Prebiotic Isomaltooligosaccharide Provides an Advantageous Fitness to the Probiotic Bacillus subtilis CU1

Round 1

Reviewer 1 Report

The study entitled “Prebiotic isomaltooligosaccharide provides an advantageous fitness to the probiotic Bacillus subtilis CU1” by Villeger et al., defines the role of IMOS on survival of Bacillus and its potential use in synbiotics. Overall the manuscript is well written and would definitely be readers interest to further evaluate the impact on IMOS on Bacillus survival in vivo. However, there are few points that needs to addressed and are mentioned below:

1. Since the experiment is major experiment to show that Bacillus consume IMOS effectively, please provide the picture in main figure or supplementary figure.

2. What is the reason behind sudden decrease in DP2 concentration and increase in glucose concentration (Fig 2C) in between 10-12 hrs. Justification for this can be explained in discussion part.

3. Authors have reported ” Moreover, the results showed……………….cytosol of B. subtilis CU1 after growth on glucose (Figure 2D)”, however figure shows basal level of α-glucosidase activity and activity is increased with IMOS. So please modify the sentence.

4. In fig 4A, there are only two curves i.e with glucose and IMOS both supplemented with bile salt. There is no data in absence of bile salts. However, in result section, author mentioned that in presence of bile lag time is increased in glucose supplemented medium. (“In glucose medium, bile increases the lag-time and reduces the growth rate, but the growth profiles are quite similar compared to results in absence of bile salts”). It is better to include all data (with and without bile) in this figure to see an affect of bile on growth of bacterium.

5. Did the authors measured the release of α-glucosidase in presence of bile with IMOS and glucose, to support their hypothesis that more DP2 is released with bile?

 

Author Response

COMMENTS REVIEWER 1

The study entitled “Prebiotic isomaltooligosaccharide provides an advantageous fitness to the probiotic Bacillus subtilis CU1” by Villeger et al., defines the role of IMOS on survival of Bacillus and its potential use in synbiotics. Overall the manuscript is well written and would definitely be readers interest to further evaluate the impact on IMOS on Bacillus survival in vivo. However, there are few points that needs to addressed and are mentioned below:

We gratefully thank reviewer 1 for critically reviewing our manuscript, giving us encouraging comments, and helping us to improve our paper. We have modified the main text as suggested (Track Changes, attached file).  We sincerely hope that we have clearly addressed your concerns, and that our new version of the manuscript will reach your expectations.

Please find our answers to your questions below.

1. Since the experiment is major experiment to show that Bacillus consume IMOS effectively, please provide the picture in main figure or supplementary figure.

Thank you to reviewer 1 for its suggestion. We sincerely apologize, we have forgotten to cite the Table 1 in the main text, and this has been corrected. This table include the results obtained for each strain with every single prebiotic substrate. Moreover, we have included the pictures of growth experiments obtained with B. subtilis CU1 in Supplementary Information (Figure S1).

2. What is the reason behind sudden decrease in DP2 concentration and increase in glucose concentration (Fig 2C) in between 10-12 hrs. Justification for this can be explained in discussion part.

Thank you to reviewer 1 for this question. As mentioned in the results section, we supposed that increase of DP1 around 12 h is due to extracellular hydrolysis of higher DP oligosaccharides, including DP3 and higher DP oligosaccharides. This explanation has been added to the discussion (Track Changes).

3. Authors have reported ” Moreover, the results showed……………….cytosol of B. subtilis CU1 after growth on glucose (Figure 2D)”, however figure shows basal level of α-glucosidase activity and activity is increased with IMOS. So please modify the sentence.

We thank reviewer 1 for reporting this mistake. We have modified the sentence as suggested.

4. In fig 4A, there are only two curves i.e with glucose and IMOS both supplemented with bile salt. There is no data in absence of bile salts. However, in result section, author mentioned that in presence of bile lag time is increased in glucose supplemented medium. (“In glucose medium, bile increases the lag-time and reduces the growth rate, but the growth profiles are quite similar compared to results in absence of bile salts”). It is better to include all data (with and without bile) in this figure to see an affect of bile on growth of bacterium.

Thank you to reviewer 1 for the suggestion. We have included the data in absence of bile in Figure 4A and 4B. We have modified the results section in accordance to the new data (Track Changes).

5. Did the authors measured the release of α-glucosidase in presence of bile with IMOS and glucose, to support their hypothesis that more DP2 is released with bile?

Thank you to reviewer 1 for this interesting suggestion. Indeed, the release of α-glucosidase in presence of bile has not been measured. Unfortunately, we do not have these samples anymore, and we can’t add this result to the paper. We sincerely apologize for this inconvenience.

Author Response File: Author Response.docx

Reviewer 2 Report

This study elucidated the specific IMSO utilization capacity of B. subtilis CU1 through detection and analysis of substrate utilization, growth, enzyme activity, and proteomics. And confirmed that IMSO provides a fitness advantage to B. subtilis CU1 for bile tolerance. These results suggest that IMOS could be a good candidate to be associated with B. subtilis CU1 in a synbiotic product. It is an interesting finding, but the following questions need to be addressed before publication:

1.         It is suggested to divide the first paragraph of Introduction into two paragraphs according to the background and current research status.

2.         The unit form in the manuscript should be harmonized, such as "g.L-1" and "mg/mL", "mL/min" and "mL / min", or some units appearing in the axes of the figures.

3.         The content of the first paragraph of Results needs supplementary data/graphics support, and the last sentence of this paragraph seems to be wrong in form.

4.         The monitoring periods in Fig. 4A and 4B are inconsistent, and in particular, the data presented in Fig. 4A do not appear to have reached a plateau.

Author Response

COMMENTS REVIEWER 2

 

This study elucidated the specific IMSO utilization capacity of B. subtilis CU1 through detection and analysis of substrate utilization, growth, enzyme activity, and proteomics. And confirmed that IMSO provides a fitness advantage to B. subtilis CU1 for bile tolerance. These results suggest that IMOS could be a good candidate to be associated with B. subtilis CU1 in a synbiotic product. It is an interesting finding, but the following questions need to be addressed before publication:

We gratefully thank reviewer 2 for its very positive comments, for critically revising our manuscript and helping us to improve our paper. We have changed the text as suggested (Track Changes, attached version). We sincerely hope that we have clearly addressed your concerns, and that our new version of the manuscript will reach your expectations.

Please find our answers below.

 

1. It is suggested to divide the first paragraph of Introduction into two paragraphs according to the background and current research status.

Thank you to reviewer 2 for its suggestion. As suggested, we have divided the introduction into two paragraphs.

2. The unit form in the manuscript should be harmonized, such as "g.L-1" and "mg/mL", "mL/min" and "mL / min", or some units appearing in the axes of the figures.

Thank you for reporting these mistakes. We have harmonized the units in the manuscript and the figures.

3. The content of the first paragraph of Results needs supplementary data/graphics support, and the last sentence of this paragraph seems to be wrong in form.

We sincerely apologize, we have not cited the Table 1 in the first paragraph. We have corrected this oversight. Moreover, we have added the pictures of B. subtilis CU1 growth on solid medium with single prebiotic oligosaccharides (Figure S1).

4. The monitoring periods in Fig. 4A and 4B are inconsistent, and in particular, the data presented in Fig. 4A do not appear to have reached a plateau.

Thank you for your comment. Figure 4A focuses on the growth of vegetative strains. In order to compare to data in absence of bile, we have kept the time for the growth (24 h). Regarding Figure 4B, we have followed the growth of the bacteria in presence of bile for 72 h but the bacteria have reached the plateau and start to decline after 48 h, explaining why we focused on the first 48 h of growth.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

This manuscript has been sufficiently improved to warrant publication in Applied Sciences

Author Response

Dear Reviewer,

 

Thank you a lot for your beneficial suggestions on our manuscript and supporting our publication.

My warmest regards,