Comparative analysis of proteomics and transcriptomics reveals novel mechanism underlying the antibacterial activity and immune-enhancing properties of horse milk.
Abstract: Horse milk is a highly valuable organic food that is a promising alternative to cow milk, exhibiting plenty of healthy and immune benefits to human. However, identification of proteins associated human wellness and underlying molecular mechanism in horse milk remain unclear. Unassigned: Label-free mass spectrometry-based protein quantification technology was employed to investigate protein composition of animal milk, including cow, goat, camel and horse milk. Prokaryotic expression and disk diffusion assay were applied to acquire and evaluate antimicrobial activity of candidate proteins. RAW264.7 macrophage model cell line was used to validate effect of proteins on cytotoxicity, apoptosis and immune induction. ROS probe detected cell ROS change and RT-qPCR verified expression of immune response genes induced by proteins. Microscopy was used to observe the effects of protein on the morphological characteristics of bacteria, further transcriptome analysis was performed to investigate transcriptional changes of bacteria induced by candidate proteins. Unassigned: A total of 1,335 proteins was identified in cow, goat, camel and horse milk. GO enrichment analysis showed that the proteins related to protein degradation were highly expressed in horse milk compared to other three types of milk, contributing to easier assimilation and palatability. KEGG analysis showed that horse milk contained abundant antimicrobial associated proteins relevant to pathogenic bacterial resistance, leading to the decreased risk of pathogenic diseases. A higher accumulation of proteins associated with caffeine metabolism, amino acid biosynthesis, and glycolysis/gluconeogenesis in horse milk contributes to its distinctive flavor. Notably, highly expressed proteins in horse milk were closely linked to immune signaling pathways, functioning as immune modulators. Importantly, we identified four highly expressed antimicrobial associated proteins in horse milk including LPO, B2M, CD14 and PGL, among them, PGL functioned dually by antibacterial activity and immune activation. Further transcriptome analysis demonstrated that PGL exerted significant transcriptional changes to bacteria. Enrichment analysis showed PGL could inhibit growth of and by repressing the biosynthesis of secondary metabolites. Unassigned: Comparative proteomics revealed immune enhancement and nutrient composition of horse milk compared to cow, goat and camel milk. Identification of PGL showed antibacterial activity and potential medicinal value.
Copyright © 2025 Chen, Gulbahar, Ding, Nie and Gao.
Publication Date: 2025-03-11 PubMed ID: 40135224PubMed Central: PMC11932903DOI: 10.3389/fnut.2025.1512669Google Scholar: Lookup
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Summary
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The research explores the health benefits and immune-boosting properties of horse milk, particularly its protein contents, compared to other animal milks like cow, goat and camel milk. A crucial finding is the identification and analysis of antimicrobial proteins in horse milk, especially one named PGL, which shows both antibacterial activity and immune activation potential.
Study Methods and Analysis
- The researchers undertook a comparative study of protein composition in cow, goat, camel and horse milk, using label-free mass spectrometry-based protein quantification technology. This method yields detailed insights into the kinds and quantities of proteins present.
- Prokaryotic expression and disk diffusion assays were used to acquire and evaluate the antimicrobial activity of potential immuno-enhancing proteins found in the milk.
- The impact of proteins on cytotoxicity, apoptosis and immune induction was validated using a RAW264.7 macrophage model cell line.
- To detect changes in cell ROS (Reactive Oxygen Species), a ROS probe was employed. RT-qPCR (Real-Time Quantitative Polymerase Chain Reaction) was used to verify the expression of immune response genes prompted by these proteins.
- Further explorations involved using microscopy to observe the effects of proteins on the morphological characteristics of bacteria, and transcriptome analysis to investigate transcriptional changes of bacteria induced by proteins of interest.
Key Results and Findings
- A total of 1,335 proteins were found in samples of cow, goat, camel and horse milk. The researchers also noted that proteins related to protein degradation were more highly expressed in horse milk than in the other three types of milk, suggesting easier assimilation and palatability for consumers.
- KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis highlighted that horse milk contained a large number of antimicrobial proteins, contributing to a reduced risk of pathogenic diseases for consumers and revealing a distinct advantage over the other types of milk analysed.
- A higher level of protein accumulation related to caffeine metabolism, amino acid biosynthesis, and glycolysis/gluconeogenesis was discovered in horse milk, contributing to its unique flavor profile.
- Highly expressed proteins identified in horse milk were particularly linked to immune signaling pathways, making them plausible immune modulators.
- Notably, the researchers identified four highly expressed antimicrobial proteins in horse milk, namely LPO, B2M, CD14 and PGL. Particularly, PGL stood out due to its dual functionality – offering both antibacterial activity and immune activation.
- Transcriptome analysis and enrichment analysis also demonstrated that the protein PGL could inhibit the growth of particular bacteria by suppressing their biosynthesis of secondary metabolites.
Research Implications and Conclusion
- The findings of this study illuminate the unique health benefits and immune-enhancing properties of horse milk when compared to cow, goat and camel milk.
- The research also reveals the medicinal value of antimicrobial proteins present in horse milk that can inhibit bacterial growth and boost immunity, namely the protein PGL.
- The researchers concluded that these results demonstrate the promising potential of horse milk as an alternative to other types of milk, and potentially as a source of medicinally valuable proteins.
Cite This Article
APA
Chen X, Gulbahar K, Ding H, Nie C, Gao X.
(2025).
Comparative analysis of proteomics and transcriptomics reveals novel mechanism underlying the antibacterial activity and immune-enhancing properties of horse milk.
Front Nutr, 12, 1512669.
https://doi.org/10.3389/fnut.2025.1512669 Publication
Researcher Affiliations
- School of Pharmacy, Xinjiang Medical University, Xinjiang, China.
- School of Pharmacy, Xinjiang Medical University, Xinjiang, China.
- Engineering Research Center of Xinjiang and Central Asian Medicine Resources, Ministry of Education, Xinjiang Medical University, Xinjiang, China.
- School of Pharmacy, Xinjiang Medical University, Xinjiang, China.
- School of Pharmacy, Xinjiang Medical University, Xinjiang, China.
- Engineering Research Center of Xinjiang and Central Asian Medicine Resources, Ministry of Education, Xinjiang Medical University, Xinjiang, China.
- School of Pharmacy, Xinjiang Medical University, Xinjiang, China.
- Engineering Research Center of Xinjiang and Central Asian Medicine Resources, Ministry of Education, Xinjiang Medical University, Xinjiang, China.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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