UHPLC-HRMS/MS Probes Alcohol-positive Milk

Alcohol-positive milk (APM) is a common quality problem in dairy farming. The name comes from a standard test: when this milk is mixed with alcohol, it curdles, a sign that it may not hold up well during processing. This costs dairy producers money. In a recent study, researchers set out to find the chemical fingerprint behind the problem in Holstein cows. They compared milk from 10 cows producing normal milk with milk from 10 cows producing alcohol-positive milk. They also followed 7 of the affected cows that recovered on their own, sampling their milk again once it tested normal. To analyze the milk, the team used ultrahigh-performance liquid chromatography–high-resolution tandem mass spectrometry (UHPLC-HRMS/MS). A paper based on their research was published in the Journal of Dairy Science.¹

What is Alcohol-positive Milk (APL), and Why Does It Happen?

APM is a common problem with raw milk on large dairy farms around the world. It gets its name from a simple test: when the milk is mixed with an equal amount of alcohol, it forms clumps. Milk like this is less stable, which lowers both the quality and the amount of dairy products that can be made from it. That leads to ongoing, large financial losses for everyone along the supply chain, from farmers to processing companies. APM can also be an early warning sign that a cow has an underlying health problem or that her body chemistry is out of balance. Because the quality of raw milk is the foundation of safe dairy products, it matters both for consumers' health and for the long-term future of the dairy industry worldwide.^2-4

At its root, APM happens when milk loses its ability to stay smoothly mixed. Raw milk is made up of tiny droplets of fat spread evenly through a watery liquid. What keeps everything stable is a thin protective coating around each fat droplet, called the milk fat globule membrane (MFGM).⁵If anything upsets this balance, the milk can lose its smooth, even texture, causing its main proteins, called casein, to clump together and separate out.⁶

“Disruption of this interfacial hydrophilic-hydrophobic homeostasis,” write the authors of the paper,¹ “may lead to exposure of hydrophobic regions on fat globule surfaces, facilitate non-specific hydrophobic interactions between particles, and may ultimately result in the complete collapse of milk colloidal stability. The primary objective of this study was to elucidate the pathogenic mechanisms underlying APM onset and spontaneous remission in dairy cows.”

What Chemical Changes Did the Researchers Find in Alcohol-positive Milk?

The researchers report that alcohol-positive milk showed a two-sided imbalance in its chemistry. On one side, the processes cells used to break down sugar for energy and to build important molecules were slowed down. Levels of certain water-friendly building blocks also dropped. Cells need these to make two key types of fat that form cell membranes, so production of those fats suffered. As a result, these protective, structural fats and the ingredients used to make them were all lower than normal.¹

On the other side, certain long, water-repelling fatty acids built up to unusually high levels. Fats in cell membranes were also being broken down faster than usual, which caused one of their breakdown products to pile up.¹

In short, the hallmark of alcohol-positive milk is a double imbalance: helpful, water-friendly molecules go down, while harmful, water-repelling ones go up.¹

“This imbalance,” state the authors of the paper,¹ “disrupts the intrinsic hydrophilic-hydrophobic homeostasis of milk and drives APM occurrence.”

“Our findings,” conclude the authors,¹ “provide novel insights into in APM pathogenesis, and provide theoretical support for early warning and targeted prevention of APM in intensive dairy farming.”

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References

1. Hu, R.; Luo, D.; Zhang, Z. et al. Hydrophilic-hydrophobic Imbalance is Associated with Alcohol-positive Milk in Holstein cows: An Untargeted Metabolomics Analysis. J Dairy Sci. 2026, S0022-0302 (26), 03331-X. DOI: 10.3168/jds.2026-28994
2. Goetz, C.; Sanschagrin, L.; Jubinville, E. et al. Biofilm Formation in Dairy: A Food Safety Concern-Recent Progress in Antibiofilm Strategies in the Dairy Industry. J Dairy Sci. 2025, 108 (8), 8157-8175. DOI: 10.3168/jds.2024-25554
3. Lin, L.; Ma, H.; Zhang, J. et al. Lignocellulolytic Microbiomes Orchestrating Degradation Cascades in the Rumen of Dairy Cattle and Their Diet-influenced Key Degradation Phases.Anim. Adv. 2024, 1. DOI: 10.48130/animadv-0024-0002
4. Alnadari, F.; Ibeogu, I.H.; Shoura, H.E. et al. Immunomodulatory Potential of Bovine Colostrum: A Holistic Perspective on Health, Disease Resistance, and Aging.Anim. Adv. 2025, 2. DOI: 10.48130/animadv-0025-0001
5. Yao, D.; Sun, L. C.; Zhang, L. J. et al. Emulsion Structural Remodeling in Milk and Its Gelling Products: A Review. Gels 2024, 10 (10), 671. DOI: 10.3390/gels10100671
6. Weng, J.; Lin, R.; Jiang, C. et al. O/W Emulsion Stabilized by Bovine Milk Phospholipid-Protein Nanoemulsions: Preparation, Stability, and In Vitro Digestion. J Agric Food Chem. 2021, 69 (17), 5003-5012. DOI:10.1021/acs.jafc.0c05617