The Impact of A1- and A2 β-Casein on Health Outcomes: A Comprehensive Review of Evidence from Human Studies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper discusses the implications of beta casein variants on human healts after discussing the main evidence from preclinical models. 

The paper is overall well conceived and written. However, some improvements are required.

•  A figure or a graphical abstract would be required to facilitate comprehension. For examples, the molecular differences between A1 and A2 variants could be illustrated , as well as the pathogenetic mechanisms of A1 variants. 
• Please discuss where B-casein variants can be found. Are they present in different foods? Could they be generated after specific food processing?
• With regards to the methodology section, authors should clarify whether this article is a narrative or a systematic review. First case scenario, such depth in the description of the articles retrieval could be unnecessary; second case scenario, a peer review from a methodology expert could be required.
• Line 380-382: please remove quotation and rephrase.
• Individuating an association between A1 variants and cardiovascular or neurological affections could be misleading in terms of impact. Please provide a better definition of the magnitude of effect of this alleged risk factor. Especially for paragraph discussing such relationship, more highlight is needed on the limitations of the studies mentioned. In doing so, discuss which is the evidence and literature gap on this topic. 

Author Response

Comment 1:  A figure or a graphical abstract would be required to facilitate comprehension. For examples, the molecular differences between A1 and A2 variants could be illustrated , as well as the pathogenetic mechanisms of A1 variants.

Response 1: yes we agree, a graphical abstract has been included in the revised version.

Comment 2: Please discuss where B-casein variants can be found. Are they present in different foods? Could they be generated after specific food processing?

Response 2: The following text has been included in the introduction.“While food processing does not change one variant into another, certain processing methods can influence the release of these peptides into the processed food or during digestion in the intestine. For example, the curdling and aging processes in cheesemaking can alter the structure of casein. Cross-linking and other modifications cause individual casein molecules to no longer be in equilibrium with the micelle. Transport of intact molecules through the gut may therefore be prevented or hindered [7]. However, in Zinßius et al. study, BCM-7 concentrations were higher in the A1A1 cheeses after pressing and during ripening [8]. Standard processes applied to milk, such as heat treatment and homogenization, although they can affect the overall structure of milk proteins, such as β-lactoglobulin denaturation and complex formation, do not appear to influence BCM-7 release [9].”

Comment 3: With regards to the methodology section, authors should clarify whether this article is a narrative or a systematic review. First case scenario, such depth in the description of the articles retrieval could be unnecessary; second case scenario, a peer review from a methodology expert could be required.

Response 3: The following text has been included in the methodology section. “An exploratory review was carried out to gather and evaluate the breadth of existing evidence related to the topic, aiming to map out the current state of research.”

Comment 4: Line 380-382: please remove quotation and rephrase.

Response 4: The following text has been included. “The study concluded that no cardiovascular disadvantages were found associated with the consumption of A1 casein compared to A2 casein."

Comment 5: Individuating an association between A1 variants and cardiovascular or neurological affections could be misleading in terms of impact. Please provide a better definition of the magnitude of effect of this alleged risk factor. Especially for paragraph discussing such relationship, more highlight is needed on the limitations of the studies mentioned. In doing so, discuss which is the evidence and literature gap on this topic.

Response 5: The following paragraph notes the limitations of studies linking A1 variants to cardiovascular risk:

“In summary, although some epidemiological studies have reported a positive correlation between the consumption of β-casein A1 and an increased risk of cardiovascular diseases, these findings are based on population-level analyses that do not allow for establishing a direct causal relationship. In contrast, intervention trials conducted in adults at elevated cardiovascular risk have not shown significant differences between the consumption of β-casein A1 and A2 in terms of cardiovascular outcomes. This discrepancy could be explained by confounding factors present in observational studies, such as variations in dietary patterns, genetic background, lifestyle factors, or differences in the characteristics of the studied populations. These elements, often not adequately controlled for in epidemiological research, may influence the observed associations. Therefore, the lack of effect in intervention trials suggests that the associations reported in population studies may not be causal, highlighting the need for further investigation into the underlying mechanisms through more rigorous and well-designed studies.”

Regarding possible neurological alterations, the paragraphs included in the article make it clear that the results are not conclusive and further research is needed to clarify the role of BCM-7 in the pathogenesis of these types of disorders:

“In summary, A1 β-casein and its digestion product, βCM-7, can have been implicated in the pathogenesis of neurological disorders such as autism and schizophrenia, as well as in psychomotor development. However, further research is needed to confirm these effects and elucidate the underlying mechanisms.”

Comment on Quality of English Language: The English could be improved to more clearly express the research. Response: Authors are willing to review their manuscript using MDPI services after reviewers decide if they agree with the applied changes of this revised version. 

Reviewer 2 Report

Comments and Suggestions for Authors

This paper noted “The Impact of A1- and A2 Beta-Casein on Health Outcomes: A 2 Comprehensive Review of Evidence from Human Studies”, which provides a systematic review of the health impacts of A1/A2 β-casein. The subject is innovative and holds significant public health significance. However, there are some grammatical errors and inaccurate expressions of scientific terms. The detailed comments are listed as following:

1. In line 39: Please remove the extraneous period/full stop after “milk”;
2. The format of "β" is not uniform throughout the text. It is suggested to unify the terms and format, and change "beta" and "ß" to "β".
3. In line 47, "ß-beta-casomorphin-7" is suggested to be changed to "β -casomorphin-7"; In line 111-120, it is recommended to adopt a uniform writing format.
4. Only one RCT (Kirk 2017) supported the benefits of A2 milk for muscle recovery, and did not prove the direct negative impact of A1. The conclusion needs to be expressed with caution.
5. Regarding the association between A1 and cardiovascular diseases, epidemiological studies have shown a positive correlation (such as Line 133-134), but no differences were found in intervention trials (such as Line 125-126). This article only describes the phenomena but does not analyze the causes (such as confounding factors, differences in sample characteristics).
6. Abstract and introduction propose to explore the impact of A1/A2 β -casein on health. However, the full text involves multiple fields such as gastrointestinal function, cardiovascular system, neurocognition, and immunity, lacking a core research thread. It is suggested to take "the gut-neural axis effect of BCM-7" as the main line, integrate the evidence in the fields of digestion, immunity and neurology, and avoid the dispersion of content.

Author Response

Comment 1. In line 39: Please remove the extraneous period/full stop after “milk”

Response 1: The text has been corrected.

Comment 2: The format of "β" is not uniform throughout the text. It is suggested to unify the terms and format, and change "beta" and "ß" to "β".

Response2 : The text has been corrected.

Comment 3: In line 47, "ß-beta-casomorphin-7" is suggested to be changed to "β -casomorphin-7"; In line 111-120, it is recommended to adopt a uniform writing format.

Response3 : The text has been corrected.

Comment 4: Only one RCT (Kirk 2017) supported the benefits of A2 milk for muscle recovery, and did not prove the direct negative impact of A1. The conclusion needs to be expressed with caution.

Response4 : Taking into account the recommendation, this text has been included. “However, conclusions regarding the benefits of A2 milk for muscle recovery in athletes should be drawn with caution, as current evidence is limited and does not conclusively demonstrate a direct negative effect of A1 β-casein on recovery. Further controlled and well-designed studies are needed to clarify whether there are significant differences between A1 and A2 milk in this context and to better understand their potential role in athletic performance and post-exercise recovery.”

Comment 5: Regarding the association between A1 and cardiovascular diseases, epidemiological studies have shown a positive correlation (such as Line 133-134), but no differences were found in intervention trials (such as Line 125-126). This article only describes the phenomena but does not analyze the causes (such as confounding factors, differences in sample characteristics).

Response 5: Taking into account the recommendation, this text has been included:

“In summary, although some epidemiological studies have reported a positive correlation between the consumption of β-casein A1 and an increased risk of cardiovascular diseases, these findings are based on population-level analyses that do not allow for establishing a direct causal relationship. In contrast, intervention trials conducted in adults at elevated cardiovascular risk have not shown significant differences between the consumption of β-casein A1 and A2 in terms of cardiovascular outcomes. This discrepancy could be explained by confounding factors present in observational studies, such as variations in dietary patterns, genetic background, lifestyle factors, or differences in the characteristics of the studied populations. These elements, often not adequately controlled for in epidemiological research, may influence the observed associations. Therefore, the lack of effect in intervention trials suggests that the associations reported in population studies may not be causal, highlighting the need for further investigation into the underlying mechanisms through more rigorous and well-designed studies.”

Comment 6: Abstract and introduction propose to explore the impact of A1/A2 β -casein on health. However, the full text involves multiple fields such as gastrointestinal function, cardiovascular system, neurocognition, and immunity, lacking a core research thread. It is suggested to take "the gut-neural axis effect of BCM-7" as the main line, integrate the evidence in the fields of digestion, immunity and neurology, and avoid the dispersion of content.

Response 6: In line with the reviewer’s recommendation, this review emphasizes the gut-neural axis effects of β-casomorphin-7 (BCM-7) as a central research thread. By integrating evidence from gastrointestinal, immunological, and neurological studies, the manuscript aims to provide a cohesive perspective on how BCM-7 derived from A1 β-casein may influence health through interconnected digestive and neural pathways. This approach avoids dispersion and highlights the multifaceted impact of BCM-7 on human physiology.

Taking into account the recommendation, the abstract was modified as follows:

“The digestion of A1 β-casein present in conventional milk releases β-casomorphin-7 (BCM-7), a bioactive peptide with potential implications for gastrointestinal and neurological health. A scoping review was performed to respond to the following research question: What are the health effects of consuming milk containing the A1 β-casein variant compared to the exclusive consumption of A2 variant in humans? The evidence collected in this review of human studies with different populations (i.e. children, middle-aged adults, athletes) suggests that consumption of milk containing A1 β-casein may negatively influence gut health by altering microbial composition, reducing intestinal motility, and increasing colonic fermentation, leading to elevated gas production and altered short-chain fatty acid (SCFA) profiles. The release of BCM-7 upon digestion can also compromise intestinal barrier integrity, which may exacerbate symptoms of lactose intolerance, irritable bowel syndrome (IBS), or other allergy-related sensitivities. Its ability to cross the blood-brain barrier raises concerns about potential neurological effects. In contrast, milk containing exclusively A2 β-casein is associated with improved gastrointestinal outcomes, including enhanced abundance of beneficial bacteria such as Bifidobacterium spp. and reduced inflammatory markers."

Also, in the introduction we added the text:

“This imbalance may compromise the integrity of the intestinal barrier and interfere with bile acid metabolism, leading to additional effects. Recent research has increasingly focused on the gut–brain axis as a key pathway mediating the effects of dietary peptides such as βCM-7”

And:

“This review emphasizes the gut-neural axis effects of βCM-7 as a central research thread. By integrating evidence from gastrointestinal, immunological, and neurological studies, the manuscript aims to provide a cohesive perspective on how βCM-7 derived from A1 β-casein may influence health through interconnected digestive and neural pathways.”

 

Comment on Quality of English Language: The English could be improved to more clearly express the research.

Response: Authors are willing to review their manuscript using MDPI services after reviewers decide if they agree with the applied changes of this revised version. 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript addresses an important and timely topic with clear relevance to public health, nutrition science, and consumer interest. The potential differential health effects of A1 versus A2 β-casein in milk have gained increasing attention in recent years, and a comprehensive scoping review focused on human studies is both valuable and much needed.

The structure of the paper is clear and well-organized. The objective is explicitly stated and the methodology is appropriately detailed. The authors describe the literature search and selection process transparently, which adds credibility to the findings. The inclusion of recent references throughout the manuscript strengthens the relevance and currency of the review.

Overall, the manuscript is well-written, accessible, and thorough. The findings are clearly presented and logically interpreted. However, I have a few suggestions for improvement:

1. Graphical presentation: While the paper includes a detailed summary table, I recommend adding one or more graphical elements (e.g., flowcharts, bar charts, or summary diagrams) to visually present key findings. This would greatly enhance the reader’s understanding and improve the clarity of comparisons across studies.

2. Quantitative data in the conclusions: The conclusion section is appropriately comprehensive and well-phrased. However, it would benefit from the inclusion of numerical data, such as the number of studies included in each comparison or the number of participants across studies. This would give the reader a better sense of the weight of evidence behind each conclusion.

These are minor suggestions aimed at further strengthening an already well-prepared manuscript. I believe the work makes a meaningful contribution to the field and can be recommended for publication after minor revisions.

Author Response

Comment 1: Graphical presentation. While the paper includes a detailed summary table, I recommend adding one or more graphical elements (e.g., flowcharts, bar charts, or summary diagrams) to visually present key findings. This would greatly enhance the reader’s understanding and improve the clarity of comparisons across studies.

Response 1: We agree, thanks for the comment. A graphical abstract has been included in the revised version of our manuscript.

Comment 2: Quantitative data in the conclusions. The conclusion section is appropriately comprehensive and well-phrased. However, it would benefit from the inclusion of numerical data, such as the number of studies included in each comparison or the number of participants across studies. This would give the reader a better sense of the weight of evidence behind each conclusion.

Response 2: the Conclusions have been modified according to reviewer suggestion, and the following text has been included.

“Clinical studies (30 of which 24 were interventional clinical trials, 3 epidemiological studies, and 3 non-interventional studies, including 12 studies with children and/or adolescents and multiple trials with adults) have consistently shown that milk containing only A2 β-casein is better tolerated than milk with both A1 and A2 β-caseins. Participants consuming A2 milk reported fewer gastrointestinal symptoms such as bloating, abdominal pain, and fecal urgency. Additionally, inflammatory biomarkers were lower in those consuming A2 milk compared to conventional milk.”

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Significant improvements have been made aS asked. The insertion of a graphical abstract or a figure would be auspicabile

Reviewer 2 Report

Comments and Suggestions for Authors

The authors responded the comments well. But the format of references should be uniform, such as whether the year number should be bold font?