Mulberry Leaf Dietary Supplementation Can Improve the Lipo-Nutritional Quality of Pork and Regulate Gut Microbiota in Pigs: A Comprehensive Multi-Omics Analysis

Abstract

Mulberry leaves, a common traditional Chinese medicine, represent a potential nutritional strategy to improve the fat profile, also known as the lipo-nutrition, of pork. However, the effects of mulberry leaves on pork lipo-nutrition and the microorganisms and metabolites in the porcine gut remain unclear. In this study, multi-omics analysis was employed in a Yuxi black pig animal model to explore the possible regulatory mechanism of mulberry leaves on pork quality. Sixty Yuxi black pigs were divided into two groups: the control group (n = 15) was fed a standard diet, and the experimental group (n = 45) was fed a diet supplemented with 8% mulberry leaves. Experiments were performed in three replicates (n = 15 per replicate); the two diets were ensured to be nutritionally balanced, and the feeding period was 120 days. The results showed that pigs receiving the diet supplemented with mulberry leaves had significantly reduced backfat thickness (p < 0.05) and increased intramuscular fat (IMF) content (p < 0.05) compared with pigs receiving the standard diet. Lipidomics analysis showed that mulberry leaves improved the lipid profile composition and increased the proportion of triglycerides (TGs). Interestingly, the IMF content was positively correlated with acyl C18:2 and negatively correlated with C18:1 of differential TGs. In addition, the cecal microbiological analysis showed that mulberry leaves could increase the abundance of bacteria such as UCG-005, Muribaculaceae_norank, Prevotellaceae_NK3B31_group, and Limosilactobacillus. Simultaneously, the relative levels of L-tyrosine-ethyl ester, oleic acid methyl ester, 21-deoxycortisol, N-acetyldihydrosphingosine, and mulberrin were increased. Furthermore, we found that mulberry leaf supplementation significantly increased the mRNA expression of lipoprotein lipase, fatty acid-binding protein 4, and peroxisome proliferators-activated receptor γ in muscle (p < 0.01). Mulberry leaf supplementation significantly increased the mRNA expression of diacylglycerol acyltransferase 1 (p < 0.05) while significantly decreasing the expression of acetyl CoA carboxylase in backfat (p < 0.05). Furthermore, mulberry leaf supplementation significantly upregulated the mRNA expression of hormone-sensitive triglyceride lipase and peroxisome proliferator-activated receptor α (p < 0.05) in backfat. In addition, mulberry leaf supplementation led to increased serum leptin and adiponectin (p < 0.01). Collectively, this omic profile is consistent with an increased ratio of IMF to backfat in the pig model.

Keywords: intramuscular fat; lipid profile; lipidomic; mulberry leaves; triglyceride.

Figure 1

HE staining, Oil Red O staining, and BODIPY staining. (A) Oil Red O staining (scale bar: 1000 μm), BODIPY staining (scale bar: 1000 μm) of muscle tissue, and H&E staining (scale bar: 100 μm) of backfat. (B) Determination of lipid droplet area in the muscle. (C) Determination of the diameter of adipocytes in backfat. ** p < 0.01.

Figure 2

Effect of mulberry leaves on muscle lipid profile composition. (A) PCA. (B,C) Proportions of different lipid species in the CON and ML groups. (D) Abundances of different lipid species in the CON and ML groups. (E–I) Proportions of glycerolipids (E), glycerophospholipids (F), sphingolipids (G), fatty acids (H), and saccharolipids (I) in the CON and ML groups. (J–N) Abundance of glycerolipids (J), glycerophospholipids (K), sphingolipids (L), fatty acids (M), and saccharolipids (N) in the CON and ML groups. * p < 0.05 and ** p < 0.01.

Figure 3

Effects of mulberry leaves on muscle lipid molecules. (A) Volcano plots of lipid molecules. (B) Heat map of top 20 differentially expressed lipid molecules. (C) Heat map of correlations for different lipid species. (D–J) Heat maps of (D) GL, (E) PS and PI, (F) LPC and LPE, (G) PC, (H) PE, (I) SP, and (J) other lipids in the CON and ML groups. (K) Heat map of the correlation between the acyl groups of TG. (L) Heat map of the correlation between the acyl groups of TG, IMF, and lipid species. * p < 0.05 and ** p < 0.01.

Figure 4

Effects of mulberry leaves on cecum microbiota. (A) The proportion of bacteria at the phylum level. (B) Bacterial abundance at the phylum level. (C) The proportion of bacteria at the genus level. (D) Heat map of the top 30 most abundant bacteria. (E) Heat map showing the correlation between the differential bacteria and lipid species. (F) Heat map of the correlation between the differential bacteria and acyl groups of TG. * p < 0.05 and ** p < 0.01.

Figure 5

Effects of mulberry leaves on cecal metabolites. (A) Proportional composition of cecal metabolites. (B) Volcano plot of cecal metabolites. (C) Heat map of the top 30 differentially expressed metabolites. (D–L) Heat map of various differential cecal metabolites. (M) Heat map of the correlation between differential cecal metabolites and lipid species. (N) Heat map of the correlation between differential cecal metabolites and TG acyl groups. * p < 0.05 and ** p < 0.01.

Figure 6

Effect of mulberry leaves on the expression of key genes involved in lipid metabolism and on serum indicators of lipid metabolism. (A–D) Effect of mulberry leaves on the relative expression of ACC, FASN, PPARα, and HSL in backfat. (E–J) Effect of ML supplementation on the relative expression of ACC, FASN, DGAT1, PPARγ, LPL, and FABP4 in the muscle. (K–P) Effect of ML supplementation on serum FFA, LDL-C, HDL-C, insulin, adiponectin, and leptin. * p < 0.05 and ** p < 0.01.