Updating the INFOGEST digestion method for sterol bioaccessibility by the simultaneous addition of gastric lipase and cholesterol esterase

A harmonized gastrointestinal digestion method developed in the COST action INFOGEST was adapted for the evaluation of sterol bioaccessibility in a plant sterol (PS)-enriched beverage [1]. Recent results have demonstrated that the individual addition of gastric lipase (GL) or cholesterol esterase (CE) to the INFOGEST digestion increases the bioaccessibility of cholesterol provided by the digestion reagents, thus reducing PS bioaccessibility by 18-62% [2]. In addition, the use of CE did not allow cholesterol quantification in the bioaccessible fraction [2]. In order to evaluate the effect of the simultaneous addition of these enzymes on sterol bioaccessibility, the INFOGEST digestion using GL (60 U/mL) and CE (0.075 or 2 U/mL) is applied to a PS-enriched (1%, w/v) milk-based fruit beverage. Digestion blanks (ultrapure water) are also carried out and sterols are determined by GC-FID [1]. The bioaccessibility of individual and total PS ranges between 5.1–16.6 and 6.9–7.9%, respectively, regardless of the CE enzymatic activity assayed. These values are similar to those obtained by the individual addition of GL or CE (4.2 – 17.9%) [2]. Cholesterol bioaccessibility ranged from 20.9 to 26.1%, similar to the individual addition of GL [2]. Additionally, cholesterol content in the blank is 19.2–21.2 mg/100 g water, also similar to those when GL or CE were individually added (18.9–20.4 mg/100 g) [2]. This fact indicates that the solubility of cholesterol provided by the digestion reagents is not improved by the simultaneous addition of both enzymes, resulting thus in similar values of PS bioaccessibility vs. individual addition. These results suggest that GL could favour the formation of free fatty acids and monoacylglycerols, known to improve the solubility of lipophilic compounds. In conclusion, the INFOGEST method considering the use of GL and CE is an adequate protocol for the evaluation of sterol bioaccessibility since reproduces more faithfully the physiological gastrointestinal conditions.

References: 1. Blanco-Morales et al. Food Funct, 2018, 9, 2080-2089; 2. López-García et al. 2021, pp 44. Available at: https://www.cost-infogest.eu/content/download/4051/35805/file/V-ICFD%20Book%20of%20Abstracts.pdf

Acknowledgements: Authors thank the financial support from project PID2019-104167RB-I00/AEI/10.13039/501100011033 (Ministry of Science and Innovation, Spain). Mussa Makran holds an FPU19/00156 grant from the Ministry of Universities (Spain). Gabriel López-García holds a junior doctor researcher contract within the aforementioned project (CPI-17-025).