۱- Introduction

۲- Theory

۳- Material and methods

۴- Results and discussion

۵- Conclusions

References

Abstarct

During gastric digestion, hydrolysis of proteins by pepsin contributes largely to the breakdown of protein-rich food. We hypothesized that the effect of pepsin is limited by its diffusivity, which is co-determined by the food structure and the local pH in the food during digestion. To investigate the principle mechanism of enzyme diffusion in food matrices, we used enhanced green fluorescent protein (EGFP) as probe to study the diffusivity of proteins in whey protein isolate gels, using fluorescence correlation spectroscopy (FCS). Gels made with different ionic strength showed distinctive elastic moduli but did not show differences in diffusivity of EGFP. Some models for diffusion in hydrogels yield good description of the obtained data, and can approximate the enzyme diffusion in diverse food matrices. However, the enzyme pepsin is more complicated than the probe EGFP, to yield more accurate predictions, electrostatic and enzyme-substrate interaction also need to be considered.

Introduction

The digestion kinetics of food are dependent on the structure of the food that is digested. Food structure influences the oral processing, the gastric disintegration rate, and the consequent gastric emptying towards the duodenum (Singh, Ye, & Ferrua, 2015). The gastric disintegration of food invovles physical and chemical processes, including the peristalsis of the stomach, acid hydrolysis and enzymatic reactions (Bornhorst & Singh, 2014). Among these processes, hydrolysis of proteins by pepsin contributes largely to the breakdown of protein-rich food in the stomach. This hydrolysis can limited by the diffusion of pepsin and the local pH in the solid food matrix during digestion. The hydrolysis kinetics of egg white protein gels and whey protein gels differed strongly from that of the same proteins in solution, which is likely due to the diffusion limitation in gels for both the pepsin and the hydrolysates (Luo, Boom, & Janssen, 2015). Compared to acid-induced dairy gels, a rennet-induced casein gel consists of compact protein aggregates in the acidic gastric environment, and the rennet gel had much slower proteolysis kinetics than that of acid-induced gels (Floury et al., 2018). Thus, a quantitative investigation of pepsin diffusion in food structures may contribute to the understanding of food breakdown and digestion kinetics. We previously measured the diffusivity of pepsin in whey protein isolate (WPI) gels by fluorescence correlation spectroscopy (FCS). We found that the pepsin does not penetrate deep into the gel but remains in a thin layer below the surface of the gel. A second finding was that the diffusivity of pepsin depends strongly on the concentration of the protein gels (Luo, Borst, Westphal, Boom, & Janssen, 2017). Fluorescence Correlation Spectroscopy (FCS) was used for its non-invasiveness and suitability to be used within protein gels. In FCS, a confocal laser microscope is coupled with a photon detector to measure fluorescence intensity fluctuations in a small focal volume. If these fluctuations originate primarily from the diffusion of the fluorophores through the focal volume, autocorrelation analysis can quantify the diffusion rate of the fluorophore. Whey protein gels were used before as model for protein-based solid foods (Luo et al., 2017). Whey protein gelation is generally a two-step process. After heat denaturation, protein oligomers form primary aggregates with different shapes and sizes depending on the pH and the salt concentration. These primary aggregates then form large self-similar aggregates that precipitate or gel above a critical concentration (Aymard et al., 1996; Nicolai, Britten, & Schmitt, 2011).