Comparison of lipases for in vitro models of gastric digestion: lipolysis using two infant formulas as model substrates

Abstract

The aim of this study was to find a lipase suitable as a surrogate for Human Gastric Lipase (HGL), since the development of predictive gastrointestinal lipolysis models are hampered by the lack of a lipase with similar digestive properties as HGL. Three potential surrogates for HGL; Rhizopus Oryzae Lipase (ROL), Rabbit Gastric Lipase (RGL) and recombinant HGL (rHGL), were used to catalyze the in vitro digestion of two infant formulas (a medium-chain triacylglyceride enriched formula (MC-IF) and a predominantly long-chain triacylglyceride formula (LC-IF)). Digesta were withdrawn after 0, 5, 15, 30, 60 min of gastric digestion and after 90 or 180 min of intestinal digestion with or without the presence of pancreatic enzymes, respectively. The digesta were analyzed by scanning electron microscopy and gas chromatography to quantify the release of fatty acids (FAs). Digestions of both formulas, catalyzed by ROL, showed that the extent of gastric digestion was higher than expected from previously published in vivo data. ROL was furthermore insensitive to FA chain length and all FAs were released at the same pace. RGL and rHGL favoured the release of MC-FAs in both formulas, but rHGL did also release some LC-FAs during digestion of MC-IF, whereas RGL only released MC-FAs. Digestion of a MC-IF by HGL in vivo showed that MC-FAs are preferentially released, but some LC-FAs are also released. Thus of the tested lipase rHGL replicated the digestive properties of HGL the best and is a suitable surrogate for HGL for use in in vitro gastrointestinal lipolysis models.

Free fatty acid release from vegetable and bovine milk fat-based infant formulas and human milk during two-phasein vitrodigestion

“…Previous studies investigating the in vitro lipolysis of bovine milk, human milk and IFs only determined the release of FA with a carbon number equal to or higher than six. [36][37][38][39][40][41] The extraction of C4:0 and its quantification are notoriously difficult due to its partial solubility in water, volatility and poor detection with various analytical techniques. Therefore, the level of C4:0 released from milk TAGs are often not determined during both in vitro and in vivo digestion studies.…”

In vitro Fermentation of Digested Milk Fat Globule Membrane From Ruminant Milk Modulates Piglet Ileal and Caecal Microbiota

“…At the time this study was performed, however, rabbit gastric lipase was not commercially available, and the fungal lipases from Rhizopus orizae were used. R. orizae lipase was shown to have greater lipolysis rate (46 vs. 10%) and be insensitive to FA chain length compared to human gastric enzyme, which released only C8:0 and C10:0 under both gastric and intestinal conditions (49).…”

Biological fate of food nanoemulsions and the nutrients they carry – internalisation, transport and cytotoxicity of edible nanoemulsions in Caco-2 intestinal cells

“…Gastric digestions were conducted at pH 5.5 using conditions as per Sassene et al (100 mM NaCl, 80 mM Na taurocholate, 2 mM Tris Malic acid buffer, 20 mM phospholipid (egg PClipoid)) using rabbit gastric lipase (18 TBU mL À1 ) to replicate the function of human gastric lipase. 54 Intestinal digestion was conducted at pH 6.8, in the presence of 10 mM CaCl 2 , 14 mM mixed bile salts, and pancreatin (1500 U Table 1 Initial composition (for preparation) and average particle size distribution (Z-average diameter) of set size nanoemulsions without (empty) and with the fluorescent fatty acid BODIPY® FL C 11 (loaded). Note: all nanoemulsions were diluted to 10% v/v canola oil, 4.2% v/v Tween 80 and 18.3% v/v ethanol to ensure they all had the same composition before exposure to Caco-2 cell layers Set size (nm) proteasetrypsin, chymotrypsin and elastase) as per Wooster et al 55 Fatty acid release during the gastric phase was quantied by GC-FID using acid and base methylation (GC-FAME) as per Cruz-Hernandez et al 56 Fatty acid release during the intestinal phase was quantied direct titration using a degree of fatty acid protonation of 0.6 as per Bennett et al, 57 the validity of this approach was conrmed by verifying the nal extent of digestion using by GC-FAME analysis of the nal digesta.…”

“…The gastrointestinal digestibility of example edible nanoemulsions was compared to a control phospholipid emulsion (readily digestible) using a two-step static in vitro digestion model, 8,55 which includes a validated mimic of gastric lipase. 54,60 A pH of 5.5 was used for the gastric compartment as this is the post-prandial pH found aer consumption of liquid protein beverages, and is in the pH range for maximal gastric lipase activity. 61 Intralipid was chosen as the control emulsion as it is a certied parenteral emulsion known to be readily digestible and being extensively used as a model in lipedema.…”