Liposome encapsulation for casein-derived peptides: Release behavior, in vitro digestibility, nutrient absorption, and gut microbiota

Abstract

Protein-derived bioactive peptides hold great potential for promoting health while face significant challenges during digestion, including structural degradation by gastrointestinal enzymes and limited stability, which hinder their effective utilization. Encapsulation technology offers a promising solution to protect bioactive peptides and ensure their targeted delivery. In this study, casein peptides (CP) were encapsulated into liposomes (CPL) prepared using the thin-film hydration method. The preparation conditions were optimized through response surface methodology, with the following parameters identified: a lecithin-to-cholesterol mass ratio of 3.0, a peptide solution concentration of 0.65 mg/mL, and a wall-to-core material volume ratio of 4.0. Validation experiments confirmed the optimized CPL formulation, resulting in liposomes with an average particle size of 86.13 ± 0.62 nm and an encapsulation efficiency at 87.29 ± 0.82 %. Comprehensive characterization of CPL was conducted using transmission electron microscopy (TEM), differential scanning calorimetry (DSC), and fourier transform infrared spectroscopy (FTIR) techniques. The results demonstrated that CPL provided strong protection for CP against degradation by gastrointestinal enzymes, allowing controlled release in the intestine. This targeted release facilitated interactions with gut microbiota, leading to improved nutrient absorption and modulation of gut health. These findings highlight the potential of liposomal encapsulation to enhance the bioavailability and functional properties of bioactive peptides, paving the way for their broader application in health-related formulations.

Keywords: Casein peptide; Encapsulation; Gastrointestinal stability; Liposome; Nutrient absorption.

Graphical abstract

Fig. 1

Schematic illustration of casein peptide liposomes' synthesis process.

Fig. 2

Effect of mass ratio of soy lecithin to cholesterol (A), peptide concentration (B), and core-to-wall volume ratio (C) on the encapsulation efficiency and average particle size of casein peptide liposomes. (a-d) Bars in the figure without the same superscipts differ significantly (P < 0.05).

Fig. 3

Effect of soy lecithin to cholesterol mass ratio, core-to-wall volume ratio interaction (A) and soy lecithin to cholesterol mass ratio, peptide concentration interaction (B) on liposome encapsulation efficiency.

Fig. 4

Characterization of casein peptide liposomes: A: Macroscopic and microstructure (60000×), B: microstructure (70000×) particle size and distribution (C).

Fig. 5

Differential scanning calorimetry analysis curve (A) and infrared spectra (B).

Fig. 6

Physical stability, storage stability and oxidation stability. A: Turbiscan profiles showing changes in backscattering over 24 h (right: CP, left: CPL); B: Kinetic instability curves; C: Changes in mean particle size and entrapment efficiency at different storage times. D: Changes in DPPH radical scavenging rate under different storage times. CP: casein peptides; CPL: casein peptide liposomes. Values are means ± SEs, n = 3. “*” represents P < 0.05.

Fig. 7

In vitro modeling of microstructural changes during digestion (Yellow: CPL; Green: CP; Red: liposome). CP: casein peptides; CPL: casein peptide liposomes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Changes in the average particle size (A), zeta potential (B) and release curve (C) of liposomes during in vitro digestion.

Fig. 9

A: Rarefaction curve. B: NMDS analysis. C: Venn diagram of characteristic distribution of intestinal flora in mice. Mice intestinal flora species composition analysis histogram in family level (D) and phylum level (E).