Novel Insight into the Composition Differences Between Buffalo and Holstein Milk and Potential Anti-Inflammation and Antioxidant Effect on Caco-2 Cells

Abstract

Milk is one of the most common sources of nutrients in humans, however, the composition and healthy value of the milk derived from different animals are very different. Here, we systemically compared the protein and lipid profiles and evaluated the anti-inflammation and antioxidant effect of buffalo and Holstein-derived milk on Caco-2 cells. Results showed that 906 proteins and 1899 lipids were identified in the buffalo milk and Holstein milk samples including 161 significantly different proteins (DEPs) and 49 significantly different lipids. The DEPs were mainly enriched in defense response-related terms, while the differential lipids were mainly included in fat digestion and absorption and cholesterol metabolism pathways. In addition, the Caco-2 cells co-cultured with buffalo and Holstein milk components showed significant benefits in being resistant to LPS-induced inflammation stress and H₂O₂-induced ROS stress. The qRT-PCR and ELISA results showed that the expression of TNF-α, IL-1β, and IL-6 was significantly lower (p < 0.05) in the cells co-cultured with milk components. Further analysis showed that, after H₂O₂ treatment, the expression of keap1 and Nrf-2 in the Caco-2 cells co-cultured with milk components was significantly lower (p < 0.05). In addition, being co-cultured with milk components significantly decreased the SOD, MDA, CAT, and GSH-Px content (p < 0.05) in the Caco-2 cells induced by H₂O₂. This study provides a novel insight into the differences in proteins and lipids between buffalo milk and Holstein milk, and a reference understanding of the anti-inflammation and antioxidant effect of the consumption of milk on the intestines.

Keywords: Caco-2 cell; anti-inflammation; antioxidant; lipidome; milk; proteome.

Figure 1

Proteome analysis of the buffalo and Holstein milk. (A) Principal coordinates (PCoA) analysis of data from different milk samples. (B) Identification of proteins in buffalo milk and Holstein milk. (C) Identification of differentially expressed proteins (DEPs) in the buffalo milk and Holstein milk. (D) Subcellular localization analysis of the DEPs. (E) GO analysis of the DEPs.

Figure 2

Lipidome analysis of the buffalo and Holstein milk. (A) Correlation analysis of QC samples. (B) Analysis of total lipid in the milk samples. (C) Statistics of lipid subclasses and molecular numbers in the milk samples. (D) The proportion of lipid subclasses in different milk samples. (E) Differential analysis of the lipids. (F) Pathway enrichment analysis of the differential lipids.

Figure 3

Anti-inflammatory effect of the buffalo and Holstein milk components on Caco-2 cells. (A) Caco-2 cells co-cultured with buffalo and Holstein milk components. (B) Caco-2 cells treated with LPS (after co-culturing with buffalo and Holstein milk components).

Figure 4

qRT-PCR detection of TNF-α (A), IL-1β (B), and IL-6 (C) in the Caco-2 cells treated with LPS. ELISA detection of TNF-α (D), IL-1β (E), and IL-6 (F) in the Caco-2 cells treated with LPS. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Antioxidant effect of buffalo and Holstein milk components on Caco-2 cells. (A) Caco-2 cells were treated with H₂O₂ (after co-cultured with buffalo and Holstein milk components). (B) qRT-PCR detection of Nrf-2 in Caco-2 cells treated with H₂O₂. (C) qRT-PCR detection of keap1 in Caco-2 cells treated with H₂O₂. (D) Flow cytometry detection of ROS in Caco-2 cells treated with H₂O₂. (E) SOD content in Caco-2 cells treated with H₂O₂. (F) MDA content in Caco-2 cells treated with H₂O₂. (G) CAT content in Caco-2 cells treated with H₂O₂. (H) GSH-Px content in Caco-2 cells treated with H₂O₂. * p < 0.05, ** p < 0.01, *** p < 0.001.