Ketogenic Effects of Multiple Doses of a Medium Chain Triglycerides Enriched Ketogenic Formula in Healthy Men under the Ketogenic Diet: A Randomized, Double-Blinded, Placebo-Controlled Study

Round 1

Reviewer 1 Report

I carefully read the paper entitled "Changes in gut flora and ketogenic effects of multiple doses of a medium chain........." by Kentaro Nakamura et al.
The subject matter is interesting because it attempts to establish the positive qualities, at least potential, of using the so-called ketogenic diet and ketogenic supplementation (KF) in healthy adult men recruited in a double-blinded, placebo-controlled project. 
In addition, the aim of the project was even more ambitious as it sought to find evidence of the validity of a similar diet (ketogenic) supplemented with KF, also as a potential adjuvant therapy in cases of serious diseases such as cancer.
The effort made by the authors, including the planning, is quite substantial because many measurements were carried out on the three randomised groups over the 4-5 days of the experimental trial.
Unfortunately, the data obtained, which concerned both plasma and functional indices, also referring to any harmfulness of the trial carried out, were not exciting. In fact, the significant differences were mainly referred to the plasma values of lipid metabolism, derived, in my opinion, from the excess of lipid supplementation carried out, have a purely indicative value. This is also evident from the measurements made on the microbiota analysed.
In reality, the critical points of the project are essentially three:
1- the limited size of the sample analysed, which starts from 20 recruited subjects and then drops to 16 in the formation of the three experimental groups. Under these conditions, perhaps only certain plasma values of lipid metabolism can be significant, while others are even unlikely, such as the increase in muscle strength (even if measured in different muscles) after supplementation with FK.
2- the duration of the trial analysed. Four days of ketogenic "therapy" cannot define an effect that may only be temporary or hide events that may occur after constant supplementation over a longer period. It would also be necessary to follow up the parameters measured in order to check the long-term effectiveness after stopping treatment.
3- In my opinion, it would be very important to carry out the same measurements in elderly subjects, especially taking into account the geriatric syndromes which, as age progresses, increase their appearance in an increasingly elderly but often more fragile population. 
However, I must acknowledge that the authors have anticipated in the Discussion some of the critical reflections (point one) that I have just indicated.
To summarise, the work carried out is, in my opinion, only preliminary but it deserves attention because the field of investigation has an important value, above all in order to mitigate with a correct lifestyle the ever increasing social cost in those countries where, fortunately, life expectancy is increasingly longer.
As a further piece of advice to the authors I would ask them to tone down both in the Introduction and in the Discussion the emphasis on FK supplementation as an anti-cancer therapy which, in the state of available data, does not seem to be very promising. 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The present study aimed to look at the effect of ketogenic diets to overall being. For this, a double-blind, randomized, parallel-group comparison was performed. Blood, urine and fecal sampling were performed, as well as breath analyses. The groups included a keto diet + placebo, keto diet + ketogenic formula and a control. Results showed a number of changes in metabolites and other components in the groups under the keto diets, with more profound effects in certain measurements in the keto diet + ketogenic formula. Some of these included  total ketones, acetoacetic acid and beta-hydrozybutyrate. At the microbiome level, Firmicutes seemed to decrease in the keto diet + ketogenic formula group. At the genus level, Clostridium, and Fusobacterium seem to be more represented in both keto diet groups, whereas Veillonella seemed to be more represented in the control group.

Major comments:

Overall, the study is very well organized and controlled, and the groups are easy to interpret. The parameters and measurements taken provide a very good overview to what is happening during keto diet. The only thing I was expecting to see more was a more in depth analysis of the microbiota. Specifically, how does the taxa correlate with the parameters collected? For instance, the study below showed a correlation between certain taxa and blood parameters

Lee SH, You HS, Kang HG, Kang SS, Hyun SH. Association between Altered Blood Parameters and Gut Microbiota after Synbiotic Intake in Healthy, Elderly Korean Women. Nutrients. 2020 Oct 12;12(10):3112. doi: 10.3390/nu12103112. PMID: 33053824; PMCID: PMC7650560.

Minor comments:

Line 131: Can the authors clarify if this is cow’s milk?

Line 208: Can the authors clarify if ‘normal’ temperature is room temperature?

Line 211: Why was the V1-V2 region chosen over for example, V4 or V1-V3?

Check throughout the text if taxa at the phylum level should be italicized?

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

I reviewed the authors' response and the new version of the manuscript with the minor changes made to the previous version.
I would have preferred a more thorough analysis, especially in the discussion section, however, considering the potential and importance of the topic, I think that this paper can be accepted for publication in Nutrients.

Reviewer 2 Report

While the authors seemed to have addressed the comments, it would have still be important to address a correlation between specific measurements and the microbiome between the groups.