Free Phytosterols Research Articles

Chitosan-coated soybean protein isolate/lecithin nanoparticles for enhancing the stability and bioaccessibility of phytosterol.

Phytosterols (PS) have various beneficial effects on human health, especially the property of reducing blood cholesterol. However, the low solubility and bioaccessibility of PS have greatly limited their application in functional food ingredients. To improve the bioaccessibility and stability of PS, chitosan-coated PS nanoparticles (CS-PNP) were successfully prepared by self-assembly. The properties of CS-PNP, including size, zeta potential, encapsulation efficiency (EE) and loading amount (LA) were characterised. The optimisation of CS concentration (0.4 mg mL-1) and pH (3.5) resulted in the formation of CS-PNP with an EE of over 90% and a particle size of 187.7 nm. Due to the special properties of CS chitosan, the interaction between CS and soybean protein isolate (SPI)/lecithin (SL) led to the formation of a soluble complex. CS-PNP exhibited good stability to temperature variations but was more sensitive to salt ions. During in vitro digestion, CS efficiently maintained the stability of nanoparticles against the hydrolysis of SPI by pepsin under acidic conditions. However, these nanoparticles tended to aggregate in a neutral intestinal environment. After 3 h of in vitro digestion, the bioaccessibility of PS increased from 18.2% of free PS to 63.5% of CS-PNP. Overall, these results highlight the potential of chitosan-coated nanoparticles as effective carriers for the oral administration of PS. This multilayer construction may serve as a promising for applications in food products as delivery vehicles for nutraceuticals. © 2024 Society of Chemical Industry.

Research progress on classification, source, application of phytosterol esters, and their thermal oxidation stability

Phytosterol esters can effectively decrease serum cholesterol concentration in the human body and prevent cardio-cerebrovascular diseases. It was found that phytosterol esters exhibited better solubility and bioavailability than free phytosterols. In recent years, phytosterol esters have attracted increasing attention. However, during food processing, phytosterol esters are susceptible to degradation at high temperatures, resulting in certain losses and formation of potentially harmful substances for humans. This paper reviews the relevant literatures and updates on the thermal oxidation stability of phytosterol esters in recent years from the following aspects: (i) Sources, physiological activities, and applications of phytosterol esters; (ii) Oxidation mechanism of phytosterol esters; (iii) Effects of phytosterols species, the volume of addition, food matrix, heating temperature and time, and antioxidants on the thermal loss and oxidation stability of phytosterol esters. The research progress on the safety of phytosterol esters is also discussed in detail. Additionally, the prospects for future research are highlighted.

Steam explosion pretreatment enhances free/combined phytosterol extraction and utilization in rapeseed (Brassica napus L.) and its processed products: Insights from SPE-GC approach

The study investigates the impact of steam explosion pretreatment on the distribution of free and combined phytosterols within rapeseed and its derived products. Utilizing solid phase extraction-gas chromatography (SPE-GC) analysis, we elucidated the composition and distribution of phytosterols in five rapeseed varieties and their corresponding processed oils and cakes. The results indicated that Zhongyou 516 and Xiwang 988 are richer in combined phytosterols, whereas Dadi 199, Zhongyouza 501, and Xiwang 291 have a greater concentration of free phytosterols. Steam explosion pretreatment significantly increased the extraction proportion of combined phytosterols in rapeseeds. Throughout the oil process, more than half of the total phytosterol content, specifically 57.0%, was transferred from the steam explosion-treated rapeseed into the rapeseed oil. The variety Xiwang 291 showed the highest efficiency in this transfer, achieving a rate of 61.7%. The study provides crucial data for the enhancement of rapeseed processing techniques and the efficient utilization of phytosterols. Moreover, the study highlights the potential use of the ratio of free to combined phytosterols as a discriminator for different rapeseed oil varieties, offering valuable insights for quality assurance and product differentiation in the industry.

Phytosterols: Potential Therapeutic Effects and Challenges in Food Industry.

Increases in serum total and low-density lipoprotein (LDL) cholesterol are known as hypercholesterolemia, and it is a significant risk factor for the emergence of cardiovascular illnesses. Any action strategy for lowering serum cholesterol is supported by lifestyle changes. Phytosterols are organic substances from the triterpene family. Phytosterols can lower serum LDL cholesterol levels because of their structural resemblance to cholesterol. Phytosterols are used to enrich or fortify a broad spectrum of food products. Phytosterols are quickly oxidized, just like cholesterol and unsaturated fatty acids. The utilization of free phytosterols for the manufacture of functional meals is highlighted in this chapter, which also focuses on the therapeutic effects of phytosterols and their technological concerns in the industrial field.

Impact of molecular interactions between hydrophilic phytosterol glycosyl derivatives and bile salts on the micellar solubility of cholesterol

Hydrophilic phytosterol glycosyl derivatives are synthetic phytosterol analogues by coupling with the glycosyl moiety to improve the water solubility and bioaccessibility of free phytosterols. The aim of this study is to clarify the molecular interaction of phytosterol glycosyl derivatives with bile salts and the consequent impact on cholesterol solubilization. Sharp nonlinear decrease in the micellar solubility of cholesterol and accompanying changes in particle size, zeta potential and microtopography of mixed micelles were observed when phytosterol glycosyl derivatives were introduced in cholesterol-loaded bile salt micelles. These results suggested that β-sitosterol glycosyl derivatives molecules indeed participated in the formation of mixed micelles. Further, nuclear magnetic resonance showed that the structural change of mixed micelles was caused by the insertion of β-sitosterol glycosyl derivatives via hydrogen bonds with sodium taurocholate, which resulted in the low cholesterol solubilization. Moreover, the hydrogen-bond interactions were apparently influenced by the glycosyl moiety of β-sitosterol glycosyl derivatives. These molecular mechanisms may contribute to the development of cholesterol-absorption inhibitors.

Efficacy of Submicron Dispersible Free Phytosterols on Non-Alcoholic Fatty Liver Disease: A Pilot Study.

No pharmacological treatment is yet approved for non-alcoholic fatty liver disease (NAFLD). Plant sterols have shown healthy properties beyond lowering LDL-cholesterol, including lowering triglycerides and lipoprotein plasma levels. Despite pre-clinical data suggesting their involvement in liver fat control, no clinical study has yet been successful. Testing a sub-micron, free, phytosterol dispersion efficacy on NAFLD. A prospective, uncontrolled pilot study was carried out on 26 patients with ≥17.4% liver steatosis quantified by magnetic resonance imaging. Subjects consumed daily a sub-micron dispersion providing 2 g of phytosterols. Liver fat, plasma lipids, lipoproteins, liver enzymes, glycemia, insulinemia, phytosterols, liposoluble vitamins and C-reactive protein were assessed at baseline and after one year of treatment. Liver steatosis relative change was -19%, and 27% of patients reduced liver fat by more than 30%. Statistically and clinically significant improvements in plasma triglycerides, HDL-C, VLDL and HDL particle number and C-reactive protein were obtained, despite the rise of aspartate aminotransferase, glycemia and insulinemia. Though phytosterol plasma levels were raised by >30%, no adverse effects were presented, and even vitamin D increased by 23%. Our results are the first evidence in humans of the efficacy of submicron dispersible phytosterols for the treatment of liver steatosis, dyslipidemia and inflammatory status in NAFLD.

Phytochemical Analysis and Anti-Inflammatory Potential of Acanthus mollis L. Rhizome Hexane Extract.

The rhizomes of Acanthus mollis have traditionally been used for the treatment of several ailments involving inflammation. However, to the best of our knowledge, their chemical composition and pharmacological properties have not been studied until now. As a first approach, this study analyses the A. mollis rhizome hexane extract phytochemistry and its anti-inflammatory and antioxidant capacities in HepG2 and RAW 264.7 cell culture assays. Chemical profiling was performed with gas chromatography mass spectrometry without the modification of native molecules. Free phytosterols (such as β-sitosterol) account for 70% of detected compounds. The anti-inflammatory capacity of the rhizome extract of A. mollis is mediated by the decrease in the NO production in RAW 264.7 that has previously been stimulated with lipopolysaccharide in a dose-dependent manner. Furthermore, HepG2 pre-treatment with the rhizome extract prevents any damage being caused by oxidative stress, both through ROS scavenge and through the antioxidant cellular enzyme system. In this respect, the extract reduced the activity of glutathione peroxidase and reductase, which were stimulated under oxidative stress conditions. Our results suggest that the extract from the rhizomes of A. mollis may constitute a potential source of natural products with anti-inflammatory activity and could validate the traditional use of A. mollis.

Chemical conversions of free phytosterols during the bleaching of corn oil

Phytosterols have health benefits; however, they are partially removed during the bleaching of corn oil. We evaluated the chemical conversion of free phytosterols (FPs) during bleaching. FP degradation accelerated with increased time and temperature, following a first-order kinetic model. In the n-heptane system, air and activated clay promoted the chemical conversion of the FPs. Sterenes formation was analysed under different conditions using a zero-order kinetic model. The apparent activation energies revealed sterene formation decreasing in the following order: campesta-3,5-diene ≈ stigmasta-3,5,22-triene > stigmasta-3,5-diene. Isomers of the above were not detected, indicating that these sterenes were the only primary products of FPs. The desorption test indicated that the FP loss from corn oil was not only due to FPs being adsorbed the activated clay, but also FPs adsorbed at acidic activated sites being degraded. This study presents a vital scientific foundation for retaining FPs to develop healthier and more nutritious oils.

Hydrophilic phytosterol derivatives: A short review on structural modifications, cholesterol-lowering activity and safety

Phytosterols have received extensive attention owing to their excellent cholesterol-lowering activity and the role in cardiovascular diseases prevention. However, poor solubility in both oil and water limited the application of free phytosterols in the food industry. Chemical or enzymatic modifications were effective to improve the physicochemical properties as well as the bioavailability and cholesterol-lowering activity of phytosterols. Higher oil solubility and lower melting point of phytosterols have been achieved by esterification and transesterification with fatty acids and triacylglycerols so as to enhance the bioavailability, reduce formation of precipitates, and improve the sensory quality of products. While the researches on the improvement on its water solubility is a hot topic. Hydrophilic phytosterol derivatives have promising applications in the food industry because most of foods belong to aqueous matrix. Hydrophilic modification is useful and meaningful for phytosterols in both industrial and commercial applications. This review mainly highlights the hydrophilic phytosterol derivatives in the following aspects: (i) hydrophilic modifications of phytosterols by coupling with various polar components; (ii) cholesterol-lowering activity and possible molecular mechanisms of hydrophilic phytosterol derivatives on reducing serum cholesterol level; and (iii) safety evaluation of hydrophilic phytosterol derivatives in cell-culture studies, animal models and clinical trials.

Soy proteins as vehicles for enhanced bioaccessibility and cholesterol-lowering activity of phytosterols.

The formulation of phytosterol (PS)-enriched functional foods has attracted increasing interest in the recent years, owing to its potential health effects. However, the poor solubility and bioavailability greatly limit PS application in this regard. This study investigated whether soy protein isolate (SPI) could effectively perform as a nanocarrier for improving the water solubility, bioaccessibility, and cholesterol-lowering activity of PSs. In this work, we fabricated SPI-PS nanocomplexes, which not only can enhance the stability and bioaccessibility of PS, but also improve the cholesterol-lowering ability of SPI. This improvement was mainly due to the formation of protein-active substance complexes, through hydrophobic interactions. The complexation with PSs resulted in formation of nanosized particles with greater sizes, lower ζ-potential, and higher surface hydrophobicity. The encapsulation efficiency, loading amount, and solubility of PS were closely related to the applied PS concentration in the mixed dispersions, and the maximal PS solubility in the aqueous phase reached about 1.63 mg mL-1 at the highest PS concentration (2.0mg mL-1 ). The PS molecules in the nanocomplexes were mainly present in the amorphous form. The enhanced in vitro cholesterol-lowering activity of PS nanocomplexes relative to free PS seemed to be closely related to its higher bioaccessibility. The findings are of relevance for the development of food-grade PS ingredients suitable for the formulations of PS-enriched functional foods. © 2022 Society of Chemical Industry.

Green and efficient synthesis of pine sterol oleate catalyzed by SO3H‐functionalized ionic liquid

AbstractBackgroundPhytosterol esters can be used in the food industry because of their various physiological activities and better physical properties compared to free phytosterols. However, existing synthetic methods for phytosterol esters tend to have the problems of environment unfriendliness, excessive by‐products, and difficulty in product separation and purification. Therefore, it is crucial to develop a green and efficient synthetic process for phytosterol esters.ResultsThe catalytic properties of various SO3H‐functionalized ionic liquids for the synthesis of pine sterol oleate were investigated, and [BSO3HMIm]Ts showed the best catalytic effect and reaction selectivity. Under the optimized reaction conditions: using 7% of [BSO3HMIm]Ts (based on the mass of pine sterols) as the catalyst, the molar ratio of oleic acid to pine sterols 2:1, reaction at 140 °C for 2.5 h, the yield of pine sterol oleate could reach 93.9%, while only 4.2% of dehydrated sterols by‐products could be produced. Furthermore, the esterification yield could still reach 85.0% after the catalyst [BSO3HMIm]Ts was reused six times under the optimal conditions.ConclusionThis study demonstrates that SO3H‐functionalized ionic liquid [BSO3HMIm]Ts has higher catalytic activity and selectivity than many reported chemical catalysts in the synthesis of phytosterol esters. A green, efficient, and scalable procedure for the esterification of phytosterols with fatty acids in a solvent‐free system has been developed. © 2022 Society of Chemical Industry (SCI).

Property of Phytosterols and Development of Its Containing Mayonnaise-Type Dressing.

Phytosterols are functional ingredients with known efficacy and safety. Phytosterols are found as free sterols or as their esters with fatty acids. Although phytosterol esters are soluble in oil and have been used in many commercial foods, it has been difficult to similarly use free phytosterols since they are insoluble in water and practically insoluble in oil. We have developed mayonnaise-type dressing materials using free phytosterols since people who are conscious about cholesterol intake are likely to be conscious about oil. In this review article, we summarized pieces of evidence for the development of phytosterol-containing mayonnaise-type dressing materials.

Simultaneous analysis of free phytosterols and phytosterol glycosides in rice bran by SPE/GC–MS

In this study, free phytosterols and phytosterol glycosides in rice bran were successfully separated and analyzed using solid-phase extraction (SPE) combined with gas chromatography–mass spectrometry (GC–MS). The results showed that free phytosterols in rice bran included ergosterol (129 ± 8 μg/g rice bran), campesterol (126 ± 9 μg/g rice bran), stigmasterol (106 ± 9 μg/g rice bran), β-sitosterol (305 ± 10 μg/g rice bran), cycloartenol (80.5 ± 3.9 μg/g rice bran) and 24-methylenecycloartenol (87.1 ± 2.2 μg/g rice bran), while phytosterol glycosides included campesterol glucoside (16.0 ± 1.3 μg/g rice bran), stigmasterol glucoside (99.0 ± 4.9 μg/g rice bran) and β-sitosterol glucoside (133 ± 7 μg/g rice bran). The methodological validation indicated that this method could accurately quantify free phytosterols and phytosterol glycosides in rice bran. This study provided a new direction to establish a rapid and simple method for the simultaneous determination of different forms of phytosterols in foods.

Catalyst-free synthesis of phytosterol diacid monoesters and their competitive effect on the solubilization of cholesterol in model bile mixed micelles.

Phytosterols have received an extensive attention owing to their excellent cholesterol-lowering activity and the role in cardiovascular diseases prevention. Phytosterol diacid monoesters, the important intermediates in the structural modification of free phytosterols, were usually obtained by chemical method with catalyst. The aim of this research was to provide an eco-friendly approach for the preparation of phytosterol diacid monoester and explore the possible mechanism of their hypocholesteremic effect using model bile mixed micelles. Catalyst-free synthesis of β-sitosterol with succinic anhydride, maleic anhydride, and glutaric anhydride was investigated and optimized. The yields of three β-sitosterol diacid monoesters all reached above 94% under optimum reaction conditions and their chemical structures were confirmed further by mass spectroscopy, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. Compared with β-sitosterol, β-sitosterol diacid monoesters possessed the increased thermal stability (up to an above 355.28°C) and improved solubility in binary mixed micelle system (exceeding 1.7 mM) and similar cholesterol-lowering activity (7.78%-11.05%). Moreover, β-sitosterol diacid monoesters showed the competitive effect on solubilization of cholesterol due to their enhanced micellar incorporation capacity. Overall, the competition with cholesterol in vitro gives useful information about the cholesterol-lowering activity of phytosterol diacid monoesters, which will further expand their applications in the food industry and health. PRACTICAL APPLICATION: Modified phytosterols, phytosterol diacid monoesters, were successfully synthesized in petroleum ether without catalyst. These products enhanced the thermal stability of free phytosterols and reduced the dissolution capacity of cholesterol in model bile mixed micelles due to competitive solubilization. These results indicated that phytosterol diacid monoesters represented cholesterol-lowering potential, which might further expand the applications in the food industry and health.

Оптимизация процесса омыления таллового пека методом планирования эксперимента

Sustainable recycling of wood chemical production wastes is one of the current issues of modern technology. As a by-product of the pulp and paper industry, tall oil pitch has found application as a potential source of phytosterols. Phytosterols or plant sterols are natural compounds, polycyclic alcohols structurally similar to steroids. Due to their biological activity, phytosterols are used in such areas as pharmaceutics and cosmetology, as functional products, etc., which makes them commercially attractive. This paper considers the stage of saponification of tall oil pitch, which yields free phytosterols from their esters with fatty and resin acids. A central composite design complemented by six star points was chosen to determine the combined effect of the three factors and to plan the minimum number of experiments; the response surface methodology was used to determine the optimal values of the variables. Regression models showing the impact of the basic technological factors (excess alkali, temperature and duration of saponification) on the degree of saponification of phytosterols in saponified tall oil pitch and esters in the obtained extract were developed based on the experimental data. A statistical analysis of the models was carried out. Their validity has been proved by means of analysis of variance. The experimental and predicted values closely correlated. The developed mathematical models in the regression polynomial form enable to find the optimal values of the input process variables using the Minitab software while simultaneously reaching the maximum degree of saponification (98,1 %) with the minimum value of the ether number in the extract (4 mg KOH/g): saponification temperature – 121.7 °C, process duration – 3.18 h, and excess alkali – 100 %. Thus, reliable models for predicting the degree of saponification of phytosterols and the ester number in the extract were obtained. These models can be used in industrial saponification of tall oil pitch. For citation: Korshunov A.O., Lavrenteva E.A., Lazarev M.A., Radbil’ A.B. Optimization of Tall Oil Pitch Saponification by Experimental Design. Lesnoy Zhurnal [Russian Forestry Journal], 2022, no. 1, pp. 173–187. DOI: 10.37482/0536-1036-2022-1-173-187

Nanostructured lipid carriers for delivery of free phytosterols: Effect of lipid composition and chemical interesterification on physical stability

The objective of this study was to develop new nanostructured lipid carrier (NLC) formulations loaded with free phytosterols (FPs) using interesterified and simple lipid phases. The simple and interesterified lipid phases were composed of soybean oil as liquid lipid and fully hydrogenated palm (HPO) and crambe (HCO) oils s solid lipids. High-pressure homogenization was the preparation method, with soy lecithin as a natural emulsifier. The characteristics of NLCs were influenced by chemical interesterification and the composition of solid lipid, mainly with respect to the fatty acid chain size. All developed FP-loaded NLCs displayed an appropriate nanoscale size from approximately 167 to 330 nm (d32) and appropriate physical stability with zeta potential values ranging from − 29.83 to − 43.70 mV after 30 days of storage. The changes observed in these parameters were influenced by the lipid phase of the NLCs and the crystallization of FP on the surface of the NLCs. FP-loaded NLCs formulated with interesterified blends displayed smaller particle size values, with less evidence of particle agglomeration and higher absolute zeta potential values than NLCs formulated with simple blends. These results indicated that NLCs developed with interesterified lipid blends were more stable than those developed with single blends NLCs produced from solid lipids with fatty acids with larger chain sizes (HCO) had larger particle sizes but were less likely to flocculate and coalesce. Thermal behavior study revealed that interesterification caused a reduction in the melting temperature and recrystallization index of the NLCs. These findings indicated decreased crystallinity and a less organized structure. This behavior favored the highest encapsulation efficiency and load capacity observed for NLCs developed with interesterified blends.

Comparative analysis of free/combined phytosterols--degradation and differential formation of oxidation products during heating of sunflower seed oil

Sunflower seed oil was subjected to heating under 90, 180, and 220 °C for up to 180 min. A decrease in free phytosterols (FPS) and combined phytosterols (CPS) as well as a concurrent formation of individual free and combined phytosterol oxidation products (POPs) were determined and evaluated. During heating, the FPS decreased and were more susceptible to oxidation (a total of 22.8% of FPS vs. 19.2% of CPS was lost, respectively, after heating at 220 °C for 180 min), while lesser amount of the lost FPS (about 13.0% vs. 45.6%, after heating at 220 °C for 180 min) were transformed into typical oxidation derivatives compared to their corresponding combined forms. The absolute contents of individual POPs derived from FPS and CPS increased with heating temperature and time. We discovered that 7α-OH-, 7β-OH-, 5,6β-epoxy- and 5,6α-epoxy-PS derived from CPS were generated faster and at higher levels during heating at 180 °C and 220 °C for 180 min compared to those derived from the corresponding FPS, although the initial content of CPS (695.8 μg/g of oil) were far less than that (1545.5 μg/g of oil) of corresponding FPS.

Molecular insights into the loss of phytosterols during the neutralisation of corn oil

Herein, the physical migration and chemical conversion of phytosterols during the alkali neutralisation process of corn oil were investigated with molecular insights. A significant decrease in the phytosterol content was attributed to the migration from oil to the soapstock, along with slight chemical conversions between free phytosterols and phytosteryl esters in the heterogeneous system. Moreover, the migration of phytosterols was positively correlated with the water content in a model system through low-field nuclear magnetic resonance spectroscopy, which was attributed to the hydrogen bonding between free phytosterols and water. Compared to sodium oleate and sodium linoleate, sodium palmitate caused a higher loss of phytosterols due to its low solubility. X-ray diffraction analysis confirmed the formation of a bilayer structure of the sodium salt of fatty acids, suggesting the aggregation of the sodium salt of fatty acids and phytosterols at neutralisation temperatures. Small-angle X-ray scattering analysis suggested that the concentration of the NaOH solution affected the structure of sodium oleate and the area of the polar head base, which was associated with the loss of free phytosterols. The study presents an important fundamental scientific basis for the retention of phytosterols toward the development of more healthy and nutritious edible oils.

A novel phytosterols delivery system based on sodium caseinate-pectin soluble complexes: Improving stability and bioaccessibility

In this study, sodium caseinate (NaCas)/pectin-based phytosterols (NCP-PSs) nanoparticles were successfully prepared by emulsification evaporation and complex coacervation techniques to improve the bioaccessibility of phytosterols. The optimal mass ratio of NaCas to pectin in NCP-PSs nanoparticles was 2:1. Under these conditions, phytosterols were successfully loaded in NCP-PSs nanoparticles with high encapsulation efficiency (91%) and loading capacity (21%). The nanoparticle size was about 240 nm and the polydispersity index was less than 0.3. Fourier transform infrared spectroscopy indicated the existence of electrostatic interaction between NaCas and pectin and the formation of hydrogen bonds between phytosterols and NaCas. X-ray diffraction analyses proved that the phytosterols changed from an ordered crystalline state to a disordered amorphous state by nano-encapsulation. Circular dichroism demonstrated that the encapsulation of phytosterols induced conformational changes of NaCas. Fluorescence spectroscopy revealed the occurrence of hydrophobic interactions between phytosterols and NaCas. The results of storage experiment illustrated that NCP-PSs nanoparticles were stable against dissociation and thus can provide excellent encapsulation and protection for phytosterols. The bioaccessibility of phytosterols encapsulated in NCP-PSs nanoparticles was increased by at least 43.8% compared to free phytosterols and phytosterols encapsulated in NaCas-based nanoparticles, indicating that the presence of pectin improves the bioaccessibility of phytosterols. Consequently, this study provided a novel phytosterols delivery system based on NaCas-pectin soluble complexes, which has a broad prospect in the processing of food, nutrition, and pharmaceuticals.

Effect of agitation and added cholesterol esterase on bioaccessibility of phytosterols in a standardized in vitro digestion model

There is great interest in reaching a consensus method for in vitro digestion of foods. The COST INFOGEST network of researchers has developed a standardized protocol, which is appropriate for basic digestibility studies, but may be insufficient for more complex studies, such as micronutrient bioaccessibility. In the present study, the bioaccessibility of phytosterols (PS) was evaluated using the standardized and modified versions of the in vitro digestion protocol. The INFOGEST protocol was used without modification for 2% solutions of either free phytosterols (FPS) or esterified phytosterols (EPS) in soybean oil. The effect of two variables which have yet to be explored using the INFOGEST protocol was subsequently evaluated. Those variables are: (1) changing the mode of agitation from orbital shaking to head over heels (HOH) rotation and (2) adding pancreatic cholesterol esterase (CE) during the intestinal phase. Bioaccessibility of FPS was greater than that of EPS, and HOH rotation caused a further increase in bioaccessibility of FPS but not EPS. Addition of CE caused a slight, but significant improvement in bioaccessibility of EPS, though hydrolysis was incomplete. Our findings highlight a need for further evaluation of the standardized in vitro digestion protocol, especially for complex studies such as lipid bioaccessibility.