Fermented Cultured Wild Ginseng Roots (Panax ginseng C.A. Meyer) Using Limosilactobacillus fermentum HY7303 Enhances the Intestinal Barrier by Bioconversion of Ginsenosides and Extracellular Vesicle Production

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper is of good methodological quality. The obtained data have significant scientific significance. However, the choice of statistical methods raises questions. The authors used the parametric Student's t-test, while n=3. This is an extremely small sample size, for which it is difficult to ensure normal distribution, which is a necessary condition for the use of parametric statistical methods. The authors should use non-parametric analogues, such as the Mann-Whitney test. Alternatively, they should demonstrate the normality of the distribution.

Author Response

Full details are included in the attached file.

Comments 1

The paper is of good methodological quality. The obtained data have significant scientific significance. However, the choice of statistical methods raises questions. The authors used the parametric Student's t-test, while n=3. This is an extremely small sample size, for which it is difficult to ensure normal distribution, which is a necessary condition for the use of parametric statistical methods. The authors should use non-parametric analogues, such as the Mann-Whitney test. Alternatively, they should demonstrate the normality of the distribution.

 

Answer 1

Thank you for your insightful comments on statistical analysis methods. These are answer to questions about the statistical analysis method we chose. The reason we set n=3 to check the reproducibility of the experiment. Because the number of experiment repetitions was small, normality was tested using the Shapiro-Wilk normality test. As a result of the Shapiro-Wilk normality test, it was confirmed that the p values ​ were all greater than 0.05, and all appeared to satisfy normality. We presented statistical significance through Student’s t-test.

Shapiro-wilk normality test (p-value)
	normal	control	CWG	FCWG	CWG-EVs	FCWG-EVs
TEER	0.6955	0.1912	0.6421	0.7575	0.7854	0.5979
Papp	0.5455	0.6841	0.9715	0.4477	0.67	0.7019
ZO-1	0.2822	0.8098	0.5545	0.1288	0.7683	0.4614
ZO-2	0.3298	0.9244	0.2675	0.4307	0.1854	0.3804
OCLN	0.3004	0.4442	0.2014	0.6755	0.8891	0.7662

 

In addition, we will attach several manuscripts that show the statistical significance of experiments with a small number of replicates, such as ours, using Student’s t-tests.

1. Polo, A., Tlais, A. Z. A., Filannino, P., Da Ros, A., Arora, K., Cantatore, V., ... & Di Cagno, R. (2023). Novel Fermented Ice Cream Formulations with Improved Antiradical and Anti-Inflammatory Features. Fermentation, 9(2), 117.
2. Bae, S. E., Bae, S., Park, S. J., Lee, P., & Hyun, C. G. (2024). Microbial Consortium of Jeju Traditional Fermented Foods and Their Cosmetic Ingredient Potential.

(This is the latest manuscript of Fermentation, which will be published on 1 July 2024)

I hope my answer will help resolve the reviewer's questions.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled “Fermented Cultured Wild Ginseng Roots (Panax ginseng C.A. 2 Meyer) using Limosilactobacillus fermentum HY7303 Enhances the Intestinal Barrier by Bioconversion of Ginsenosides and Extracellular Vesicles Production” is an interesting paper with relevant information to the area of Molecular Microbiology. Nevertheless, I have questions on some of the methods used. I recommend that authors explained deeply the novelty of this paper. I also have written some comments to improve the manuscript.

 

General comments

 

Abstract and conclusion should be controlled.

PLEASE EXPLAIN THE NOVELTY AND RELEVANCE OF THE WORK CONDUCTED MORE CLEARLY.

In some sections, older references were used. The new source should be used.

Please improved all figures since they are hard to read.

 

Comments in the Methods

Ln-77-78: Please provide a reference for the inoculation of lactic acid bacteria.

Ln-104: Please justify why Ginsenoside was used as internal standard in the HPLC analysis.

Is Ginsenoside present in the sample?

Ln- 125: Why CaCo2 cells were used for the Evaluation of barrier improvement efficacy?

Please justify … References???

 

Ln-167: Please provide primers for the qRT-PCR.

References for the tight junctions analyzed in the evaluation of barrier improvement efficacy.

Please justify…

 

comments in Results

in general, this section is quite brief when it comes to the discussion of results compared with previous studies

please expand on this discussion, what did previous author found on the results and what were the implications?

Author Response

The attached file contains the full text and comments for you.

Comments 1-1

Abstract and conclusion should be controlled.

PLEASE EXPLAIN THE NOVELTY AND RELEVANCE OF THE WORK CONDUCTED MORE CLEARLY.

 

Answer 1-1

Thank you for your general comments.

We have added content to highlight our research results in abstract and conclusion.

 

Let us tell you about the novelty of our research.

We highlighted that fermentation of CWG with HY7303 resulted in a remarkable increase in minor ginsenoside content that are ginsenosides that exists in very trace amounts in nature. FCWG and FCWG-EVs equally showed increased cell thickness, decreased permeability, and increased expression of mRNA expressing tight junction proteins. This clearly suggests that FCWG and the produced EVs have a positive effect on improving the intestinal barrier.

 

And, let me comment on the relevance of our study to the results.

Through fermentation using HY7303, the trace ginsenoside content of FCWG increased compared to CWG. Additionally, FCWG-EV clearly demonstrated superior functionality compared to CWG-EV. From these results, we discovered common characteristics that positively change the intestinal barrier during the fermentation process using HY7303. This suggests increased functional benefit due to the increase in trace ginsenoside content and the resulting synergistic effect of EVs. We believe that additional research should be conducted into the molecular mechanisms that support the possibility of dramatically improving the intestinal environment through consumption of wild ginseng.

 

Comments1-2

In some sections, older references were used. The new source should be used.

 

Answer 1-2

I listed old references from before the 2000s. And we replaced it with recent studies that were similar but slightly more improved. We are truly glad that your comments have made our reference more reliable.

 

[8] Akao T, Kanaoka M, Kobashi K. Appearance of compound K, a major metabolite of ginsenoside Rb1 by intestinal bacteria, in rat plasma after oral administration--measurement of compound K by enzyme immunoassay. Biological & pharmaceutical bulletin. 1998;21:245-9.

⇒ [8] KANG, Soowon; MIN, Hyeyoung. Ginseng, the 'immunity boost' : the effects of Panax ginseng on immune system. Journal of ginseng research, 2012, 36.4: 354.

 

[29] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical chemistry. 1959;31:426-8

This paper is a major reference for the DNS method, cited 36,418 times. Instead of replacing it, I will attach recent papers that use that reference.

1. KONAPPA, Narasimhamurthy, et al. GC–MS analysis of phytoconstituents from Amomum nilgiricum and molecular docking interactions of bioactive serverogenin acetate with target proteins. Scientific reports, 2020, 10.1: 16438.
2. DESHAVATH, Narendra Naik, et al. Pitfalls in the 3, 5-dinitrosalicylic acid (DNS) assay for the reducing sugars: Interference of furfural and 5-hydroxymethylfurfural. International journal of biological macromolecules, 2020, 156: 180-185.

[33] Park J. Recent studies on the chemical constituents of Korean ginseng (Panax ginseng CA Meyer). Korean J Ginseng Sci. 1996;20:389-415

⇒ [33] Jang W, Jang Y, Kim NH, Waminal NE, Kim YC, Lee JW, Yang TJ. Genetic diversity among cultivated and wild Panax ginseng populations revealed by high-resolution microsatellite markers. J Ginseng Res. 2020 Jul;44(4):637-643.

 

Comments1-3

Please improved all figures since they are hard to read.

 

Answer 1-3

We increased the size of each axis shown in the figure to improve readability for our readers. Additionally, the content of the processed sample was listed in the figure legend.

 

Comments 2

Ln-77-78: Please provide a reference for the inoculation of lactic acid bacteria.

 

Answer 2

Thank you for your comments. Limosilactobacillus fermentum HY7303 is managed under the accession number KCTC 15628BP, and other bacteria belong to the bacterial library of hy.Co., Ltd.

I will attach a reference where we used 5 brix CWG medium. According to Mo et al. (2021), it was shown that lactic acid bacteria can grow in 5 brix red ginseng medium. So, we also CWG to 5 brix and used it as a medium. I will attach a reference paper. I hope this provides an appropriate answer to your question.

1. Mo, S. J., Nam, B., Bae, C. H., Park, S. D., Shim, J. J., & Lee, J. L. (2021). Characterization of novel Lactobacillus paracasei HY7017 capable of improving physiological properties and immune enhancing effects using red ginseng extract. Fermentation, 7(4), 238.

 

Comments 3

Ln-104: Please justify why Ginsenoside was used as internal standard in the HPLC analysis. Is Ginsenoside present in the sample?

 

Answer 3

Thank you for your comments. Rather than using an internal standard, we simply created a ginsenosides standard and then measured the ginsenosides inside the sample. I will also attach a reference containing information about the ginsenosides we measured. I hope I can help you understand your question.

1. Ye, X. W., Li, C. S., Zhang, H. X., Li, Q., Cheng, S. Q., Wen, J., ... & Li, X. R. (2023). Saponins of ginseng products: a review of their transformation in processing. Frontiers in Pharmacology, 14, 1177819.

 

Comments 4

Why CaCo2 cells were used for the Evaluation of barrier improvement efficacy? Please justify … References???

 

Answer 4

Thank you for your comments. The reason for choosing Caco-2 cells is that this line is an immortalized cell line of human colorectal adenocarcinoma cell. So, it is primarily used as a model of the intestinal epithelial barrier. In intestinal epithelial cells, tight junctions regulate the permeability of ions, nutrients, and water while maintaining the barrier. Therefore, we confirmed the mRNA expression of tight junction protein in Caco-2 cells to prove that the sample had an effect on intestinal barrier. I attach the paper for your reference.

1. Sambuy, Y., De Angelis, I., Ranaldi, G., Scarino, M. L., Stammati, A., & Zucco, F. (2005). The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell biology and toxicology, 21, 1-26.
2. Hyrslova, I., Drab, V., Cihlar, J., Krausova, G., Mrvikova, I., Kana, A., ... & Musilova, S. (2023). Functional and probiotic characterization of newly isolated strains from infant feces and breast milk. Fermentation, 9(11), 960.

 

Comments 5

Please provide primers for the qRT-PCR. References for the tight junctions analyzed in the evaluation of barrier improvement efficacy. Please justify.

 

Answer 5

Thank you for your comments. Information about the primers used in qRT-PCR is inserted in Table 2 (line 186).

The intestinal barrier system depends on the interaction of various components, including intercellular tight junctions. Intercellular tight junctions are formed by the assembly of several proteins in epithelial cells. Tight junction proteins constitute the main physical contributors to the intestinal barrier system. Many previous papers have described the link between the intestinal barrier system and tight junction proteins. I'm attaching a reference that may be helpful to your comment.

1. Suzuki, T. (2020). Regulation of the intestinal barrier by nutrients: The role of tight junctions. Animal Science Journal, 91(1), e13357.
2. Van Itallie, C. M., Tietgens, A. J., & Anderson, J. M. (2017). Visualizing the dynamic coupling of claudin strands to the actin cytoskeleton through ZO-1. Molecular biology of the cell, 28(4), 524-534.
3. Kim, H., Yoo, M. S., Jeon, H., Shim, J. J., Park, W. J., Kim, J. Y., & Lee, J. L. (2023). Probiotic properties and safety evaluation of Lactobacillus plantarum hy7718 with superior storage stability isolated from fermented squid. Microorganisms, 11(9), 2254.

 

Comments 6

in general, this section is quite brief when it comes to the discussion of results compared with previous studies. please expand on this discussion, what did previous author found on the results and what were the implications?

 

Answer 6

Thank you for your key comments. These thoughts are further described in the two paragraphs of Discussion section.

 

We use data from Seong et al. (2015) to explain the possibility as to why FCWG-EVs have a positive effect on Caco-2 cells.

 [line 392-399] The added parts are as follows:

“Previous studies have shown that milk-derived extracellular vesicles (mEV) alleviate epithelial tight junction disruption and have therapeutic potential in metabolic diseases re-lated to the gut-liver axis [68]. In previous study, cellular uptake of mEVs by Caco-2 cells was confirmed, supporting the possibility of cellular uptake for the functionality shown by FCWG-EVs in our study. Additionally, by comparing the results of CWG-EVs and FCWG-EVs, it suggests that fermentation using HY7303 makes a significant difference in the production of EVs of cultured wild ginseng roots and the resulting ability to improve the intestinal barrier.”

 

We followed Tong et al. (2023), we confirmed the expression of more tight junction proteins and emphasized that this is the first study to evaluate the ability to improve the intestinal barrier using FCWG-EVs.

 [line 414-422] The added parts are as follows:

“In a previous study, the recovery of ZO-1 lost when fermented wild ginseng was orally administered to mice with colitis was reported through fluorescent staining [72]. Our study provides additional evidence for the intestinal barrier recovery ability of fermented wild ginseng by not only confirming the recovery of ZO-1 as confirmed in previous stud-ies, but also the recovery of ZO-2 and OCLN. In addition, we ruled out the possibility of external factors affecting protein expression and confirmed that FCWG has an effect on intestinal cells by confirming the expression of mRNA. And our study is, to the best of my knowledge, the first study on the intestinal barrier improvements of EVs derived from fermented wild ginseng.”

 

We sincerely appreciate your comments, which have improved the quality of the discussion section of our study.

 

I hope my answer will help resolve the reviewer's questions.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

In this study, Mo et al. investigated the effects of fermenting cultured wild ginseng roots (CWG) with Limosilactobacillus fermentum HY7303, which enhanced intestinal barrier functions by increasing minor ginsenosides and producing extracellular vesicles (EVs). The fermentation process resulted in a significant increase in minor ginsenosides known for better absorption and pharmacological effects, and the EVs produced were shown to improve the intestinal epithelial barrier, suggesting potential benefits for inflammatory intestinal conditions. The topic of this study is fascinating and may be of interest to readers, but there are some issues. Here are some comments on this study:

 

1.        Methods section, please define the abbreviations when they appears.

2.        Line 191 “The initial inoculation CFU of bacteria was 1.78 × 106 ± 1.23 × 106 on average”, would it make more sense to control for consistent concentrations of inoculum CFUs?

3.        It would be useful for the authors to briefly describe the methodology for preparing CWG-EVs and FCWG-EVs (line 257).

4.        Line 363 “and identified the 362 peak of EVs size (20 to 250 nm) in FCWG using a size analyzer (Figure 3 and Table 4)[61]”, no need to cite a reference for experimental results.

 

5.        There were multiple typos in the text, such as format of lines 83-84, line 135 “cm2”, and space is required between numbers and units.

Author Response

 The attached file contains the full text and comments for you.

Comments 1

Methods section, please define the abbreviations when they appear.

 

Answer 1

Thanks for your comment. We have carried out a comprehensive revision of the material and method part. I sincerely thank you for your advice in improving the quality of paper.

• Cultured wild ginseng roots (CWG), Fermented cultured wild ginseng roots (FCWG)
• Lactic acid bacteria (LAB)
• Tangential flow filtration (TFF)
• Lipopolysaccharide (LPS)

 

Comments 2

Line 191 “The initial inoculation CFU of bacteria was 1.78 × 106 ± 1.23 × 106 on average”, would it make more sense to control for consistent concentrations of inoculum CFUs?

 

Answer 2

Thank you for your comment. We have removed ambiguous standard errors that are confusing to read (line 200). In order to conduct experiments with the same initial inoculum amount, CFU could be confirmed through MRS agar before inoculation into CWG medium, but the activated bacteria were immediately added to CWG medium to be used as fermentation strains.

We will strive to devise more accurate experimental methods by reflecting your opinions. Thank you for the sound advice.

 

Comments 3

It would be useful for the authors to briefly describe the methodology for preparing CWG-EVs and FCWG-EVs (line 257).

 

Answer 3

Thank you so much for your comment. We have added a methodology for sample preparing and processing in the materials and methods section. Thank you for the advice on improving the quality of the paper.

 

(line 145-149)

“CWG and FCWG were diluted to a concentration of 10ug/mL, and CWG-EVs and FCWG-EVs dissolved in DMSO were treated at 10ug/mL with 1ug/mL of Lipopolysaccharides (LPS, Sigma-Aldrich, St. Louis, USA) for 24 hours. In all subsequent experiments, cells were treated with the same concentration of LPS and samples.”

 

Comments 4

Line 363 “and identified the 362 peak of EVs size (20 to 250 nm) in FCWG using a size analyzer (Figure 3 and Table 4)[61]”, no need to cite a reference for experimental results.

 

Answer 4

Thank you for your comments. I really appreciate for pointing out the unnecessary reference and contributing to improving the quality of the paper. The reference has been removed from the manuscript.

 

Comments 5

There were multiple typos in the text, such as format of lines 83-84, line 135 “cm2”, and space is required between numbers and units.

 

Answer 5

I really appreciate your comment. Typos in unit entries throughout the paper have been corrected. In particular, the spacing of units has been completely revised. I really appreciate your advice on the quality of paper.

 

I hope my answer will help resolve the reviewer's questions.

 

Author Response File: Author Response.docx