authors have performed genome sequencing, metabolic analysis of Lactiplantibacillus pentosus P7 Isolated, they have characterized EPS production from L. pentosus. These findings would be helpful for industrial applications in the production of EPS, and similar compounds, in future, the manuscript was well organized and written. However, there were some comments about it.

(1) Authors have performed genome sequencing using Illumina NovaSeq platform PE150 model, how about did the results? Authors didn’t provide detail information about it, for example, did it produce single sequence? Or many contigs or scaffold sequences. In addition, in many microorganism genomes sequencing, single molecule sequencing, such as PacBio or Nanopore sequencing were well used for producing high quality genome sequences.

(2) 16S rRNA gene sequence of P7 was retrieved from its genome, did authors perform PCR sequence to validate this sequence?

(3) In EPS gene identification process, authors have adopted “An e-value <10-5, identity >30% and coverage >50%” (line 137) in annotation pipeline, the identify was so low, which would be loose for blast search process.

(4) The raw data of short reads would be also released by public database.

(5) In figure 1C, the figure captions were also needed to clearly explain how different colors were calculated and displayed, with detailed descriptions required.

(6) In Figure 1A, the authors had assembled all contigs in whole genome, however, they did not specify the number of contigs nor describe how these contigs were assembled. If other genomes were used as references to assemble the P7 genome, it was recommended that this process be elaborated on in both the Methods section, and the Results section.

(7) Figure 2A and 2B had some overlapping content. It was suggested to adjust them or remove one of the figures.

Author Response

In the manuscript named “Functional Genomic and Phenotypic Analysis of Lactiplantibacillus pentosus P7 Isolated from Pickled Mustard Greens Reveals Capacity for Exopolysaccharide, B-Vitamin, and Lactic Acid Production”, authors have performed genome sequencing, metabolic analysis of Lactiplantibacillus pentosus P7 Isolated, they have characterized EPS production from L. pentosus. These findings would be helpful for industrial applications in the production of EPS, and similar compounds, in future, the manuscript was well organized and written. However, there were some comments about it.

(1) Authors have performed genome sequencing using Illumina NovaSeq platform PE150 model, how about did the results? Authors didn’t provide detail information about it, for example, did it produce single sequence? Or many contigs or scaffold sequences. In addition, in many microorganism genomes sequencing, single molecule sequencing, such as PacBio or Nanopore sequencing were well used for producing high quality genome sequences.

# Thank you for your suggestions. We have added information on the number of raw paired-end reads, the number of contigs in the assembled genome, and the completeness of the assembled genome of strain P7 in the Results section. We know that long read sequencing led by PacBio and ONT offers several advantages over short read sequencing. However, their high cost and limited accessibility currently make them less practical for this study. Instead, our group has gained extensive experience with the Illumina platform, which provides high accuracy and reliability for genome sequencing and annotation, and has been successfully applied in our previous studies. Nevertheless, we recognize the added value of long-read sequencing, and plan to incorporate PacBio or Nanopore technologies in future studies to achieve more comprehensive genome assemblies.

References

Quach NT, Nguyen TTA, Vu THN, Ta TTT, Phi QT, Trieu TA, Van Thuoc D. Genome mining and physiological analyses uncover adaptation strategies and biotechnological potential of Virgibacillus dokdonensis T4.6 isolated from high-salt shrimp paste. Arch Microbiol. 2024 Jun 19;206(7):309. doi: 10.1007/s00203-024-04049-6.

Quach NT, Vu THN, Nguyen TTA, Le PC, Do HG, Nguyen TD, Thao PTH, Nguyen TTL, Chu HH, Phi QT. Metabolic and genomic analysis deciphering biocontrol potential of endophytic Streptomyces albus RC2 against crop pathogenic fungi. Braz J Microbiol. 2023 Dec;54(4):2617-2626. doi: 10.1007/s42770-023-01134-8.

(2) 16S rRNA gene sequence of P7 was retrieved from its genome, did authors perform PCR sequence to validate this sequence?

# Thank you for your question. To validate the 16S rRNA gene sequence retrieved from the genome assembly, the 16S rRNA gene sequence was already amplified by PCR using the primr pair 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′), followed by Sanger sequencing. The validated sequence was found to be 100% identical to the sequence obtained from the whole-genome shotgun (WGS) NGS data.

(3) In EPS gene identification process, authors have adopted “An e-value <10-5, identity >30% and coverage >50%” (line 137) in annotation pipeline, the identify was so low, which would be loose for blast search process.

# We thank the reviewer for the suggestion. For genome mining of genetic determinants related to EPS, folic acid, riboflavin, and pyridoxine production, we have to use low similarity to obtain as much as annotated and putative genes. After that, these genes were extensively analyzed at the different databases to confirm their function. Regarding the genes associated with EPS, all exhibited high sequence similarity to reference genes, with identities of at least 65%.

(4) The raw data of short reads would be also released by public database.

# We would like to thank the reviewer for the valuable comment and have taken it on board. The raw short-read sequencing data was already deposited in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1304875 and BioSample SAMN50574712. We have added this information to the revised manuscript.

(5) In figure 1C, the figure captions were also needed to clearly explain how different colors were calculated and displayed, with detailed descriptions required

# We agreed to the reviewer’s valuable comment and corrected Fig. 1 caption.

(6) In Figure 1A, the authors had assembled all contigs in whole genome, however, they did not specify the number of contigs nor describe how these contigs were assembled. If other genomes were used as references to assemble the P7 genome, it was recommended that this process be elaborated on in both the Methods section, and the Results section.

# We appreciate the comment made by the reviewer. The missing information related to sequencing and assembly data was added in the sub-section 3.2 as “The whole-genome sequence of strain P7 was obtained using the Illumina NovaSeq sequencing 150PE platform, generating 5,546,849 paired-end reads with an average length of 150 bp. After quality trimming with Trimmomatic, 4,313,170 reads remained and were used for genome assembly with SPAdes software. The resulting draft genome of strain P7 comprised 123 contigs, with a total length of 3,749,478 bp, a GC content of 46.5%, and no plasmids detected (Fig 1A)”

In addition, we performed whole-genome de novo assembly of the P7 genome without using any reference genomes.

(7) Figure 2A and 2B had some overlapping content. It was suggested to adjust them or remove one of the figures.

# Many thanks. We eliminated Figure 2A

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Thanks for authors’ works, the manuscript had been well revised, most of my comments were well addressed in revision. There is no new comment about it, it was recommended to be accepted, good luck.