Characterization of an Acidogenic Bacterial Consortium as Probiotic and Its Effect on Rumen Fermentation In Vitro and In Vivo
, Diego Cardoso-Carmona 1, Laura González-Dávalos 2, Carlos Lozano-Flores 3, Allan Páez-Trejo 4, Armando Shimada 2 and Ofelia Mora 2,*Round 1
Reviewer 1 Report
This is very interesting research and very useful for production under no antibiotic conditions in feeds, but still need to be improved.
Firstly, please provide more background and include all relevant references in the introduction, meanwhile, please give us this research meaningful reason.
Secondly, in this paper, the methods is not adequately described, please supplementary more detail methods, will be repeated by others researchers.
Thirdly, the figure 2 and figure 3 is not clear, should be color, will be beautiful for readers. The figure 4 and figure 5 is also not clear, please improve the pixel of pictures.
To sum up, this research is very good, but still need to minor revised.
Further modifications and improvements are needed in English grammar and individual vocabulary usage.
Author Response
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Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript appears to report a study on evaluating efficacy and safety of PBC, however this does not come as clear message and readers will struggle to understand that. The topic is interesting, and such information is necessary when progressing to development of novel feed additives.
However, the report is poorly written, with so many flaws and missing information throughout. It does not follow the rules of scientific writing. All sections are defect, from Introduction to Discussion, and need significant revision and improvement before it can be revised again. The Introduction is quite general, without mentioning the issues that will be addressed in the study. and there is no indication on why authors choose to work on PBC, what are the research gaps and why the study was needed.
In Materials and Methods, the methodology is just thrown int here, without real explanation what it was used for. This needs to be clearly stated in the aims and objectives, as well as in some sort of ‘experimental overview’ in here.
What was the purpose of 2.3 and 2.4?
How many animals were in the in vivo trial? What was the purpose of collecting blood and rumen fluid? I don’t understand ‘to confirm bacteria genus…’
Results
3.1 – why is this reported, was this the aim of the study and what was the hypothesis?
I don’t understand the use of P. acidipropionici as a control. Why it was used in some tests (i.e. susceptibility to antibiotics), and not in the others (i.e. pH resistance)?
Table 2 and 3 can be joined in one
3.2.9- How was this assessed?
3.5 – follow scientific reporting rules on quoting tables and reporting results
First half of the Discussion is actually Introduction. The Discussion is not really a discussin of results of the current study, but rather an extensive literature review on some relevant and some irrelevant matters.
There are so many subheadings that should be combined under just two or three (efficacy, safety). There are also too many tables that should again be combined in just few ones. There is also a general sense that this manuscript (or parts) was/were written by the aid of AI. Authors may need to revisit these parts and rewrite in their own words.
Detailed comments
L15- probably can remove first sentence, as readers should know what probiotics are
L19 - optimal dose for what?
L19-L23 - needs to be rewritten to meet scientific standard reporting
L24; L38-L46 – where are the references for these statements
Section 2.1 – is the description really necessary if this is a commercial nutrient medium?
L79 – how did you prepare agar plates, how was the culture prepared?
L9 – what is the full name of P. acidipropionici?
L209 – the reference is wrong
L203 – how was anaerobiosis achieved and maintained?
L322 – must define word ‘antibiogram’
L327 – L329 – is this something that is reported earlier, as this is used as a control? Was this expected, or is this a new finding that needs to be reported here? Same goes for 3.2.2
L328-329 – this is Discussion, not Results
L366 – delete
L366-L367 – belongs to Materials and Methods
L404 – the PBC does not have capacity to report
L444 -L445 – belongs to Materials and Methods
L463-L465 - belongs to Materials and Methods
Often poorly written. Parts of MS look like to be written with aid of AI.
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 3 Report
A potential probiotic of propionogenic bacterial consortium was investigated by in vitro and in vivo methods. Some issues are needed to clarify before considering for publication.
1. The bacterial consortium should be introduced in detail even though it was described in other reference. How the bacterial community of such a mixed bacterial consortium maintained consistent in various batches of incubation and multiplication. Are all the microorganisms in the consortium propionogenic bacteria? Which is the core bacteria?
2. The experimental design should be more detailed to give the number of replication, which is the determinant of reasonable statistic analysis. As mentioned in the section of Statistical analysis Line 288-290 “Data were obtained in triplicate (antibiotic susceptibility, bile salts resistance, resistance to pH, temperature tolerance, and antagonistic activity) or duplicate (in vitro adhesion assay) and expressed as means ± standard deviation (SD).” So was the replication number of “Relative quantification of Lactobacillus and Propionibacterium” and “Disappearance of dry matter and VFA production in vitro”? How many jars and FN-57 bags were used in in vitro incubation? Line 223-224 “For the quantification of VFA (acetate, propionate, butyrate), two samples were collected per jar, in the three time points (0, 24 and 48 h)” How the corresponding data could be analyzed?
3. Line 290 read “...expressed as means ± standard deviation (SD);...” but SEM was used in all the Tables. For Table 4, the annotation read “Different superscripts in the same row indicate significant differences (P < 0.0001, n = 3)”, if the comparison of 24 h and 48 h was done? The same in Table 8. In Table 5, how could the SEM was larger than the largest mean? Why the statistic analysis of Table 6 could not be done? Why two corresponding SEM were used and no SEM was for total VFA in Table 9? The same in Table 10, the SEM of parameter DMI might be not true. In Table 12, no SEM was for total VFA, how the statistic comparison was done?
4. In Figure 1, what is the meaning of the formulas “GT=...”. Why the OD could not reach a plateau phase?
5. Why “Relative quantification of Lactobacillus and Propionibacterium” was done?
6. The Discussion is suggested to separate into several sections with indication of subtitles. The statistic indication P value is not needed in discussion and too much short paragraph should be avoided.
7. A brief conclusion was missed in the Abstract.
ok
Author Response
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Reviewer 4 Report
1. Please introduce why use propionogenic bacterial consortium as probiotics. two reference are suggested :
L265 performed? Table 8 and Table 9, why dry matter disappearance increased by 6%, but the total VFA increased by 73%?
L529-533, Where you want the PBC survive in?
Moderate editing of English language required
Author Response
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Round 2
Reviewer 3 Report
Introduction can be more brief in avoid too many short paragraphs.
Author Response
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Reviewer 4 Report
My two main concerns have not been answered
1. Please introduce why increase propionate. the two suggestedreferences have the answer :
Li, Z., . 2021. . Journal of Animal Science and Biotechnology 12:125. doi:10.1186/s40104-021-00645-4.
Shabat, 2016. ISME J. doi:10.1038/ismej.2016.62.
Table 8 and Table 9, The dry matter disappearance increased by only 6%, but the total VFA increased by 73%. Please verify and explain the conflicting data
Minor editing of English language required
Author Response
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Round 3
Reviewer 4 Report
Accept in present form
Author Response
The reviewer made no further corrections
