In Vitro Fermentation of Different Indigestible Glucans with Varying Physico-Chemical Properties by Human Fecal Microbiota

Round 1

Reviewer 1 Report

In the present manuscript

“In vitro fermentation of different indigestible glucans varying physico-chemical properties by human fecal microbiota”

the authors investigated in vitro fermentation characteristics of different glucans with varying physicochemical properties (barley β-glucan, laminarin, yeast β-glucan, pachyman, resistant starch and litesse). The impact of in vitro fermentation of the different glucans on pH-values, SCFA concentrations, degradation of glucans and bacteria composition were analyzed.

Overall, the authors present some interesting findings in their manuscript. The manuscript is well written and well-structured. The manuscript is worth publishing because it would contribute further insights to the role of glucans on its fermentability and impact on microbial community which in turn could exert health-promoting effects.

However, there are some major concerns that must be addressed before publishing.

 

Major points are:

1.    The authors should state what new findings are presented in their work.  In vitro fermentation with glucans has done before. The authors say that are only a few studies that investigated fermentation profile of different glucans. In the discussion some examples should be given and compared to the own results if applicable. And the authors should state in the abstract and introduction what is new about their work.

2.    Line 115: The authors state that they used yeast extract in the fermentation medium. Does this yeast extract contain yeast glucan? If so, does the yeast mask some effects? This point should be addressed by the authors.

3.    Line 119. IGs were sterilized using heat. How can the authors exclude that this step causes physico-chemical changes in the investigated glucans? Such changes should then affect fermentability. Please clarify.

4.    Method section 2.3: Can the authors exclude that the feces donors have consumed dietary fiber rich food or even glucan-rich food such as barley or oat that could influence fermentation? Please clarify.

5.    The authors should discuss the validity of their findings compared to physiological conditions. They used a batch-fermentation system without a pre digestion of the samples. How transferable are the obtained results to the in vivo situation.

6.    Discuss the in vitro obtained findings regarding SCFA concentrations with in vivo findings. Concentrations and Molar ratios etc. What results were obtained by other authors investigating in vitro fermentation of glucans. Are the results comparable to the in vivo situation or to results obtained by others?

7.    Results: Please chose one presentation of results and avoid redundant presentation of the same results. For example, Figure 2 C is redundant to A and B. Is it possible to prefer one presentation? Maybe the other can be presented in the Supplemental? Also Figure 3, here again pH-values are presented, they were already given in Figure 2. Figure 3 B and C may also be redundant because total SCFA can be seen in Figure 3 A also. These are the same results but in other forms of presentations. It would be better to prefer one presentation with the most important or meaningful results. Maybe the results would prefer from a tabularly arrangement. Or transfer some results to the Supplemental section. The same with Figures 8 and 9. Are these results redundant?

 

 

Minor points:

1.    Title: check spelling

2.    Line 24: “The targeted intervention” …This is not an intervention study. This implicates that the authors performed an intervention. This was not the case. Please change.

3.    Line 40: “…on the its…”: check spelling

4.    Line 57: “…, etc[…” à etc.

5.    Figure 1 legends: please insert a reference from which the structures were adopted.

6.    Line 151 and in general: please use “x g” instead “rpm”

7.    Figure 2: Description of A and B is mixed up. Please correct this mistake.

8.    Line 271 and in general: “… BG and L…” groups. Why do the authors speak of groups?

9.    Figure 4 and 5: Did the authors perform statistical analyses for these results?

10.  All Figure and Legends: Please explain all abbreviations.

11.  Line 317: What is ASV?

12.  Lines 318 and 319: What is C0 and C24?

13.  Figure 8: the green colors are difficult to distinguish.

14.  Line 348: Please check sentence.

15.  Line 393: “significantly positive correlated” à please check spelling.

16.  Line 411: Insert some examples/references for the few reports that studied fermentation of different glucans

17.  Line 424: is BY a soluble glucan? Please check. Can you insert some information about solubility of the different glucans?

18.  Lines 426-430: Please check sentence, there are some mistakes.

19.  Lines 457…: Please discuss in a bit more detail how this study fits physiological conditions and what is different à absorption in the small intestine à monosaccharides in the gut?

20.  Lines 482….: Please discuss conditions of SCFA under physiological conditions. What are the results obtained by other using in vitro fermentation of similar samples? Are the results comparable?

21.  Line 503: one main function of butyrate à Histone deacetylase inhibitor, chemoprevention regarding colon cancer, this could be mentioned

22.  Please define: What are beneficial microbes. Please discuss in a bit more detail, why the observed shift in microbial community could be beneficial (maybe shift of ratio F/B?).

23.  Is there a relationship between shift of pH-values and abundance of bacteria? How do the observed effects regarding SCFA formation, pH reduction and bacteria abundance might interact?

Comments for author File: Comments.pdf

Author Response

please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The topic of the paper is interesting, with a broad experimental plan and a great amount of results collected. However, results are shown in a confusing way, discussions need to be improved and more detailed, and the English form needs to be reviewed. Moreover, the final considerations and potential application of this research were not stated by the authors; so I suggest the following major revisions before the publication.

 

Abstract

The abstract looks quite chaotic. Reading is difficult due to the number of conditions tested and to English sentences not always being correctly written.

In particular:
Lines 16-18: the sentence is not clear

Line 18: pH of what?

Line 22-24: do the authors mean that, after fermentation, on BG the main species found was Faecalibacterium prausnitzzi? And so on

 

Introduction

The introduction is well written and clearly presented. Maybe a brief description of each IG used in the present study could improve the section

Line 40: please remove “the” (to the its..)

 

Materials and methods

Did the three fecal sample mixed and used together for fermentation?

Line 134: “and was only in quadruplicate at 24h”: the sentence, as presented is not clear

Lines 135-138: this part describes analyses and not the fermentation. Authors could create an ad hoc paragraph

Line 142: please delete “with” (with slightly modified )

Lines 145-146: Please, add more details about the Ionic chromatography applied for residual sugar detection. Didn’t you use any standard?

Paragraphs 2.5, 2.6 and 2.7: could the authors provide more details on the methods? (eluent, flow..)

Paragraph 2.8

Lines 158-159: “proper amount”, could the authors explain better?

 

Results

Paragraph 3.1:

Lines 225: Also BY should be added to the IGs list as it shows a decreasing trend throughout the fermentation, that seems to overlap the trend of Lit in Figure 1A.

Lines 228-230: At time 48 h, significant differences were observed between all IG groups, except for BY and Lit. Can it be stated also for the previous points (5, 11, and 24 h)?

Figures 1: Abbreviations used for IG groups(BY, BG, Lit, L, R, PAC) in the Figures should be specified in the corresponding caption.

Moreover, cite in the text Figure 1C when you discuss the statistical significance of the pH and gas accumulation results.

Figure 1C: Dimensions and resolution should be improved.

Paragraph 3.2:

Lines 251-252: I suggest writing “.. remained stable after 24 h, mainly due to the constant levels of propionic- and butyric acids” or “.. “due to the unchanged level of propionic- and butyric acids between 24 and 48 h”.

In general, a very confusing result presentation can be noticed. The description of the SCFA results should be schematized/ simplified.

Figures 3: Abbreviations used for IG groups(BY, BG, Lit, L, R, PAC) in these Figures should be specified in the corresponding caption.

In my opinion, Figure 3b is superfluous and redundant: all informations are already shown in 3A and 3C. Which is the purpose of highlighting the SCFAs concentrations only at 5 and 48 h?

Figure 4: Specifications of IG group abbreviations must be specified in the caption.

Lines 275-276: Is the decreasing tract (from 11 h to 24 h) of lactic acid concentration in L group statistically significant? Error bar of the point 24 h seems to suggest a constant trend from 11 h to 48h.

Please correct the name of y -axis in Figures 3A and 4 (Concentration instead of “concentraction”).

Paragraph 3.3

Please, pay attention in this section to the English grammar.

Figure 5: Abbreviations used for IG groups(BY, BG, Lit, L, R, PAC) should be specified in the corresponding caption.

Paragraph 3.4

The description of the fermentation degree results is very confusing and difficult to follow.

In particular, Figure 6 shows an increase of fermentation degree for BG after 24 h while in the text you stated a decreasing trend from time 11 h; moreover, you stated a decreasing trend for BY starting from 24 h but in the figure it starts at time 11h.

Figure 6: Abbreviations used for IG groups(BY, BG, Lit, L, R, PAC) should be specified in the corresponding caption.

Paragraph 3.6

Figure 10: It is necessary to specify all the group abbreviations. The legend above is superfluous.

Paragraph 3.7

The Spearman’s rank correlation method should be previously introduced in materials and methods.

Moreover, probably, a greater number of subjects from which collect faecal samples could increase the reliability of the results

Discussions

Lines 426-443: This part is badly written and chaotic. Please, review it.

In general, pay attention to the punctuation.

Discussion on pH and gas production during fermentation with different IGs are missing.

Lines 482-495: Discussion regarding the SCFAs is unsatisfactory, as it seems a repetition of the results previously shown.

Lines 500-503: these considerations are more suitable for the introduction section, as they don’t represent an effective discussion of results.

Lines 505-508: Why hasn’t there been a significant pH reduction during the fermentation, despite a certain production of SCFAs? Why has there been a gas accumulation during the process?

In general, when literature studies are mentioned, please discuss more about them and do possible comparisons.

 

Conclusions are missing and should be added to the paper for completeness.

Author Response

please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Thank you very much for the corrections. I my opinion, the authors have done nice work in improving their manuscript.

But, I still have a problem with the statement that in vitro fermentation studies of different glucans with different physicochemical properties are lacking....this is not quite true. Therefore, the aim should be reworded in some kind of "Until now, only a few studies are availabe, that investigate...." and more research is needed....

Please check the manuscript. There are still some mistakes in spelling.

 

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf