Lactic Acid Bacteria as Probiotics Improve Bioactive Compounds in Radix Angelica gigas (Danggui) via Solid-State Fermentation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript under review, entitled “Lactic acid bacteria as probiotics that improve bioactive compounds in Radix Angelica gigas (Danggui) via solid-state fermentation,” reports interesting data. The authors studied the effect of various probiotic strains, and among them of the lactic acid bacteria (the most commonly used probiotics) on the bioactive profile of Radix Angelica gigas under solid-state fermentation conditions. The plant Angelica gigas has been chosen by the authors more likely due to its consideration as a traditional herb with therapeutic properties in east Asia. However, and according to the authors and the cited references, low bioactivity and potentially toxicity have been observed using the aforementioned herb for therapeutic purposes. The present study of the authors focuses on improving the bioactivity and safety profile of Radix Angelica gigas.

The text is not followed well and, therefore authors should proceed with extensive proofreading of the English language of their text. All methods are well described. Nevertheless, the present version of this manuscript is not publishable. Authors should prepare a revised version of their manuscript in accordance with the following comments and suggestions. They should also provide acceptable answers to reasonable questions that arise while reading their manuscript.

(1) Subsection 2.5. Statistical analysis: The authors state that they carried out triplicate experimental measurements and their data were analyzed with one-way and two-way ANOVA tests “followed by Duncan’s multiple range test” in order to “determine significant differences at p < 0.05” through the use of the statistical software SPSS. I think the authors ignore the terms of use of a statistical software, as well as the prerequisites for using the aforementioned statistical tests. Both one-way and two-way ANOVA tests require at least normally distributed residuals of response variables, equal population variances, and normally distributed independent errors having the same variance. Furthermore, Duncan’s multiple range test is applied to a sample of observed means that have been independently drawn from a number of normal populations with true means. All three of the aforementioned statistical tests are parametric. In cases where there are only a few observations (for whatever reason), non-parametric alternatives should be used, such as one-way Kruskal-Wallis ANOVA and/or other tests based on randomizations [e.g., Korean J Anesthesiol. 28, 2016, 8-14]. Moreover, I did not find any comments on the statistical analyses in the "Results", "Discussion" and "Conclusions" sections.

(2) Lines 172-177: How did the authors reached at these results? What statistical methodology was followed? Can the authors show their statistical treatments? Do they are referred to the results of Table 1? There are no triplicates (values) anywhere in the text, which are mentioned in subsection 2.5; there are no appendices and/or supplementary material!!! Authors should provide scientifically sound answers.

(3) Lines 178-180: Similar comments and answers as in (2).

(4) Figure 1: In the figure legend, as well as in the text of “Results”, the authors should explain the complexity (in Figure 1A), and the obscure (to me), but significant for them, differences (in Figure 1B).

(5) Table 1: The authors should also explain the statistically significant differences in the section “Results”.

(6) Figure 2: It should be redrawn. The graphs are confusing. Why aren't error bars provided in all cases?

(7) Figure 3: Why in Figures 1 and 2 the experiments were performed at incubation periods of 0, 2, 4, 6, and 8 days whereas in Figure 3 the experiments were performed only at the limited periods of 0, 4 and 6 days?

(8) Figure 4: Similar inquiries as above about the incubation periods (?)

(9) The Discussion and Conclusion sections are based on the Results section. Therefore, authors should rewrite the Results, Discussion, and Conclusion sections based on their responses to my comments and questions mentioned above. And they should be convincing according to their results (speculation will not be accepted).

Overall: The current version of this manuscript under review cannot be published in the journal Fermentation. I suggest that the authors prepare and resubmit a properly revised version of their manuscript, where their results and conclusions will be based on statistically correct and proven differences.

Comments on the Quality of English Language

The authors should proceed with extensive proofreading of the English language of their text.

Author Response

Author's Reply to the Review Report (Reviewer 1)

 

(1) Subsection 2.5. Statistical analysis: The authors state that they carried out triplicate experimental measurements and their data were analyzed with one-way and two-way ANOVA tests “followed by Duncan’s multiple range test” in order to “determine significant differences at p < 0.05” through the use of the statistical software SPSS. I think the authors ignore the terms of use of a statistical software, as well as the prerequisites for using the aforementioned statistical tests. Both one-way and two-way ANOVA tests require at least normally distributed residuals of response variables, equal population variances, and normally distributed independent errors having the same variance. Furthermore, Duncan’s multiple range test is applied to a sample of observed means that have been independently drawn from a number of normal populations with true means. All three of the aforementioned statistical tests are parametric. In cases where there are only a few observations (for whatever reason), non-parametric alternatives should be used, such as one-way Kruskal-Wallis ANOVA and/or other tests based on randomizations [e.g., Korean J Anesthesiol. 28, 2016, 8-14]. Moreover, I did not find any comments on the statistical analyses in the "Results", "Discussion" and "Conclusions" sections.

Reply: We appreciate your detailed comment regarding the statistical analysis.

We have carefully re-examined our statistical methodology and have revised the manuscript. We have added specific mentions of statistical significance in the Results and Discussion sections where appropriate, including relevant p-values.

 

(2) Lines 172-177: How did the authors reached at these results? What statistical methodology was followed? Can the authors show their statistical treatments? Do they are referred to the results of Table 1? There are no triplicates (values) anywhere in the text, which are mentioned in subsection 2.5; there are no appendices and/or supplementary material!!! Authors should provide scientifically sound answers.

Reply: We thank the reviewer for raising these comments.

We were conducted in triplicate in all treatment at each time of incubation. Duncan’s multiple range test was used to identify differences between temperature treatments. Statistical significance was set at P < 0.05. We have rewritten 5.2 in detail to reflect the above.

To avoid complexity, we added descriptions of Figures 1(A) and (B) to the main text of “Results”. Also, the words and sentences used in the results were reconstructed and written.

 

(3) Lines 178-180: Similar comments and answers as in (2).

Reply: We thank the reviewer for raising these comments.

We were conducted in triplicate in all treatment at each time of incubation. Duncan’s multiple range test was used to identify differences between temperature treatments. Statistical significance was set at P < 0.05. We have rewritten 5.2 in detail to reflect the above.

To avoid complexity, we added descriptions of Figures 1(A) and (B) to the main text of “Results”. Also, the words and sentences used in the results were reconstructed and written.

 

(4) Figure 1: In the figure legend, as well as in the text of “Results”, the authors should explain the complexity (in Figure 1A), and the obscure (to me), but significant for them, differences (in Figure 1B).

Reply: We thank the reviewer for raising these comments.

To avoid complexity, we added descriptions of Figures 1(A) and (B) to the main text of “Results” and slightly revised the titles of the figures for clarity, as following. Also, the statistics were reflected in the picture and redrawn to reflect more clarity.

 

The effects of culture temperature and duration on the growth of L. buchneri in SSF of Radix Angelica gigas are shown in Figure 1 (A).

The pH changes during SSF using L. buchneri is shown in Figure 1 (B).

 

 Figure 1. Effects of culture temperature and duration on the growths of L. buchneri (A) and the pHs (B) in SSF of Radix Angelica gigas. ^a,b,c Means in the same period with different superscripts are significantly different (p < 0.05). 25°C, 30°C and 35°C mean temperature of incubation.

 

(5) Table 1: The authors should also explain the statistically significant differences in the section “Results”.

Reply: We appreciate your thorough comment, which has significantly contributed to the improvement of our manuscript.

We have carefully revised the manuscript according to the reviewer’s comment. The corresponding changes have been made in the “Results” part.

 

(6) Figure 2: It should be redrawn. The graphs are confusing. Why aren't error bars provided in all cases?

Reply: We thank the reviewer for raising this comment.

To avoid confusion in the figure 2, the image has been revised to make it clearer and rearrange the ligands. We also provided a margin of error in all cases.

 

(7) Figure 3: Why in Figures 1 and 2 the experiments were performed at incubation periods of 0, 2, 4, 6, and 8 days whereas in Figure 3 the experiments were performed only at the limited periods of 0, 4 and 6 days?

Reply: We thank the reviewer for raising this question.

We have carefully revised the results according to the reviewer’s comment, as follow;

1. acidophilus, L. buchneri, L. plantarum, and L. reuteri in the tested LAB showed enhanced growth in the screening experiment compared to the other strains (Table 1). To compare the growth in SSF of Radix Angelica gigas, the four strains were cultured at 30°C and 37°C for 8 days (Figure 2).

 

(8) Figure 4: Similar inquiries as above about the incubation periods (?)

Reply: We thank the reviewer for raising this question.

We have carefully revised the results according to the reviewer’s comment, as follow;

1. acidophilus, L. buchneri, L. plantarum, and L. reuteri in the tested LAB showed enhanced growth in the screening experiment compared to the other strains (Table 1). To compare the growth in SSF of Radix Angelica gigas, the four strains were cultured at 30°C and 37°C for 8 days (Figure 2).

 

 (9) The Discussion and Conclusion sections are based on the Results section. Therefore, authors should rewrite the Results, Discussion, and Conclusion sections based on their responses to my comments and questions mentioned above. And they should be convincing according to their results (speculation will not be accepted).

Reply: We appreciate your detailed comment

We rewrite the overall content of the Discussion and Conclusion sections based on the Results section.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

 

In this study, through solid-state fermentation (SSF) technology, the enhancing effects of lactic acid bacteria (LAB) and other probiotics on the bioactive components of Radix Angelica gigas were explored. A variety of LAB strains (including Lactobacillus rhamnosus, L. acidophilus, L. buchneri, L. reuteri and L. plantarum) and auxiliary microorganisms (Bacillus) were selected for the study Fermentation was carried out under different temperatures (25°C, 30°C, 35°C) and fermentation times (2-8 days) of subtilis and Saccharomyces cerevisiae, and their effects on the free radical scavenging ability of DPPH and ABTS, as well as the contents of total phenols and flavonoids were evaluated. The results showed that L. buchneri grew best at 30° C. After 4 days of fermentation, the colony count reached its peak (706.76 × 10⁵ CFU/g), while the pH decreased significantly. The antioxidant activity of Angelica sinensis after fermentation was significantly enhanced. Among them, the free radical scavenging activity of DPPH increased by up to 230% (35°C), the activity of ABTS increased by 111% (30°C), and the contents of total phenols and total flavonoids increased by 137% and 133% respectively. Multi-strain fermentation (such as the mixture of L. acidophilus and L. plantarum) shows a stronger antioxidant enhancement effect than single-strain fermentation, indicating that there may be a synergistic effect among the strains. This study confirmed that LAB-mediated SSF can effectively enhance the biological activity of Angelica sinensis, providing a scientific basis for the modern application of traditional herbal medicine. Future research can further optimize the fermentation conditions and explore their application potential in functional foods or animal feeds

 

 

1. Why does L. buchneri exhibit superior growth performance in SSF compared to other LAB strains? Is its metabolic mechanism unique?
2. What are the specific regulatory mechanisms of 30°C and 35°C on the growth of LAB and the synthesis of antioxidant substances? Is there a better temperature window?
3. How does multi-strain fermentation (such as L. acidophilus and L. plantarum) enhance antioxidant activity through interaction? Is there a competitive or symbiotic effect?
4. Why does antioxidant activity increase most significantly in the early stage of fermentation (such as on the 4th day)? Is the change in activity in the later stage related to the depletion of substrate or the inhibition of metabolites?
5. How is the correlation between pH decline and the growth of LAB and the accumulation of antioxidant substances? Can the fermentation effect be further optimized by adjusting the pH?
6. How to control the low-cost advantage of SSF (such as pH and temperature control)? How can large-scale production be achieved in the future?

Author Response

Author's Reply to the Review Report (Reviewer 2)

 

• Why does L. buchneri exhibit superior growth performance in SSF compared to other LAB strains? Is its metabolic mechanism unique?

Reply: We thank the reviewer for raising this question.

Added to 'Discussion' as follows (Line 296-303):

Also, we found that L. buchneri exhibit superior growth performance in SSF compared to other LAB strains. As similar research results, [30,31] reported that L. buchneri out-grows all other species within a short period on plant materials such as grass silage. Metabolic equipment of L. buchneri is unique as it is capable to convert lactic acid to acetic acid under aerobic as well as anaerobic conditions [32,33]. Additionally, L. buch-neri is highly resistant against external perturbations such as the presence of compet-ing microorganisms, oxygen, high lactic acid or ethanol concentrations, and low pH compared to other lactic acid bacteria [33].

 

• What are the specific regulatory mechanisms of 30°C and 35°C on the growth of LAB and the synthesis of antioxidant substances? Is there a better temperature window?

Reply: We thank the reviewer for raising this question.

Added discussions with reference in ‘Discussion’ as follows (Line 326-335):

Maintaining the optimal temperature during fermentation results in improved mi-crobial growth and enzymatic activity, and consequently, the benefits of herbal fer-mentation are improved. Particularly, the release of antioxidative compounds was ac-celerated by the increased degradation of the plant cell walls, but extremely high or low temperatures reduced antioxidant activity during fermentation [36]. A fermenta-tion temperature of 30°C significantly increased polyphenols (followed by fermenta-tion temperatures of 34 and 38°C); the lowest levels are obtained at 24 and 42°C in bush tea [41]. Also, the β-glucosidase activity of L. casei was maximal at 35°C [42]. β-glucosidase activity during microbial fermentation causes the hydrolysis of phenolic glycosides and the release of free aglycones, which can have a high antioxidant activi-ty..

 

• How does multi-strain fermentation (such as L. acidophilus and L. plantarum) enhance antioxidant activity through interaction? Is there a competitive or symbiotic effect?

Reply: We thank the reviewer for raising this question.

Added discussions with reference in ‘Discussion’ as follows (Line 372-379):

Multi-strain fermentation (such as L. acidophilus and L. plantarum) can increase the production of beneficial compounds and promote a synergistic effect between the bac-teria, leading to a greater overall antioxidant [45,53,54]. The mixed cultures enhanced the growth of L. plantarum strains, which relate to the production of nutrients, such as vitamins, by other strains [55]. The antioxidant activity of multi-strain fermentation enhanced due to an increase in the solubility of phenolic compounds in the guava leaf tea and due to the production of more phenolic compounds with higher bioactivities, such as kaempferol and quercetin [56].

 

• Why does antioxidant activity increase most significantly in the early stage of fermentation (such as on the 4th day)? Is the change in activity in the later stage related to the depletion of substrate or the inhibition of metabolites?

Reply: We thank the reviewer for raising this question.

Added discussions with reference in ‘Discussion’ as follows (Line 344-352):

These findings showed that antioxidant activity is most efficiently enhanced in the early fermentation period and continues to rise with extended incubation. This can occur due to the release and increase in the concentration of phenolic compounds and flavonoids during the early stages of fermentation and depletion of substrates or inhi-bition by metabolites, as the fermentation process progresses and the environment shifts in the later stages [43-45]. This phenomenon was also confirmed in Table 3, where DPPH, total phenols, and total flavonoid showed a significant positive correlation with cell growth in the early stage of fermentation (days 0–4), and a significant negative correlation thereafter.

 

• How is the correlation between pH decline and the growth of LAB and the accumulation of antioxidant substances? Can the fermentation effect be further optimized by adjusting the pH?

Reply: We thank the reviewer for raising this cpmments.

We analyzed Added the correlation between pH decline and the growth of LAB and the accumulation of antioxidant substances (Table 3), and added related description in Materials and Methods, Results, and Reference, as follow (Line 214-236, 244-352):

In Results;

Correlation analyses between bacterial growth or pH and antioxidant levels (DPPH, ABTS, phenol, and flavonoid) were performed under three temperature conditions (25 °C, 30 °C, and 35 °C) (Table 3). At all temperatures, DPPH measured during days 0–4 (hereafter referred to as the “0–4 day group”) exhibited a significant positive correlation with bacterial growth, whereas, DPPH showed a significant negative correlation with growth during days 6–8 (hereafter referred to as the “6–8 day group”). Phenol levels measured at 30 °C and 35 °C were significantly positively correlated with growth in the 0–4 day group across all temperature conditions. In contrast, phenol measured at 25 °C displayed a significant negative correlation with growth in the 6–8 day group at all temperatures, with the magnitude of the negative correlation diminishing as temperature increased. Flavonoid concentrations measured at 30 °C and 35 °C also exhibited significant positive correlations with growth in the 0–4 day group across all temperatures, and the strength of these positive correlations increased with rising temperature. Conversely, in the 6–8 day group, flavonoids measured at 30 °C and 35 °C showed significant negative correlations with growth across all temperatures, with stronger negative correlations observed at higher temperatures. Although DPPH, phenol, and flavonoids demonstrated either positive or negative correlations with bacterial growth under specific temperature–time conditions, ABTS did not exhibit any statistically significant correlation with growth. Regarding pH, both the 0–4 day and 6–8 day groups showed significant negative correlations between pH and DPPH under most temperature conditions. Phenol measured at 30 °C and 35 °C exhibited a significant negative correlation with pH in the 0–4 day group across all temperatures, while phenol measured at 25 °C displayed a significant negative correlation with pH during days 6–8 at every temperature. Moreover, most flavonoid measurements at 30 °C and 35 °C were significantly negatively correlated with pH in both the 0–4 day and 6–8 day groups across all temperatures. Similar to the growth correlation results, ABTS did not show any significant correlation with pH under any of the tested conditions.

In Discussion;

These findings showed that antioxidant activity is most efficiently enhanced in the early fermentation period and continues to rise with extended incubation. This can occur due to the release and increase in the concentration of phenolic compounds and flavonoids during the early stages of fermentation and depletion of substrates or inhibition by metabolites, as the fermentation process progresses and the environment shifts in the later stages [43-45]. This phenomenon was also confirmed in Table 3, where DPPH, total phenols, and total flavonoid showed a significant positive correlation with cell growth in the early stage of fermentation (days 0–4), and a significant negative correlation thereafter.

 

• How to control the low-cost advantage of SSF (such as pH and temperature control)? How can large-scale production be achieved in the future?

Reply: We thank the reviewer for raising this question.

In solid-state fermentation where the substrate is not fluid, it is considered difficult to control and maintain the appropriate pH during fermentation. Therefore, it is considered advantageous to adjust the pH of the solution to the appropriate level when controlling the moisture content of the solid substrate and to ferment it at an appropriate temperature for a certain period of time. In preliminary test on various pH and substrate moisture, we used distilled water (pH 7.0) to adjust moisture and mixed it with the raw material in a 1:1 ratio, which resulted in good results for the growth of lactic acid bacteria, and applied it to this experiment. For mass production, it is thought that fermentation should be conducted based on the results of this study, and verification and supplementation are necessary to increase efficiency. I'm sorry I can't give you a sufficient answer as this part is still under research.

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The article reflects the results of important research that is necessary for the development of biotechnology and agriculture. The authors have done a lot of experimental work, as well as analyzed and summarized the result at a very high level. At the same time, I would like to make some recommendations and ask some questions to the authors:

Line

34: I would like to draw the attention of the authors of the manuscript to the fact that, in accordance with the International Code of Nomenclature of Algae, Fungi and Plants (ICN), family-level taxa are not written in italics.

Line

48: I recommend that the authors indicate the full name of the abbreviated term CRP at its first mention, so that it would be easier for the potential reader to understand the essence.

 Line 113: I think it would be more correct to indicate the full name of dimethyl sulfoxide (DMSO), as it was done for DPPH (on line 115) and for ABTS (on line 121)

 Lines 152-161: Did the authors carry out a correlation analysis of the dependence of the growth rate of microorganisms on pH? The results of this analysis could enhance the significance of the results obtained.

Lines

259-276: In this part, it would be nice to give a biological explanation of why it was at a temperature of 30 degrees that the highest growth rate of L. buchneri cells was observed. 

Author Response

Author's Reply to the Review Report (Reviewer 3)

 

Line 34: I would like to draw the attention of the authors of the manuscript to the fact that, in accordance with the International Code of Nomenclature of Algae, Fungi and Plants (ICN), family-level taxa are not written in italics.

Reply: We thank the reviewer for noble comments.

‘Danggui, is the dried root of Angelica gigas Nakai (Umbelliferae)’ has been changed by Umbelliferae.

 

Line 48: I recommend that the authors indicate the full name of the abbreviated term CRP at its first mention, so that it would be easier for the potential reader to understand the essence.

Reply: We thank the reviewer for noble recommendation.

‘lowered endotoxin and C-reactive protein (CRP) levels’ has been changed by C-reactive protein (CRP).

 

Line 113: I think it would be more correct to indicate the full name of dimethyl sulfoxide (DMSO), as it was done for DPPH (on line 115) and for ABTS (on line 121)

Reply: We thank the reviewer for noble recommendation.

‘The supernatant was filtered, freeze-dried, and dissolved in dimethyl sulfoxide (DMSO)’ has been changed by dimethyl sulfoxide (DMSO).

 

Lines 152-161: Did the authors carry out a correlation analysis of the dependence of the growth rate of microorganisms on pH? The results of this analysis could enhance the significance of the results obtained.

Reply: We thank the reviewer for noble recommendation

We performed a correlation analysis of the dependence of the growth rate of microorganisms on pH, and added related description in Materials and Methods, Results, and Reference, as follow.

 

In Statistical and correlation analysis.

All statistical analyses and data handling were performed using R (version 4.2.1) in a UNIX-compatible environment [21]. Pearson’s correlation coefficients were cal-culated for all variable pairs using the R package with cor() function. To assess statis-tical significance, two-tailed p-values were obtained via R package with cor.test() for each pair of variables, applying default settings to test the null hypothesis of zero cor-relation [21].

 

In Results

These four strains also showed a greater decrease in pH values post-fermentation, which correlated with their enhanced cell growth (p < 0.05). A Pearson’s correlation analysis between bacterial growth and pH change for seven species (L. rhamnosus, L. acidophilus, L. buchneri, L. plantarum, L. reuteri, Bacillus subtilis, and S. cerevisiae) yielded a correlation coefficient of 0.925 (p = 0.003).

 

Lines 259-276: In this part, it would be nice to give a biological explanation of why it was at a temperature of 30 degrees that the highest growth rate of L. buchneri cells was observed.

Reply: We thank the reviewer for noble recommendation.

Rewritten in 'Discussion' and added reference as follows:

The optimal growth temperature for most LAB ranges from 30 to 45°C [34]. Other studies have also shown that the optimal growth conditions for Lactobacillus spp. are 30–40℃ temperature and 5.5–6.2 pH [35]. 30℃ falls within this optimal range, and allows the optimal conditions for L. buchneri's enzymes and cellular processes to func-tion most efficiently, resulting in the highest growth rate similar to many other LAB strains [36, 37].

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors of this manuscript entitled “Lactic acid bacteria as probiotics that improve bioactive compounds in Radix Angelica gigas (Danggui) via solid-state fermentation” submitted an improved revised version (compared to the original). Furthermore, and although most of the authors' responses to my comments and suggestions regarding the initial version of their manuscript are satisfactory, there are still some that remain essentially unanswered. For example, my comments on Figures 3 and 4, related to different (limited) incubation periods (0, 4 and 6) versus those of 0, 2, 4, 6 and 8 days in Figures 1 and 2, and/or the comments related to the error bars in Figures 1B (37°C), 2 and 3.

Overall: The revised version of this manuscript under review can be published in the journal Fermentation. Nevertheless, I would suggest that authors be more demanding and rigorous with the quality of both their own results and presentations when preparing future manuscripts. Statistical analysis is not a simple process of using a statistical package and “publishing” the results. Your future studies and their results may not be treated with leniency.

Reviewer 2 Report

Comments and Suggestions for Authors

no comments