Mixing of Apples and Oranges in Milk Research: A Cohort Analysis of Non-Fermented Milk Intake and All-Cause Mortality
Round 1
Reviewer 1 Report
In my opinion the work is interesting, especially because of the large number of participants included in the study. The authors claim to give a different approach to data treatment. However, in the manuscript, in my opinion, this approach is not clear. The treatment of the data is not sufficiently well explained, making it difficult to understand the final conclusions.
Authors should better explain the statistical analysis performed. Likewise, they should explain more and better the results presented.
I also consider that the discussion is quite poor and could be improved.
Comments for author File: 
Comments.pdf
Author Response
AU: We thank the reviewer for the comments and understand that the presentation should be improved to ease understanding. Accordingly, we have now made attempts to better introduce the reader to our design concept (including a new figure) and to present the Results and Discussion parts in a more pedagogic way.
Reviewer 2 Report
Review of manuscript ID: nutrients-799449
Title: Mixing of apples and oranges in milk research: a cohort analysis of non-fermented milk intake and all-cause mortality
Authors: Karl Michaëlsson and Liisa Byberg
General comment:
The investigators present mortality rates by intake of non-fermented milk fat content type and examine the degree of bias when other fat content types of non-fermented milk are kept in the reference category. For this purpose, they used a longitudinal cohort consisting of 61,433 Swedish women who had been administered food frequency questionnaires in 1987-1990 and in 1997, and analyzed time to death.
The authors provided convincing epidemiological evidence that an increase of non-fermented milk intake, irrespective of fat content, is related to all-cause mortality in a dose-dependent pattern.
Material and Methods
There is recent evidence that thermal processing of milk plays an important role in milk´s biological signaling of milk exosomes and their microRNA cargo such as microRNA-21, which is an oncogenic microRNA with identical sequence homology between humans and bovine species (Bos taurus).
In pasteurized milk, exosomes stay functional and bioavailable (Benmoussa A, Lee CH, Laffont B, Savard P, Laugier J, Boilard E, Gilbert C, Fliss I, Provost P. Commercial dairy cow milk microRNAs resist digestion under simulated gastrointestinal tract conditions. J Nutr. 2016 Nov;146(11):2206-2215; Manca S, Upadhyaya B, Mutai E, Desaulniers AT, Cederberg RA, White BR2, Zempleni J. Milk exosomes are bioavailable and distinct microRNA cargos have unique tissue distribution patterns. Sci Rep. 2018 Jul 27;8(1):11321; Zempleni J, Sukreet S, Zhou F, Wu D, Mutai E. Milk-derived exosomes and metabolic regulation. Annu Rev Anim Biosci. 2019 Feb 15;7:245-262; Benmoussa A, Laugier J, Beauparlant CJ, Lambert M, Droit A, Provost P. Complexity of the microRNA transcriptome of cow milk and milk-derived extracellular vesicles isolated via differential ultracentrifugation. J Dairy Sci. 2020 Jan;103(1):16-29). Ultraheat treatment (UHT) of milk (Kirchner B, Pfaffl MW, Dumpler J, von Mutius E, Ege MJ. microRNA in native and processed cow's milk and its implication for the farm milk effect on asthma. J Allergy Clin Immunol. 2016 Jun;137(6):1893-1895.e13) and bacterial fermentation of milk (Yu S, Zhao Z, Sun L, Li P. Fermentation results in quantitative changes in milk-derived exosomes and different effects on cell growth and survival. J Agric Food Chem. 2017 Feb 15;65(6):1220-1228) inactivates milk exosomes and reduces their microRNA content such as microRNA-21.
It is thus of critical concern to obtain data on the percentage of pasteurized milk consumption related to UHT milk consumption in Sweden (for instance in form of annual sales data) during the milk exposure period (1987-1990) of this epidemiological investigation. This could be an important and overlooked pathological factor that may differ from country to country (Sweden versus USA).
Thus, the authors should provide data on the percentage of pasteurized versus UHT-milk consumed in Sweden during the data collection time of their cohort.
Discussion
This paper misses biochemical aspects of milk´s physiology. Milk is not food, but has been appreciated as a signaling system promoting mTORC1-dependent growth and anabolism that operates during a restricted lifetime of all mammals excluding Neolithic humans (Wiley AS. Cow milk consumption, insulin-like growth factor-I, and human biology: a life history approach. Am J Hum Biol. 2012 Mar-Apr;24(2):130-8; Melnik BC, John SM, Schmitz G. Milk is not just food but most likely a genetic transfection system activating mTORC1 signaling for postnatal growth. Nutr J. 2013 Jul 25;12:103; Melnik BC. Milk--A nutrient system of mammalian evolution promoting mTORC1-dependent translation. Int J Mol Sci. 2015 Jul 27;16(8):17048-87).
As the authors demonstrated that milk´s fat fraction is not the explanation for milk´s mortality-increasing effect, milk´s essential branched-chain amino acids as well as milk-derived exosomes – all components of the fat-free milk serum - are potential effectors of milk´s life shortening effects recently reviewed elsewhere (Melnik BC, Schmitz G. MicroRNAs: Milk's epigenetic regulators. Best Pract Res Clin Endocrinol Metab. 2017 Aug;31(4):427-442; Melnik BC, Schmitz G. Exosomes of pasteurized milk: potential pathogens of Western diseases. J Transl Med. 2019 Jan 3;17(1):3); Benmoussa A, Provost P. Milk MicroRNAs in health and disease. Comprehensive Reviews in Food Science and Food Safety. 2019; 18(3):703-722). The destruction or inactivation of milk exosomes by bacterial fermentation (yogurt processing) are a plausible explanation for less long-term toxicity of fermented milk products compared with unfermented milk products (Yu S, Zhao Z, Sun L, Li P. Fermentation results in quantitative changes in milk-derived exosomes and different effects on cell growth and survival. J Agric Food Chem. 2017 Feb 15;65(6):1220-1228).
This biochemical information, which has been presented by researchers in the fields of medicine, nutritional sciences and molecular biology should be included to improve the quality of the authors´ important epidemiological results that will stimulate future research activities in this progressing field of clinical nutrition.
References
Need further biochemical updates as outlined above.
Author Response
AU: Regarding consumption of UHT milk in Sweden. Sales of UHT milk in Sweden is very modest but the actual consumption UHT-milk versus pasteurized milk is for us unknown and has to our knowledge not been investigated. UHT-milk has mainly been introduced to the Swedish market during the last decade. We have now in the Methods mentioned that both consumption of raw milk and UHT milk is low in Sweden (line 76).
AU: Regarding biological mechanisms explaining why high milk consumption will lead to higher rates of death. Our intention with our study was not to present possible pathogenic behind the association between milk consumption and mortality. Nevertheless, as suggested by the reviewer, some of the conceivable mechanisms are now briefly mentioned in the second last paragraph of the Discussion.
