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From gastric to small intestinal lipid digestion: the role of oil-in-water emulsion design to affect in vitro lipolysis kinetics

Book - Dissertation

The digestion of lipids in the human body has several health and nutritional implications. On the one side, a balanced lipid intake provides energy and essential fatty acids. On the other side, lipid overconsumption increases the risk of developing metabolic related diseases such as obesity, diabetes or cardiovascular diseases. In addition, lipid-based products could be employed as delivery systems for lipophilic functional compounds or as food intake control strategy. All of the latter functions depend on the lipid digestion process occurring in the gastrointestinal tract. Lipid digestion is an interfacial phenomena meaning that the water-soluble lipase needs to first adsorb to the oil-water interface before enzymatic conversions can start. Therefore, lipids are organized as colloidal structures, mainly as oil-in-water (o/w) emulsions, which can be designed during food processing or structured during digestion. From a food design perspective, different in vitro studies have demonstrated that the kinetics of lipid digestion can be influenced by emulsion properties. However, most of these studies did not include a physiological relevant gastric lipase to simulate lipolysis in that compartment. Some of them have also only measured the extent of lipid digestion at the end of a digestion step. Another research gap is related to the quantification of lipolysis products in nutritional functionality of emulsions studies. In the past, nutritionists and food scientists have been mainly quantified lipolysis products using the pH-stat technique, which is not specific for the lipid digestion analytes generated during digestion. Related to the lipid quantification, few studies have included the analysis of multiple digestion species. In addition, limited studies applied advanced statistical techniques either to describe the lipolysis behavior of emulsions or to elucidate the lipid digestion mechanism. For the reasons previously exposed, this PhD work aimed (i) to evaluate the gastrointestinal lipolysis kinetics as affected by the o/w emulsion droplet size and interfacial composition, and (ii) to elucidate the molecular mechanism of lipolysis. To fulfill the research objective, a static in vitro digestion method was employed. This method included a relevant substitute of human gastric lipase, namely rabbit gastric lipase. Multiple lipid digestion products (triolein, sn-1,2/2,3-diolein ; sn-1,3-diolein; sn-2-monoolein; sn-1/3-monoolein and oleic acid) were quantified as a function of time (kinetic approach) using for this purpose a developed HPLC-Charged aerosol detector (CAD) technique. Single-response modeling was employed to compare the digestion behavior of the different emulsions. Additionally, the advanced multi-response modeling allowed us to obtain mechanistic insights into the lipolysis phenomena. In this research, the in vitro lipolysis behavior of two important interrelated emulsion design properties was studied: oil droplet size and interfacial composition (emulsions with single and mixed emulsifiers). Hereof, emulsions were formulated with 5% (w/w) triolein, 0.00625-1% (w/w) emulsifiers of different nature such as sodium taurodeoxycholate (NaTDC), Tween 80 (TW80), soy lecithin (LEC), soy protein isolate (SPI), and citrus pectin (CP). These emulsions were subjected to in vitro gastric and small intestinal digestion according the protocol of the international network INFOGEST. Digested samples taken as a function of time were characterized in terms of oil droplet properties (droplet size, microstructure visualization, and droplet charge), and lipolysis products content (HPLC-CAD). Detailed insights were obtained into the lipolysis kinetics in the gastric and small intestinal phase due to the use of different modeling approaches. It was observed that the evaluated levels of emulsion properties resulted in diverse lipolysis kinetics in the gastric and small intestinal phase. For instance, the effect of oil droplet size behavior on in vitro gastric lipolysis was analyzed by digesting emulsions prepared with NaTDC. These emulsions were prepared to present different initial droplet sizes (fine: 0.58 μm; medium: 1.82 μm; and large: 4.00 μm). In this study, for the first time, multiple lipolysis products, including diolein and monoolein regioisomers, were quantified. We encountered an inverse relation between the droplet size and the initial rate and final extent of lipolysis. After understanding the role of the droplet size behavior, the effect of the interfacial composition on in vitro lipolysis was analyzed. Emulsions stabilized by 1% (w/w) of small surfactants NaTDC, LEC, and TW80, as well as 1% (w/w) of biopolymers SPI and CP, were digested under in vitro gastric and small intestinal conditions. In the gastric phase, biopolymer-stabilized emulsions reached the highest extents and initial rates of lipolysis compared to surfactants, which presented diverse extents of lipolysis. In the ensuing small intestinal phase, gastric emulsion stability played a dominant role in the lipolysis kinetics, because even a low gastric lipolysis extent seemed to trigger lipid digestion in the small intestine. Based on these insights on physical stability and in vitro lipid digestion behaviors, CP and TW80 were selected to form and in vitro digest emulsions with a mixed interface (1% CP and TW80 0.00625-0.1%). In the gastric phase, the kinetic analysis revealed that lipid digestion was modulated by the TW80 concentration in the initial emulsions: lower TW80 concentrations led to higher magnitudes of rate constants and extent of lipolysis. In the small intestinal phase, all emulsions were fully digested, and the rate constant depended on the emulsion gastric stability, as observed for the emulsions stabilized by single emulsifiers. The in vitro digestion of emulsions with a mix of emulsifiers (CP and TW80) in the gastric phase generated an intermediate hypothesis about competitive adsorption phenomenon potentially taking place. This hypothesis was tested through the in vitro digestion of single and mixed interfaces using a modified pendant drop technique. In this case, the kinetics of interfacial tension were highly correlated with the lipolysis kinetics of the o/w emulsions. In addition, the interfacial tension and interfacial rheology measurements confirmed that competitive adsorption (e.g. orogenic displacement) played a major role during the lipolysis of emulsions stabilized by CP and/or TW80. Furthermore, the quantification of multiple lipolysis products, including intermediate products regioisomers, in the gastric and small intestinal phase allowed the elucidation of the in vitro lipolysis mechanism. A first proof of this mechanistic insight was performed with 2 data sets generated from the in vitro gastric digestion of emulsions with different droplet sizes. This mechanistic model of in vitro gastric lipolysis model included a reaction scheme with enzymatic and isomerization reactions. Interesting outcomes of this mechanistic insight include (i) a certain activity of gastric lipase over the sn-2 position of the glycerol backbone apart from the higher activity towards the sn-1/3, and (ii) the detection of fatty acids migration from the sn-2 towards the sn-1/3 positions (isomerization). A validation of the gastric lipolysis mechanism was performed with 6 independent data sets produced in the following in vitro gastric digestion experiments. These extra data sets were produced at a different gastric pH and lipase activity, which reflects the robustness of the mechanistic model. Another mechanistic model was obtained from only one data set, but this time in the small intestinal phase which mirrors the activity of both gastric and pancreatic lipases. This mechanistic model represents an update of a previous reaction scheme proposed in our research unit. Enzymatic and isomerization reactions were included in the final reaction scheme. Lipase activity over the three positions of the glycerol moiety was detected. The sn-2 position cleavage may be an indication of a certain extent of gastric lipase activity during in vitro small intestinal digestion. This PhD research demonstrated that modulation of in vitro lipid digestion kinetics was possible through the manipulation of emulsion design properties. This work may be a starting point for the production of emulsion-based products with specific nutritional functionalities. Moreover, the combination of advanced lipid quantification and statistical modeling techniques permitted a deep comprehension of the molecular lipolysis mechanism in both gastric and small intestinal phase.
Publication year:2021
Accessibility:Closed