Novel Lactic Acid Bacteria Strains from Regional Peppers with Health-Promoting Potential

Round 1

Reviewer 1 Report

In this work entitled “Novel Probiotic for Peppers Fermentation with Safe and Health-Promoting Potential”, authors performed many experiments to test the strains isolated from peppers. 

Although authors obtained some valuable data. However, there are still many problems.

1. The title for the manuscript should be revised. For pepper fermentation, the safety tests like coagulase, amino acid decarboxylase, and DNA enzyme activities should be tested. On the other hand, authors highlighted the word “Novel”, however, according to Table 1, the homology were all greater than 97%, suggesting that the isolates are not “novel” strains.

2. A positive control LAB strain should be used to compare with the isolates, to better demonstrate the activities of isolates.

Author Response

Reviewer 1

Thank you for your feedback on our manuscript ID fermentation-2920664. We greatly appreciate the constructive comments provided. Additionally, we would like to express our gratitude for the time and effort invested in reviewing our manuscript. The suggested corrections have been duly noted and implemented in the revised version of the manuscript (highlighted in yellow).

In this work entitled “Novel Probiotic for Peppers Fermentation with Safe and Health-Promoting Potential”, authors performed many experiments to test the strains isolated from peppers.

Although authors obtained some valuable data. However, there are still many problems.

1. The title for the manuscript should be revised. For pepper fermentation, the safety tests like coagulase, amino acid decarboxylase, and DNA enzyme activities should be tested. On the other hand, authors highlighted the word “Novel”, however, according to Table 1, the homology were all greater than 97%, suggesting that the isolates are not “novel” strains.

Answer: We acknowledge this observation; however, due to time constraints, we revised the title and redefined the focus of this study. We now emphasize the beneficial properties of lactic acid bacteria, particularly the functional and technological capabilities of bacterial strains isolated from regional peppers. The revised title reads: "Novel Lactic Acid Bacteria Strains from Regional Peppers with Health-Promoting Potential". Furthermore, we have appended the following paragraph to the end of this article: "Overall, these characteristics suggest that the five selected strains hold promise as probiotics, with functional merits suitable for application in the fermentation of bell peppers. However, further studies are necessary for these strains to qualify definitively as probiotics." Regarding the term "Novel," our study identified nine known species with high identity scores ranging from 97.38% to 100%. Nonetheless, the isolates from regional peppers represent new bacterial strains that have not been previously explored concerning the functional and technological properties under investigation.

2. A positive control LAB strain should be used to compare with the isolates, to better demonstrate the activities of isolates.

Answer: Lactiplantibacillus plantarum ATCC 10241 isolated from sauerkraut was used as a reference strain.

Reviewer 2 Report

In this study, lactic acid bacteria (LAB) were isolated from peppers. The stress tolerance, digestive properties, adhesion capacity and other functional properties of LAB were systemically investigated. The topic is interesting and may provide beneficial for nutritious and healthy probiotic fermentation products. But the manuscript still needs to be improved, and the specific comments are as follows:

Lines 194-198. Please add the digestive enzyme activity (pepsin and pancreactin) in the experimental method.

Lines 529-532. Data from the compatibility test may be submitted in the form of supplementary data.

Please note the significant difference between the data in figure 2, 3, 4, 5 and table 6, 8.

Author Response

Reviewer 2

Thank you for your feedback on our manuscript ID fermentation-2920664. We greatly appreciate the constructive comments provided. Additionally, we would like to express our gratitude for the time and effort invested in reviewing our manuscript. The suggested corrections have been duly noted and implemented in the revised version of the manuscript (highlighted in yellow).

In this study, lactic acid bacteria (LAB) were isolated from peppers. The stress tolerance, digestive properties, adhesion capacity and other functional properties of LAB were systemically investigated. The topic is interesting and may provide beneficial for nutritious and healthy probiotic fermentation products. But the manuscript still needs to be improved, and the specific comments are as follows:

1-Lines 194-198. Please add the digestive enzyme activity (pepsin and pancreactin) in the experimental method.

Answer: To determine bacterial resistance to gastrointestinal tract conditions, the bacteria were subjected to sequential exposure to simulated gastric and intestinal juices (subsection 2.2.3) containing, among other things, pepsin and pancreatin, respectively.

2-Lines 529-532. Data from the compatibility test may be submitted in the form of supplementary data.

Answer: A figure S2 with the compatibility results was added.

In our study, no inhibition halos were observed in any case, indicating compatibility among the selected strains (Lp. plantarum LVP 40, P. pentosaceus LVP 43, Lt. curvatus LVP 44, Lp. plantarum LVP 46, and Lv. brevis LVP 41).

3-Please note the significant difference between the data in figure 2, 3, 4, 5 and table 6, 8.

Answer: In the revised version of our manuscript, we have highlighted the statistically significant differences observed in all experiments.

Reviewer 3 Report

The primary focus of this study is to examine the probiotic properties of lactic acid bacteria isolated from peppers. Therefore, it seems necessary to explain why pepper is important as the origin of lactic acid bacteria isolation in the Introduction.

 1.      The characteristics of microorganisms appear differently depending on the strains. It has strain specificity. The strain selected should be accurately stated. Please specify the five pepper bacterial strains in the Abstract.

2.      The picture in Figure 1 is unclear and does not match the strain name.

3.      The strains presented in Tables 4 and 5 are not identical.

4.      The results in Tables 2, 3, 4 and Figure 2 do not include a control strain.

5.      Line 649 : Brevis → brevis

Author Response

Reviewer 3

Thank you for your feedback on our manuscript ID fermentation-2920664. We greatly appreciate the constructive comments provided. Additionally, we would like to express our gratitude for the time and effort invested in reviewing our manuscript. The suggested corrections have been duly noted and implemented in the revised version of the manuscript (highlighted in yellow).

The primary focus of this study is to examine the probiotic properties of lactic acid bacteria isolated from peppers. Therefore, it seems necessary to explain why pepper is important as the origin of lactic acid bacteria isolation in the Introduction.

Answer: We think the next paragraph of the introduction explains the importance: “The search for new probiotic strains with unique properties continues to be an area of great interest despite the availability of numerous well-characterized probiotic strains worldwide. In this sense, there is a growing interest in exploring the probiotic and biotechnological potential of microorganisms already adapted to the food matrices in which they are found naturally. This is especially relevant in the case of bacteria isolated from red and green peppers (Capsicum annum L.), which have the advantage of being already adapted to this specific microbiome”.

1.     The characteristics of microorganisms appear differently depending on the strains. It has strain specificity. The strain selected should be accurately stated. Please specify the five pepper bacterial strains in the Abstract.

Answer: The five selected strains are specified in the abstract.

2. The picture in Figure 1 is unclear and does not match the strain name.

Answer: Figure 1 was corrected.

3. The strains presented in Tables 4 and 5 are not identical.

Answer: In the updated version of our manuscript, we included Lp. plantarum LVP 46 and Lp. plantarum ATCC 10241 in Table 4 to align both tables.

4. The results in Tables 2, 3, 4 and Figure 2 do not include a control strain.

Answer: Lactiplantibacillus plantarum ATCC 10241 as a reference strain was now included.

5. Line 649 : Brevis → brevis

Answer: ¨Brevis¨ was replaced by ¨brevis¨.

Reviewer 4 Report

In this work the authors have analyzed the characteristics of 15 strains of lactic acid bacteria. It is a very complete study, but they have to correct a couple of things for them to be accepted for publication:

-line 182, there is an error in the bile tolerance reference values

-page 12, the table 4 is over the text, and I couldn´t read it.

- Why is each treatment done at a different bacterial concentration (10⁷, 10⁸ or 10⁹ cfu/mL)?

Author Response

Reviewer 4

Thank you for your feedback on our manuscript ID fermentation-2920664. We greatly appreciate the constructive comments provided. Additionally, we would like to express our gratitude for the time and effort invested in reviewing our manuscript. The suggested corrections have been duly noted and implemented in the revised version of the manuscript (highlighted in yellow).

In this work the authors have analyzed the characteristics of 15 strains of lactic acid bacteria. It is a very complete study, but they have to correct a couple of things for them to be accepted for publication:

-line 182, there is an error in the bile tolerance reference values

Answer: Thank you, the mistake was corrected.

“The LAB strains were classified as resistant above 67%, tolerant between 34.0–66.9%, and sensitive below 33.9%, according to Vera-Mejía et al. (2018) [12].”

-page 12, the table 4 is over the text, and I couldn´t read it.

Answer: It was corrected

- Why is each treatment done at a different bacterial concentration (10⁷, 10⁸ or 10⁹ cfu/mL)?

Answer: The choice of different bacterial concentrations stems from the observation that the ability to adsorb mutagens may vary depending on the concentration of bacteria in the medium. For this fact, the bacterial concentration is a variable to investigate. However, the effect is not always dose dependent.

Round 2

Reviewer 1 Report

The authors carefully revised the manuscript.

Author Response

Thank you for your feedback on our manuscript ID fermentation-2920664.

Reviewer 2 Report

Subsection 2.2.3, please add this information about the source (e.g. from porcine gastric mucosa) and activity (U/mL or U/g) of pepsin and pancreatin. This is very important for the reproducibility of the experiment.

Author Response

This information was added in the new revised version of the manuscript (highlighted in color).

Reviewer 3 Report

1.     Generally, well-known strain with excellent probiotic properties, such as L. rhamnosus GG, is used as a reference strain. L. plantarum ATCC 10241 doesn't seem like that. An explanation is needed why the selection was made.

2.     In Figure 1, the band of Rep-PCR still doesn't look clear.

Author Response

Comments and Suggestions for Authors

1. Generally, well-known strain with excellent probiotic properties, such as L. rhamnosus GG, is used as a reference strain. L. plantarum ATCC 10241 doesn't seem like that. An explanation is needed why the selection was made.

Answer: Lactobacillus plantarum ATCC 10241 was isolated from sauerkraut (fermented cabbage) and is also known as LMG 9208, ATCC 10241, NCFB 343, NCIMB 7220, NCIMB 8184, NCTC 7220, NCDO 343, NCIB 7220, NCIB 8184, JCM 1551, CCRC 12251, CECT 749, CIP 71.39, KCTC 3104, BCRC 12251, NCIM 2593, DSM 2601 (https://bacdive.dsmz.de/strain/6493). This strain is widely available in different culture collections, making it easy for researchers and scientists to access.

- L. plantarum ATCC 10241 was used as reference in several studies for technological characterization of lactic acid bacteria isolated from fruits and vegetables (Fessard and Remize, 2019), and for isolation and identification of L. plantarum from vegetable samples (Khemariya et al., 2016). Furthermore, it has been specifically mentioned by Mis-Solval et al. (2019) as a microorganism frequently identified as a probiotic (¨L. plantarum NRRL B-1927 also known as ATCC 10241 is frequently identified as a probiotic¨).

Fessard A, Remize F. 2019. Genetic and technological characterization of lactic acid bacteria isolated from tropically grown fruits and vegetables. Int. J. Food Microbiol., 301, 61-72, ISSN 0168-1605, https://doi.org/10.1016/j.ijfoodmicro.2019.05.003

Khemariya P, Singh S, Jaiswal N, Chaurasia NS. 2016. Isolation and identification of Lactobacillus plantarum from vegetable samples, Food Biotechnol., 30(1), 49-62, DOI: 10.1080/08905436.2015.1132428

Mis-Solval KE, Jiang N, Yuan M, Joo KH, Cavender GA. 2019. The effect of the ultra-high-pressure homogenization of protein encapsulants on the survivability of probiotic cultures after spray drying. Foods, 8, 689. https://doi.org/10.3390/foods8120689

- L. plantarum DSM2601 used as a starter culture for sausage fermentation has the ability to enrich the conjugated linoleic acid (CLA) content of this food (Özera and Kılıçb, 2020).

Özer CO, Kılıç B. 2020. Utilization of optimized processing conditions for high yield synthesis of conjugated linoleic acid by L. plantarum AB20-961 and L. plantarum DSM2601 in semi-dry fermented sausage. Meat Sci., 169, 108218. doi: 10.1016/j.meatsci.2020.108218.

- This bacterial strain also has very good antimicrobial properties, and mainly anti-quorum sensing (QS) capacity against Pseudomonas aeruginosa (Valdez et al., 2005). In addition, the metabolites responsible of its anti-QS ability were previously reported by one of the authors of this article (Dr. Mario Arena) (Ramos et al., 2015). Even more recently we have published an article where L. plantarum ATCC 10241 is used as a reference probiotic strain (Verni et al., 2024).

Valdez JC, Peral MC, Rachid M, Santana M, Perdigón G. 2005. Interference of Lactobacillus plantarum with Pseudomonas aeruginosa in vitro and in infected burns: the potential use of probiotics in wound treatment. Clin. Microbiol. Infect., 11, 472-479. doi: 10.1111/j.1469-0691.2005.01142.x

Ramos AN, Sesto Cabral ME, Arena ME; Arrighi CF, Arroyo Aguilar AA, Valdez. 2015. Compounds from Lactobacillus plantarum culture supernatants with potential pro-healing and anti-pathogenic properties in skin chronic wounds. Pharm. Biol., 53 (3), 350-358. doi: 10.3109/13880209.2014.920037 

Verni MC, Matos TS, Alberto MR, Blázquez MA, Sussulini A, Arena ME, Cartagena E. 2024. UHPLC-MS/MS and GC-MS metabolic profiling of a medicinal Flourensia fiebrigii chemotype. Chem. Biodivers., e202301978. doi: 10.1002/cbdv.202301978

- Another reason why we also used Lactobacillus plantarum ATCC 10241 as a reference strain was because the majority of our isolates corresponded to this species.

2. In Figure 1, the band of Rep-PCR still doesn't look clear.

Answer: Figure 1 was corrected.