Mechanistic insights into cholesterol and micellar components insolubilisation by cowpea bioactive peptides in a model system

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Marques, Neus Vilanova Garcia, Gustavo Guadagnucci Fontanari, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7612609/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cowpea beans are widely recognised for their hypocholesterolemic potential, which is largely attributed to their high protein content and derived bioactive peptides. This study explored the mechanistic effects of hydrolysed cowpea proteins and sequence-defined synthetic peptides on cholesterol micelle structure and solubilisation in vitro. Synthetic peptides consistently reduced micelle size by at least 6 nm, whereas the ≤ 3 kDa hydrolysate fraction induced a threefold increase in micelle radius. This hydrolysate fraction also decreased cholesterol solubility and phosphatidylcholine content in a dose-dependent manner, forming insoluble aggregates through 50–60% complexation with phosphatidylcholine and thereby disrupting micellar organisation. The three synthetic peptides reduced cholesterol solubility by 10–20% relative to untreated micelles, effects attributed to altered bile acid and phosphatidylcholine composition, as well as potential direct peptide–cholesterol interactions. These findings indicate that micellar competition between peptides and non-cholesterol constituents is a central mechanism modulating cholesterol solubilisation. By examining both complex hydrolysate fractions and precise peptide sequences, this study provides detailed mechanistic insight into cowpea protein activity. These results not only enhance our understanding of peptide–micelle interactions but also establish an in vitro foundation for future validation studies, informing potential dietary or therapeutic strategies aimed at modulating cholesterol absorption. Cowpea beans cholesterol micelles protein bioactive peptides Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Diet is an important source of cholesterol, and nutritional recommendations aim to control dyslipidaemia and atherosclerosis. A Westernised human diet typically provides approximately 300–400 mg of cholesterol per day. An additional ~ 1000 mg of cholesterol enters the intestinal lumen via bile secretion, most of which facilitates lipid absorption. Consequently, dietary cholesterol intake at levels close to the recommended amounts has only a modest impact on serum cholesterol. In contrast, there is substantial evidence that bioactive food compounds, such as dietary fibres and phytosterols, can reduce the intestinal absorption of both dietary and biliary cholesterol, thereby lowering blood cholesterol levels [ 1 , 2 ]. In this context, the absorption of cholesterol and other lipids depends on their ability to form micelles within the intestinal lumen after food ingestion. Such micelles are formed when lipids from bile (i.e. phospholipids, cholesterol, bile salts such as taurocholate) and dietary lipids (triglycerides, phospholipids, cholesterol, phytosterols) reach a critical concentration and aggregate. Marrink and Mark [ 3 ] proposed a radial core–shell model to describe the structure of micelles at pH 7, comprising a hydrophobic interior and a hydrophilic exterior. Phospholipid molecules are oriented radially, whereas bile salt molecules align parallel to the micelle surface, filling the gaps between phospholipid head groups. These groups protrude substantially into the aqueous phase, allowing the phosphate–nitrogen dipole to adopt an almost parallel orientation, thereby enabling cholesterol solubilisation. The efficiency of cholesterol absorption has been extensively studied and is correlated with total cholesterol and low-density lipoprotein (LDL) serum concentrations in both humans and animals[ 4 ]. The concentrations and ratios of bile acids, phospholipids, and cholesterol in the gallbladder must remain within specific limits to avoid gallstone formation and to optimise micelle formation in the intestinal lumen. Assessing cholesterol solubility in bile salt micelles is critical for understanding its availability for intestinal absorption and for developing strategies to reduce epithelial internalisation. This topic has been the focus of some studies, as dietary compounds can reduce cholesterol absorption either by binding to bile acids or by disrupting micelle formation, thereby decreasing plasma cholesterol levels and lowering the risk of associated diseases[ 5 ]. Historically, dietary proteins have been valued primarily for their amino acid content, essential for cellular maintenance, growth, and energy supply. A protein is considered of high biological value when it provides amino acids in proportions that closely match the body’s requirements. In addition to these traditional roles, bioactivity is now recognised as a significant attribute. Bioactive peptides derived from dietary proteins are typically short amino acid sequences ranging from 2 to 30 residues, capable of exerting one or more physiological effects in the human body. However, these sequences are latent within the native protein structure and exhibit bioactivity only after being released through processes such as enzymatic hydrolysis, fermentation, or food processing [ 6 ]. Many of the sources of bioactive peptides come from plant sources such as cowpea beans. Cowpea ( Vigna unguiculata ) is a well-established staple and has also been described as a functional food due to its protein and dietary fibre content. Although it has been consistently demonstrated that cowpea protein reduces the low-density lipoprotein (LDL) and total blood cholesterol levels in both hamsters and humans, the mechanisms underlying these effects remain nuclear[ 7 ]. One of the hypotheses is that peptides released from incomplete digestion of cowpea proteins can reduce the micellar solubilisation of cholesterol in the intestinal lumen, thereby limiting its reabsorption [ 8 , 9 ]. Our previous studies also indicated that cowpea protein, following gastrointestinal enzymatic action, can inhibit 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), a key enzyme in hepatic cholesterol biosynthesis[ 10 ]. In silico analyses further suggested that the cowpea peptide Gln–Gly–Phe (QGF) has a significant probability (p ≤ 0.100) of interacting with HMGCR [ 11 ]. However, the overall magnitude of cholesterol reduction observed in vivo cannot be fully explained by inhibition of the biosynthetic pathway alone, raising further mechanistic questions. It is well established that phenolic compounds and plant sterols can alter the composition of cholesterol micelles, often increasing micellar size within the intestinal environment. The physicochemical interactions responsible for these changes, however, are not fully understood. Alterations in micelle size may affect their recognition by enterocyte brush-border receptors, hindering absorption and promoting faecal excretion[ 12 ]. Similarly, changes in micellar composition may reduce their efficiency in solubilising cholesterol and other lipids. For instance, hydrophobic peptides may compete with cholesterol for micellar solubilisation sites, thereby reducing or blocking cholesterol absorption in the intestine[ 13 ]. Thus, the purpose of this study was to investigate the effect of natural and synthesized peptides from cowpea beans crop on the structure and composition of artificial cholesterol micelles. 2. Material and Methods Cowpea seeds ( Vigna unguiculata L. Walp), cultivar BRS Milênio, were supplied by EMBRAPA (Brazilian Agricultural Research Corporation, Brazil). The seeds were milled (M 20 universal mill, IKA®, Germany) and sieved (0.42 mm sieve) to obtain cowpea flour. 2.1. Protein preparation 2.1.1. Protein isolation Cowpea protein isolate was obtained through conventional methods of alkaline solubilisation and isoelectric precipitation, as described previously[ 7 ]. The cowpea flour was first defatted with hexane (1:6, w/v) during 4 hours and dried overnight for solvent evaporation. The defatted flour was stirred for 2 h at 25 ◦C in ultra-pure water (1:10, w/v), at pH 8.5, adjusted by addition of NaOH (1 mol.L -1 ), and centrifuged (10,000 × g for 20 min, at 4 ◦C). Afterwards, the supernatant was precipitated at pH 4.5 (corresponding to the average isoelectric point) by addition of HCl (1 mol.L -1 ) and centrifuged at 10,000 × g for 20 min (4 ◦C). The insoluble fraction, representing the cowpea isolated protein, was collected. Targeting improved purity, the protein isolates were defatted again according to Bligh and Dyer method[ 14 ]. The moisture and protein content were analysed according to the method 950.46 and 923.03 from AOAC[ 15 ]. To calculate the total nitrogen, the conversion factor 6.25 was used. 2.1.2. Cowpea protein hydrolysis The protein cowpea isolate was hydrolysed using an enzyme/substrate ratio of 1:1,000 (w/w of protein) in 2% protein solution (w/v) according to Megías et al.[ 16 ], with modifications. This ratio was chosen based on preliminary tests that minimized the presence of peptides from the proteolytic enzymes. Pepsin (SIGMA p-7012 activity ≥ 2,500 units/mg protein) was used first (37°C for 2 hours, pH = 2) and, subsequently, pancreatin (from porcine pancreas, SIGMA p-7545, activity equivalent to 8 x U.S.P. specifications) was added to the hydrolysis medium (37°C for 2 hours, pH = 7). The sample was centrifuged (10,000 × g /15 min), the supernatant collected and filtered through a 3 kDa molecular weight cut-off (MWCO) membrane to isolate the low molecular weight peptides. The protein content was analysed according to the method 923.03from AOAC[ 15 ]. To calculate the total nitrogen, the conversion factor 6.25 was used. The ≤ 3kDa hydrolysate fraction was used in the in vitro assays. 2.1.3. Cowpea peptides synthesis Our previous study [ 8 ] on the in silico prediction of the biological activity of several short peptides in the ≤ 3 kDa fraction of the cowpea hydrolysate, revealed three peptides that are prone to alter cholesterol homeostasis: Leu-Leu-Ans-Pro-Asp-Asp-Glu-Gln-Leu (LLNPDDEQL); Phe-Phe-Phe-Gly-Gln-Asp-Gly-Gly-Ser-Lys-Gly-Glu-Glu (FFFGQDGGSKGEE) and Leu-Asn-Leu (LNL) (Table 1 ) . They were synthesized by Aminotech Research and Development (Diadema, SP, Brazil) with a prity greater than 95 % without further modifications. The structure of the synthetic peptides was confirmed by mass spectrometry (supplementary material). Table 1 Peptide sequences present in cowpea bean ≤ 3 kDa hydrolysate fraction and their potential biological activity, physical-chemical properties and protein source (adapted from Marques, et.al. 2015a). Peptide a Observed molecular weight /theoretical molecular mass (Da) e Possible biological activities b Total hydrophobic ratio (%) c Iso-electric point e Net charge at pH 7 e Cowpea protein source d FFGQDGAVVAGSC 1256.5/1257.3 ACE inhibitor; neuropeptideinhibitor; dipeptidyl-aminopeptidase IV inhibitor 53.84 pH 3.1 -1 Zeaxanthinepoxidase; Glutathione reductase LLNPDDEQL 1055.5/1056.1 ACE inhibitor; dipeptidyl-aminopeptidase IV inhibitor; Glucose uptake stimulating peptide 30.00 pH 2.87 -3 Acetyl-CoA carboxylasecarboxyltransferase;Phospholipase D alpha 1 LNL 358.2/358.4 ACE inhibitor 66.66 pH 6.01 0 No significant similarity found a Peptides with PEAKS ALC score of 50% or greater b Determined using BIOPEP c Calculated the percentage of hydrophobic residues (I, V, L, F, C, M, A, W) in the peptide sequence. d Determined using BLAST tool e Determined using Innovagen´s peptide property calculator Next, the impact was tested of the ≤ 3 kDa hydrolysate fraction and the three synthesized peptides on cholesterol solubilisation, micelle size, bile acid binding, phosphatidylcholine binding and in vitro cholesterol crystal formation. 2.2. Preparation of in vitro cholesterol micelles Micelles were prepared according to the method of Zhang et al.[ 17 ]. The lipids (final concentrations in the aqueous buffer: 0.5 mM cholesterol, 1 mM 9- cis ,12- cis -linoleic acid and 2.4 mM phosphatidylcholine from egg: 1,2-Diacyl- sn -glycero-3-phosphocholine) were dissolved in methanol and added to tubes of 2 mL and dried under N 2 flow. The dried lipid mixture was combined with 500 µL of 15 mM sodium phosphate monobasic buffer containing 6.6 mM taurocholic acid sodium salt and 132 mM NaCl at pH 7.4. Thus, the [phosphatidylcholine]/([ phosphatidylcholine] + [bile acids]) ratio was 0.26. This ratio is considered the Cholesterol Saturation Index (CSI) and it was calculated based on Carey’s critical tables[ 18 ]. This index represents the maxima amount of cholesterol dissolved in bile acids in an equilibrium of solubility. All the chemicals were purchased from Sigma-Aldrich®. The suspension was submitted to sonication in an ice bath for 15 min and incubated for a further 30 min at 37°C to form the micelles. When required, ≤ 3 kDa hydrolysate fraction (ranging from 1 to 200 mg/mL in protein) and peptides (0.3 mg/mL to 1 mg/mL) were added and the mixture was submitted to sonication for 1 min and incubated for 1 h at 37°C. The solution was then centrifuged at 1,000 x g for 10 min and filtered through a 0.20 µm Millex-GP filter (Millipore, Bedford, MA, USA). All analyses were performed after 2 days to ensure the system stabilization. The micelle formation was verified by fluorescence spectroscopy described below. 2.3. Fluorescence spectroscopy Following the work of Greenspan and Fowler[ 19 ], we used the solvatochromic dye Nile Red as a reporter of micelle formation and changes in the micelles upon addition of the ≤ 3 kDa hydrolysate and peptides. To this end, samples were prepared by adding 0.83 µL of a 0.1 mg/mL Nile Red solution in acetone to 0.5 mL of a micellar solution. Fluorescence emission spectra were recorded at 25°C in a wavelength range from 575 to 700 nm upon excitation at 549 nm on a Varian® Cary Eclypse fluorimeter equipped with a xenon lamp. The normalized fluorescence intensity of the samples was obtained after subtraction of the buffer solution spectrum (blank). For that, this experiment monitored the micelle formation with 0.5 mM cholesterol (standard micelle as shown in above section) and without cholesterol (buffer plus taurocholate without cholesterol) used as a control of spontaneous micelle formation. The baseline containing only phosphate buffer (without taurocholate and without cholesterol) was also controlled. In the same micelle with cholesterol (0.5 mM cholesterol), it was added hydrolysate and peptides to test the variation on Nile Red spectra, searching for blue shift. All samples tested were containing the same amount of Nile Red. 2.4. Cholesterol solubilisation in micelles The impact of the ≤ 3 kDa hydrolysate fraction and peptides on cholesterol solubilisation into micelles was examined spectroscopically. The compounds were separately added to a micelle solution, which was sonicated in a bench top ultrasound bath (Transsonic Digital S Elma) during 1 min, and subsequently incubated for 1 h at 37°C. The solution was then centrifuged at 1,000 x g for 10 min and the supernatant filtered through 0.20 µm Millex-GP filters (Millipore, Bedford, MA, USA) to exclude other non-micelles fractions. After filtration, 50 µL of this solution was collected, and its cholesterol concentration was determined using Amplex® Red Cholesterol Assay Kit (Invitrogen, Paisley, UK) by fluorescence according to the manufacturer instructions. Excitation wavelength was 555 nm and emission detection was at 590 nm, measured with SpectraMax M5 equipment (Molecular Devices, Sunnyvale, CA, USA). Cholesterol standard curves were obtained using calibration standards. 2.5. Micelle sizing by dynamic light scattering The apparent hydrodynamic radii of the micelles were measured by dynamic light scattering (DLS). The experiments were performed on an ALV/CGS-3 MD-4 compact goniometer system equipped with a Multiple Tau digital real time correlator (ALV-7004) and a solid-state laser (λ = 532 nm; 40 mW). Typical experiments cover scattering angles from 60 to 120°, averaging over 3 x 20 sec runs at 20°C. As a control, the sodium phosphate monobasic buffer containing 6.6 mM sodium taurocholate salt and 132 mM NaCl at pH 7.4 was also measured; here, spontaneous micelle formation was not detected. 2.6. Phosphatidylcholine solubilisation in micelles To evaluate the interaction between micellar phosphatidylcholine and the ≤ 3 kDa hydrolysate fraction and peptides, the micelle solution was prepared and stored at 37°C. Two hundred microliters of two different concentrations of each peptide (0.3 and 1 mg/mL) were added to the 6 mL micellar solutions. The mixture was incubated for 1 h at 37°C and then centrifuged at 25,000 × g for 1 h. In order to exclude other non-micelles fractions, the supernatant was passed through a 0.20 µm Millex-GP filter (Millipore, Bedford, MA, USA) syringe filter, and the phosphatidylcholine solubilisation in the presence and absence of peptides was quantified according to a phospholipids assay kit (n° cat. MAK122, Sigma-Aldrich ® ) in the filtrate, according to the manufacturer instructions[ 20 ]. This method is based on the enzymatic hydrolysis of the choline fraction present in the phospholipids and their subsequent spectrophotometric detection at 570 nm. 2.7. Bile acid binding assay The interaction between the ≤ 3 kDa hydrolysate fraction and peptides with micellar bile acids was assessed according to Yoshie-Stark and Wasche[ 21 ]. Different concentrations of the ≤ 3 kDa hydrolysate fraction and peptides were added in phosphate buffer solution (0.1 M) containing sodium taurocholate 2 mM at pH 7. After incubation at 37°C for 2 h, each sample was centrifuged, and the supernatant was transferred to a volumetric flask. A further 1 mL of 0.1 M sodium phosphate buffer at pH 7.0 was added to the sediment, thoroughly mixed and centrifuged. The supernatant was removed and combined with the earlier supernatants. This procedure was repeated, and the supernatants were collected to the existing supernatants in a volumetric flask. The concentration of bile acids was measured spectrophotometrically at 405 nm according to the manufacturer instructions of the Total Bile Acids Assay kit (Diazyme, Poway, CA, USA). The experimental data was obtained from a calibration curve obtained using calibrated cholic acid standard solutions. Cholestyramine resin, a drug that binds bile acid and lowers cholesterol, was also evaluated for its ability to bind bile acid. All analyses were performed in triplicate. The capacity of bile acid binding was expressed as µmol of bile acids per gram of sample (precipitate after centrifugation). 2.8. Separation of micelles and cholesterol crystals 2.8.1. The intermixed micellar bile salt concentration (IMC) measurements The intermixed micellar/vesicular (non-phospholipid associated) bile salt concentration (IMC) is the sum of the concentrations of monomeric and simple micellar bile salt. This parameter was measured firstly to determine the minimum amount of bile salt (deoxycholate) required to prepare the cholesterol crystals for quantitative analysis ( vide supra ). Briefly, a 10 kDa Centrisart ultrafilter was rinsed with ultrapure water and submitted to centrifugation for 5 min at 500 × g . The remnant water after centrifugation was totally removed by a syringe. The filter was then pre-incubated during 1 h at 37°C. After the incubation, 2 mL of micellar solution without ≤ 3 kDa hydrolysate fraction or peptides were added, centrifuged in a 10 kDa Centrisart ultrafilter for 5 min at 500 × g for three times, and the filtrates pooled. In the filtrates, the amount of bile acids was quantified to determine the IMC value. 2.8.2. Separation and quantification of the amount of cholesterol crystals and micelles Aiming to evaluate whether cholesterol crystals form and if so, separate these from the micellar phase, the ≤ 3 kDa hydrolysate fraction and peptides were first added to a solution of cholesterol micelles, after which it was submitted to centrifugation for 10 minutes. After then, it was added sodium deoxycholate in a sufficient amount to desaturate the model system (cholesterol saturation index < 1)[ 22 ]. Subsequently 2 mL of taurocholate solution 197 mM were then added to a 300 kDa Centrisart ultrafilter and centrifuged for 5 minutes at 500 × g . The residues were carefully withdrawn by a syringe and discarded. The minimum concentration of deoxycholate was determined previously (197 mM), based on IMC[ 23 ], and it was checked against tabulated values to determine the cholesterol saturation index[ 18 ]. Next, each micellar solution was centrifuged at 50,000 × g (37°C for 30 minutes). The supernatant was collected and applied on a disposable filter and submitted to gravitational filtration during 1 h. The mass of cholesterol in crystals was determined in the filtered fraction using the assay Amplex ® red (Invitrogen, Paisley, UK). 2.9. Statistical analysis Statistical analyses were performed using analysis of variance (ANOVA) and when necessary, a Tukey multiple comparison post hoc test was run, with in both cases p < 0.05 signalling significant differences between the studied conditions. To compare the systems where the proteins or peptides were added to the control micellar solution, the t-Student paired test with also signalling significant differences in p < 0.05 was used. The statistical analyses were performed using software GraphPad Prism version 5 for Windows. 3. Results Aiming to advance the current understanding of the mechanisms through which hydrolysed cowpea protein, and its peptides reduce the solubility of cholesterol in cholesterol mixed micelles and thereby cholesterol absorption by the gut and cholesterol plasma concentrations, we herein study the effect of cowpea hydrolysate and peptides on the structure and composition of artificial cholesterol micelles. There are different pathways which may contribute to the way in which cowpea hydrolysate and peptides may disturb micelle formation and impair recognition of the micelles by the border-brush intestinal cell receptors. These include changes in micellar dimensions and composition, e.g., variations in the micellar content of cholesterol, phosphatidylcholine, and bile acids, as well as cholesterol crystallisation. 3.1. Impact of cowpea derived peptides on micelle formation The formation of cholesterol micelles and changes induced by hydrolysates and peptides were monitored using Nile Red fluorescence emission spectra with and without added hydrolysate and peptides (Fig. 1 ). According to Greenspan and Fowler[ 19 ], Nile Red exhibits a blue shift in emission when bound within hydrophobic environments, such as micelles, but shows negligible fluorescence in water (λ exc = 549). It shifts increasingly towards the blue if the local environment becomes more hydrophobic. In this situation, when binding to cholesterol which is internalised within the hydrophobic environment of a micelle. As expected, we observe no discernible signal in the phosphate buffer (control sample), since this does not contain any micelles. A modest signal peaked around an emission maximum at λ em = 641 nm appears when bile salt (taurocholate) but no cholesterol is added, signalling the presence of more hydrophobic domains. A marked increase in emission and blue shift of the emission maximum to λ em = 629 nm is observed for the micellar solution containing 0.5 mM cholesterol. Interestingly, the emission maximum remains fairly unaffected at λ em = 629 ± 1 nm, whilst the fluorescence intensity is noticeably reduced upon addition of 50 mg/mL and 100 mg/mL hydrolysate. Similarly, addition of either LLNPDDEQL, FFFGQDGGSKGEE or LNL also reduces the fluorescence intensity (emission maximum respectively λ em = 625 nm, λ em = 628.9 nm and λ em = 631 nm). The decrease in Nile Red fluorescence in all samples with hydrolysate and peptides signals a reduction in cholesterol solubilisation, which is presumably due to displacement by the hydrolysate and peptides of cholesterol from the micelle. 3.2. Impact of cowpea derived peptides on micellar dimensions The literature reports conflicting in vitro results regarding experiments about variations in micellar dimensions. It seems possible that larger and smaller mixed micelles could negatively impact the intestinal absorption of cholesterol[ 24 , 25 ]. To examine whether cowpea hydrolysate and peptides affect micellar dimensions to such a large extent, dynamic light scattering experiments were performed to determine the hydrodynamic radius R h of the micelles as a function of the added amount of hydrolysate and peptides (Fig. 2 ). The size of the cholesterol micelles varied little up to ≤ 3 kDa hydrolysate concentrations of 10 mg/mL, whilst the micelles grew larger in a dose-dependent manner for hydrolysate concentrations above 100 mg/mL (Fig. 2 a). Surprisingly, the addition of the analogous synthetic peptides, led to a reduction in micellar size for much lower peptide concentrations up to 1 mg/mL (Fig. 2 b). Addition of 1 mg/mL of the FFFGQDGGSKGEE and LNL peptides reduced micellar dimensions from about 65.8 ± 1.3 nm in the absence of either to 58.0 ± 1.3 nm (FFFGQDGGSKGEE) and 59.6 ± 1.4 nm (LNL), for respectively. Nonetheless, according to in vitro experiments on the cholesterol absorption, neither the up to 3-fold increase induced by the hydrolysate, nor the slight decrease due to the synthetic peptides is sufficiently large to significantly affect the cholesterol absorption through the gut[ 26 ]. 3.3. Cholesterol micellar solubilisation in vitro Since changes in micelle size alone did not account for the reduced cholesterol absorption previously reported at 1 mg/mL compound concentration[ 10 ], we next examined cholesterol solubilisation directly. Both the ≤ 3 kDa hydrolysate fraction and the synthetic peptides reduced micellar cholesterol content in a dose-dependent manner. The ≤ 3 kDa hydrolysate fraction decreased cholesterol solubility by 50–60% (Fig. 3 a), whereas peptides reduced solubility by 20–30%. Although the peptide concentrations required were relatively high, such levels are physiologically achievable in the intestinal lumen (Fig. 3 B). 3.4. Peptide-phosphatidylcholine and peptide-bile acids interaction To examine whether cowpea peptides interfere with other components in the micelles, the phosphatidylcholine and bile acid (Fig. 4 ) solubilized in the presence of the ≤ 3 kDa hydrolysate fraction and the analogue synthetic peptides were quantified. Absorbance measurements revealed an increase in phosphatidylcholine dispersion compared with controls. The ≤ 3 kDa hydrolysate fraction increased solubilised phosphatidylcholine nearly threefold (Fig. 4 a), while synthetic peptides produced a similar, though less pronounced, effect (Fig. 4 b). The Fig. 4 c and 4 d depicts the peptide-bile acids interaction with cholestyramine being used as positive control. Cholestyramine is a drug that has been used in hypercholesterolemia treatments capable of binding intestinal bile acids. The bile acids COO - tail is the main structural target for anionic swap, promoted by cationic resins, clinically used as bile acids abductors. Food proteins from soy, white rice, fishes, milk goat and others were described as bile acids abductors. They reduce the amount of bile acids recycled in the liver, and increase the bile acids synthesis by cholesterol clearance from plasma[ 27 ]. It is possible to observe that the ≤ 3 kDa hydrolysate fraction was able to bind the bile acids at 200 mg/mL. Figure 4 d shows that there were not significant differences in bile acids binding by the synthetic analog cowpea peptides, except by ≤ 3 kDa hydrolysate fraction at 200 mg/mL and the LNL peptide. 3.5. Cholesterol crystals formation Finally, the potential for cholesterol crystallisation was examined. Micelles phase containing peptides or hydrolysates were centrifuged, and crystallised cholesterol was quantified ( Fig. 4 e and 4 f ) . Only cowpea analogue synthetic peptides induced cholesterol crystal formation, whereas the hydrolysate fraction did not. (Fig. 4 f ) . 4. Discussion The maximum emission spectra of Nile Red depend on the environment polarity. Variations in fluorescence spectra of the solubilised Nile Red indicate the presence of different association complexes in solution (i.e. micelles, bilayers membranes, vesicles)[ 28 ]. This study corroborates previous findings [ 29 ] that is possible simulate a stable human micelle in vitro with a hydrophobic internal core filled with cholesterol. A direct relationship was also observed between the cholesterol content of micelles and the fluorescence signal intensity. It is known that enzymatic digestion of proteins leads to extensive chemical modifications in the final products and may alter micellar dimensions. Reynier and co-workers [ 26 ] attested that the diffusion rate of the micelles through the unstirred water, layer decreases as micellar size increases, suggesting a decrease in intestinal absorption of huge cholesterol micelles. Our earlier study showed that 1 mg/mL of the ≤ 3 kDa hydrolysate fraction sufficed to impair cholesterol solubilisation, though micellar size was not assessed at that time[ 10 ]. As a result, clearly the ≤ 3 kDa hydrolysate fraction could not alter the micelle radius at lower concentrations tested (less than 100 mg/mL) as compared, for example, with other compounds such as phytosterols[ 30 ]. Significant increases in micelle size were observed only at hydrolysate concentrations above 100 mg/mL of ≤ 3 kDa hydrolysate fraction, far above that where significant reduction in cholesterol solubilisation was observed. This suggests that the reduced cholesterol solubilisation at hydrolysate concentrations below 100 mg/mL was mediated by mechanisms other than micellar size modification. Considering Khoshakhlagh [ 31 ] report on a drug-delivery system based on cholesterol micelles, these results raised the possibility of a non-linear relationship between solubilisation of cholesterol and the increase or decrease in the micelle size in the presence of protein hydrolysate as it is common with drugs in a micellar system. Similarly, Hu and colleagues [ 13 ] did not verify apparent change in the size distribution data at different dosages of β -sitosterol glycosyl derivatives in presence of cholesterol micelles. They concluded that β -sitosterol glycosyl derivatives preferred to interact with bile salts at the interface of micelles rather than being incorporated into the hydrophobic cores of micelles. The reasons are due to their amphiphilic structure, comprising hydrophilic polar groups and a hydrophobic β -sitosterol tail. This decrease in size supported the interaction of β -sitosterol glycosyl derivatives with cholesterol-loaded bile salt micelles Nevertheless, the cholesterol solubility was found reduced by about 50–60% after adding the ≤ 3 kDa hydrolysate fraction in a dose-dependent manner. Although relatively high concentrations of cowpea peptides were required, such levels are physiologically attainable following legume consumption and digestion. Comparable reductions in micellar cholesterol solubility have been reported for milk casein (four peptides at 3 mg/mL decreased 32–53%) using the same protocol[ 32 ], and gluten hydrolysates < 1kDa from wera able to inhibit 47.08 ± 1.71% of micellar cholesterol solubilisation at 2 mg/mL[ 33 ]. Although the displacement of cholesterol by peptides was evident, the precise mechanism remains unclear. A possible explanation would be that hydrophobic peptides interfere with the structural formation of the micelles, competing then with cholesterol as phytosterols do [ 34 ]. However, when using peptides with distinct hydrophobicity in our study, the absolute effects of reduction of cholesterol solubility among peptides are comparable. In line with this, the pentapeptide IIAEK, derived from β-lactoglobulin, was shown to reduce cholesterol levels in an in vivo model, although it did not alter cholesterol or bile acid solubility in vitro . The authors suggested that the peptide may act as a surfactant or interfere with gene expression [ 35 ]. It is plausible that peptides act via multiple mechanisms simultaneously. Therefore, the hydrophobicity does not fully explain the peptide action. Isolated proteins and phospholipids can present synergism or antagonism in the stability of colloidal systems. Interaction between proteins and phospholipids can lead to changes in their superficial activity, in their structural conformation and in their incorporation in surfactant-based structures, such as micelles and vesicles [ 36 ]. In Brown and co-worker´s [ 37 ] study, phytosterols were also able to reduce cholesterol solubility without interfering with micelle size. Intending to clarify this behaviour, we evaluated whether cowpea peptides interact with phosphatidylcholine or bile acids. Food-derived components have previously been shown to bind phosphatidylcholine. For instance, the spectroscopic study done with curcumin to assess the peptide interaction with phosphatidylcholine suggests that an absorbance increase, is a consequence of an increase of the hydrophobic environment in the micelles [ 38 , 39 ]. The α-lactalbumin from milk was also described as being able to penetrate in phosphatidylcholine vesicles through the most hydrophobic regions of the protein[ 27 ]. Hence, the cowpea protein seems to reorganize the structure; changing the micellar composition due to effects sum between fluctuation of cholesterol and phosphatidylcholine concentrations. Thus, the cholesterol is not satisfactory solubilised, or it is inefficiently incorporated into the mixed micelle formed. Again, the ≤ 3 kDa hydrolysate fraction of cowpea was more effective than the isolated peptides from cowpea bean, which exhibit a tiny action featuring the LNL sequence; and this effect does not seem to be dose-dependent. Similarly to α-lactalbumin and hydrolysed dry beans ( Phaseolus vulgaris L.) protein, the hydrophobicity of the LNL peptide can explain the featured effect[ 40 , 41 ]. Despite of being a hydrophobic peptide, the mechanism of protein action on cholesterol micelles (prepared with phosphatidylcholine) is not the same found for other polyphenols that consider the hydrophobicity as an important chemical characteristic condition[ 42 ]. Bile acid binding represents another potential mechanism. Peptides, including those from soy and amaranth, can sequester bile acids at levels comparable to cholestyramine, reducing cholesterol solubility[ 34 , 43 ]. However, this action seems to be sensitive to many parameters such as the hydrolysis conditions (enzymes used, time, temperatures) and bile acids type. The information about the structure-function of peptides/proteins and the intestinal bile acid sequestration is scarce. A successful bile acids binding is explained for their action points towards a low digestibility and a higher amount of hydrophobic amino acids in the structure[ 44 ]. The study of Johns and Bates [ 45 ] proved that the cholestyramine has affinity dependent in part on the extent of the hydrophobic character of bile salt anions to the possibility the anionic swap. In our system the LNL (hydrophobic), in fact, could not perform this swap due to its net neutral charge at pH = 7, leading to insolubility. Moreover, the peptide was also engaged with phosphatidylcholine. The increase in bile acid solubilisation observed with LNL is consistent with models of cholesterol–bile acid crystallisation [ 46 ] and more recently corroborate by Matsuoka [ 5 ] about the crystallization mechanisms of cholesterol and bile acids. A relationship between the micellar solubilisation and the peptide-bile acid binding can be established; being this peptide responsible for the composition change of the micelles. The increase of the bile acids solubilisation in the system causes the reduction of ([phospholipid]/([bile acids + phospholipid]) ratio. Interestingly, the alteration in this micellar equilibrium (with bile acids concentration changing) is associated with a higher precipitation of cholesterol crystals. Although the ≤ 3 kDa hydrolysate fraction reduces cholesterol solubilisation, it has no effect in promoting the cholesterol crystallization. This is directly correlated with the increase of phosphatidylcholine that is solubilised in the presence of ≤ 3 kDa hydrolysate fraction. The excess of phosphatidylcholine liberated from the micelles can act as a cholesterol binder, not necessarily forming crystals. This binding capacity was demonstrated by Yang et.al.[ 47 ], who observed that egg phosphatidylcholine attenuated cholesterol solubilisation and its absorption. The strong cholesterol inhibitory effect observed is associated with the higher degree of saturation and the long chains of its acyl groups in the phosphatidylcholine molecule, able to reorganize the micellar structure. Cholesterol is critical for micelle stability, and its removal disrupts micelle self-organisation [ 12 ]. Summarizing the events, the ≤ 3 kDa hydrolysate fraction binds phosphatidylcholine and then cholesterol, forming a large complex. This rearrangement is responsible for micelle size increasing in the presence of ≤ 3 kDa hydrolysate fraction. On the other hand, in the presence of the isolated peptides, a significant precipitation of cholesterol crystals is observed. Even though this is not a dose-dependent phenomenon, these results show that the solubilisation of cholesterol is due to the alteration of ([phospholipid]/([bile acids + phospholipid]) ratio, and the crystal formation are caused by the peptides tested. Several reports with food proteins describe the various possible mechanisms of protein/peptide – micelle interaction; however, none of them details which is the main target of this interaction: cholesterol, phosphatidylcholine or bile acids[ 48 , 49 ]. The present study shows that the mechanism by which less cholesterol is incorporated into the micelles is through the competition between the micellar constituents (phosphatidylcholine or bile acids) and the cowpea derivatives, rather than specifically with cholesterol. This raises multiple possibilities for the inhibition of cholesterol absorption by the hydrolysates and peptides. 5. Conclusion This study extends our knowledge about the physical-chemical interaction between cholesterol micelle components and peptides generated from diet. Our experiments have shown that the ≤ 3 kDa hydrolysate fraction, and all tested peptides could reduce the cholesterol internalisation of in vitro micelles. However, the changes in micelle size are not related only to this lack of cholesterol inside the structure. The mechanism of lower cholesterol internalization in micelles by the ≤ 3 kDa hydrolysate fraction and LNL peptide is related to peptides’ binding to phosphatidylcholine and then to cholesterol, forming a large complex. The hydrolysate binding with bile acids was less effective. The mechanisms of cholesterol insolubilisation achieved by LLNPDDEQL, FFFGQDGGSKGEE and LNL peptides are a consequence of a combination between bile acids and phosphatidylcholine interaction with the peptides tested. However, the mechanism of the peptides additionally showed some kind of direct interaction with cholesterol due to the formation of cholesterol crystals. In all cases, the peptides are less effective than the ≤ 3 kDa hydrolysate fraction. A key contribution of the present study was to verify that, unlike polyphenols and phytosterols, the mechanism of competition for the intra-micelle space with cholesterol is better explained by an interaction of peptides with other micelle constituents rather than a change in their size or a direct interaction with the cholesterol in the micelles. Declarations Conflict of interest The authors declare that they have no conflict of interest. Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Marcelo Rodrigues Marques , Neus Vilanova Garcia and Gustavo Guadagnucci Fontanari. The first draft of the manuscript was written by Marcelo Rodrigues Marques with contribution of Ilja Karina Voets and Jose Alfredo Gomes Arêas. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgements The author MR Marques is grateful to FAPESP (Foundation for research support of the State of São Paulo, Brazil) [grant 2013/09304-2], [grant 2012/15900-4] and CNPq (National Council for Scientific and Technological Development) [PhD grant 202992/2015-2]. This research is also part of the research program of the DPI (Dutch Polymer Institute), project #772ap. The authors wish to thank Rosana A.M. Soares-Freitas and Cintia P. Silva for technical support. 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Chen, G.-W., et al., Purification and identification of cholesterol micelle formation inhibitory peptides of hydrolysate from high hydrostatic pressure-assisted protease hydrolysis of fermented seabass byproduct. International Journal of Molecular Sciences, 2021. 22 (10): p. 5295. Bao, X., et al., Flaxseed‐derived peptide, IPPF, inhibits intestinal cholesterol absorption in Caco‐2 cells and hepatic cholesterol synthesis in HepG2 cells. Journal of Food Biochemistry, 2022. 46 (1): p. e14031. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterialv2.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7612609","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":524817787,"identity":"5c24db6f-7f6b-413f-aee5-b5096a423007","order_by":0,"name":"Marcelo Rodrigues. 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The maximum of each spectra is indicated. The standard micelle is composed by 0.5 mM cholesterol, 1 mM linoleic acid, 2.4 mM egg phosphatidylcholine, 15 mM sodium phosphate monobasic buffer, 6.6 mM taurocholic acid sodium salt and 132 mM NaCl at pH 7.4. The ≤ 3 kDa hydrolysate fraction and peptides were added in this standard micelle solution to perform all tests. The buffer plus taurocholate has the same components of the standard micelle without cholesterol and tested samples. The phosphate buffer is composed by 15 mM sodium phosphate monobasic buffer and 132 mM NaCl. All the conditions were containing Nile Red at the same concentration\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7612609/v1/65faa2803ca1902b871d303c.png"},{"id":92830246,"identity":"40e1e1a9-e469-45dc-8459-ef2f493c43db","added_by":"auto","created_at":"2025-10-06 06:07:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":232086,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ≤ 3 kDa hydrolysate fraction (A) and cowpea analogue synthetic peptides (B) on micelle size (mean ± SD). (*) p \u0026lt; 0.05 compared to standard micelle (\u003cem\u003et-Student \u003c/em\u003etest p \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7612609/v1/1b3ef13bc9ab3ca0eb78022c.png"},{"id":92830254,"identity":"22a96129-eb72-41c9-8e0c-cc837f0ec2f8","added_by":"auto","created_at":"2025-10-06 06:07:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":183002,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of cholesterol solubilisation in the presence of ≤ 3 kDa hydrolysate fraction (A) and in the presence of cowpea analogue synthetic peptides (B), compared to standard micelle (without hydrolysate or peptides). (Mean ± SD). (*) p \u0026lt; 0.05 by \u003cem\u003et-Student \u003c/em\u003etest\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7612609/v1/38c453a51ea9163986e3241a.png"},{"id":92830256,"identity":"a08b1f6d-71a3-4808-aa6d-7852d2e94166","added_by":"auto","created_at":"2025-10-06 06:07:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":789076,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of phosphatidylcholine solubilised in the presence of ≤ 3 kDa hydrolysate fraction (A) and in the presence of cowpea analogue synthetic peptides (B) compared to standard micelle (0 mg/mL). Cholesterol micelles bile acids binding results. (C) Interaction with ≤ 3 kDa hydrolysate fraction. (D) Interaction with cowpea analogue synthetic peptides. The cholestyramine was used as positive control (mean ± SD). Quantification of cholesterol crystals formed in the presence and in the absence of ≤ 3 kDa hydrolysate fraction (E) and the cowpea analogue synthetic peptides (F) (mean ± SD). (*) p \u0026lt; 0.05 compared to standard micelle (without hydrolysate or peptides) by \u003cem\u003et‑Student \u003c/em\u003etest\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7612609/v1/72a19705859c8cd1863ba06d.png"},{"id":100379880,"identity":"3d138da5-310d-4152-ad10-c12fbca1e350","added_by":"auto","created_at":"2026-01-16 09:51:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2227318,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7612609/v1/e5e058a9-8ebe-44ad-93b4-090e0cb10f9f.pdf"},{"id":92830244,"identity":"c3a5ebf8-3e7a-45b0-bed8-54df386a0ca7","added_by":"auto","created_at":"2025-10-06 06:07:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":186455,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterialv2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7612609/v1/d1d2130cdf08adfcea6afa40.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanistic insights into cholesterol and micellar components insolubilisation by cowpea bioactive peptides in a model system","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDiet is an important source of cholesterol, and nutritional recommendations aim to control dyslipidaemia and atherosclerosis. A Westernised human diet typically provides approximately 300\u0026ndash;400 mg of cholesterol per day. An additional\u0026thinsp;~\u0026thinsp;1000 mg of cholesterol enters the intestinal lumen via bile secretion, most of which facilitates lipid absorption. Consequently, dietary cholesterol intake at levels close to the recommended amounts has only a modest impact on serum cholesterol. In contrast, there is substantial evidence that bioactive food compounds, such as dietary fibres and phytosterols, can reduce the intestinal absorption of both dietary and biliary cholesterol, thereby lowering blood cholesterol levels [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this context, the absorption of cholesterol and other lipids depends on their ability to form micelles within the intestinal lumen after food ingestion. Such micelles are formed when lipids from bile (i.e. phospholipids, cholesterol, bile salts such as taurocholate) and dietary lipids (triglycerides, phospholipids, cholesterol, phytosterols) reach a critical concentration and aggregate. Marrink and Mark [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] proposed a radial core\u0026ndash;shell model to describe the structure of micelles at pH 7, comprising a hydrophobic interior and a hydrophilic exterior. Phospholipid molecules are oriented radially, whereas bile salt molecules align parallel to the micelle surface, filling the gaps between phospholipid head groups. These groups protrude substantially into the aqueous phase, allowing the phosphate\u0026ndash;nitrogen dipole to adopt an almost parallel orientation, thereby enabling cholesterol solubilisation.\u003c/p\u003e\u003cp\u003eThe efficiency of cholesterol absorption has been extensively studied and is correlated with total cholesterol and low-density lipoprotein (LDL) serum concentrations in both humans and animals[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The concentrations and ratios of bile acids, phospholipids, and cholesterol in the gallbladder must remain within specific limits to avoid gallstone formation and to optimise micelle formation in the intestinal lumen. Assessing cholesterol solubility in bile salt micelles is critical for understanding its availability for intestinal absorption and for developing strategies to reduce epithelial internalisation. This topic has been the focus of some studies, as dietary compounds can reduce cholesterol absorption either by binding to bile acids or by disrupting micelle formation, thereby decreasing plasma cholesterol levels and lowering the risk of associated diseases[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHistorically, dietary proteins have been valued primarily for their amino acid content, essential for cellular maintenance, growth, and energy supply. A protein is considered of high biological value when it provides amino acids in proportions that closely match the body\u0026rsquo;s requirements. In addition to these traditional roles, bioactivity is now recognised as a significant attribute. Bioactive peptides derived from dietary proteins are typically short amino acid sequences ranging from 2 to 30 residues, capable of exerting one or more physiological effects in the human body. However, these sequences are latent within the native protein structure and exhibit bioactivity only after being released through processes such as enzymatic hydrolysis, fermentation, or food processing [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMany of the sources of bioactive peptides come from plant sources such as cowpea beans. Cowpea (\u003cem\u003eVigna unguiculata\u003c/em\u003e) is a well-established staple and has also been described as a functional food due to its protein and dietary fibre content. Although it has been consistently demonstrated that cowpea protein reduces the low-density lipoprotein (LDL) and total blood cholesterol levels in both hamsters and humans, the mechanisms underlying these effects remain nuclear[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOne of the hypotheses is that peptides released from incomplete digestion of cowpea proteins can reduce the micellar solubilisation of cholesterol in the intestinal lumen, thereby limiting its reabsorption [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Our previous studies also indicated that cowpea protein, following gastrointestinal enzymatic action, can inhibit 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), a key enzyme in hepatic cholesterol biosynthesis[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. \u003cem\u003eIn silico\u003c/em\u003e analyses further suggested that the cowpea peptide Gln\u0026ndash;Gly\u0026ndash;Phe (QGF) has a significant probability (p\u0026thinsp;\u0026le;\u0026thinsp;0.100) of interacting with HMGCR [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, the overall magnitude of cholesterol reduction observed in vivo cannot be fully explained by inhibition of the biosynthetic pathway alone, raising further mechanistic questions.\u003c/p\u003e\u003cp\u003eIt is well established that phenolic compounds and plant sterols can alter the composition of cholesterol micelles, often increasing micellar size within the intestinal environment. The physicochemical interactions responsible for these changes, however, are not fully understood. Alterations in micelle size may affect their recognition by enterocyte brush-border receptors, hindering absorption and promoting faecal excretion[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Similarly, changes in micellar composition may reduce their efficiency in solubilising cholesterol and other lipids. For instance, hydrophobic peptides may compete with cholesterol for micellar solubilisation sites, thereby reducing or blocking cholesterol absorption in the intestine[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThus, the purpose of this study was to investigate the effect of natural and synthesized peptides from cowpea beans crop on the structure and composition of artificial cholesterol micelles.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCowpea seeds (\u003cem\u003eVigna unguiculata\u003c/em\u003e L. Walp), cultivar BRS Mil\u0026ecirc;nio, were supplied by EMBRAPA (Brazilian Agricultural Research Corporation, Brazil). The seeds were milled (M 20 universal mill, IKA\u0026reg;, Germany) and sieved (0.42 mm sieve) to obtain cowpea flour.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Protein preparation\u003c/h2\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\u003ch2\u003e2.1.1. Protein isolation\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCowpea protein isolate was obtained through conventional methods of alkaline solubilisation and isoelectric precipitation, as described previously[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The cowpea flour was first defatted with hexane (1:6, w/v) during 4 hours and dried overnight for solvent evaporation. The defatted flour was stirred for 2 h at 25 ◦C in ultra-pure water (1:10, w/v), at pH 8.5, adjusted by addition of NaOH (1 mol.L\u003csup\u003e-1\u003c/sup\u003e), and centrifuged (10,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 20 min, at 4 ◦C). Afterwards, the supernatant was precipitated at pH 4.5 (corresponding to the average isoelectric point) by addition of HCl (1 mol.L\u003csup\u003e-1\u003c/sup\u003e) and centrifuged at 10,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 20 min (4 ◦C). The insoluble fraction, representing the cowpea isolated protein, was collected. Targeting improved purity, the protein isolates were defatted again according to Bligh and Dyer method[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The moisture and protein content were analysed according to the method 950.46 and 923.03 from AOAC[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To calculate the total nitrogen, the conversion factor 6.25 was used.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.1.2. \u003cem\u003eCowpea protein hydrolysis\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe protein cowpea isolate was hydrolysed using an enzyme/substrate ratio of 1:1,000 (w/w of protein) in 2% protein solution (w/v) according to Meg\u0026iacute;as et al.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], with modifications. This ratio was chosen based on preliminary tests that minimized the presence of peptides from the proteolytic enzymes. Pepsin (SIGMA p-7012 activity\u0026thinsp;\u0026ge;\u0026thinsp;2,500 units/mg protein) was used first (37\u0026deg;C for 2 hours, pH\u0026thinsp;=\u0026thinsp;2) and, subsequently, pancreatin (from porcine pancreas, SIGMA p-7545, activity equivalent to 8 x U.S.P. specifications) was added to the hydrolysis medium (37\u0026deg;C for 2 hours, pH\u0026thinsp;=\u0026thinsp;7). The sample was centrifuged (10,000 \u0026times; \u003cem\u003eg\u003c/em\u003e/15 min), the supernatant collected and filtered through a 3 kDa molecular weight cut-off (MWCO) membrane to isolate the low molecular weight peptides. The protein content was analysed according to the method 923.03from AOAC[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To calculate the total nitrogen, the conversion factor 6.25 was used. The \u0026le;\u0026thinsp;3kDa hydrolysate fraction was used in the \u003cem\u003ein vitro\u003c/em\u003e assays.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.1.3. Cowpea peptides synthesis\u003c/h2\u003e\u003cp\u003eOur previous study [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] on the \u003cem\u003ein silico\u003c/em\u003e prediction of the biological activity of several short peptides in the \u0026le;\u0026thinsp;3 kDa fraction of the cowpea hydrolysate, revealed three peptides that are prone to alter cholesterol homeostasis: Leu-Leu-Ans-Pro-Asp-Asp-Glu-Gln-Leu (LLNPDDEQL); Phe-Phe-Phe-Gly-Gln-Asp-Gly-Gly-Ser-Lys-Gly-Glu-Glu (FFFGQDGGSKGEE) and Leu-Asn-Leu (LNL) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. They were synthesized by Aminotech Research and Development (Diadema, SP, Brazil) with a prity greater than 95 % without further modifications. The structure of the synthetic peptides was confirmed by mass spectrometry (supplementary material).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePeptide sequences present in cowpea bean\u0026thinsp;\u0026le;\u0026thinsp;3 kDa hydrolysate fraction and their potential biological activity, physical-chemical properties and protein source\u003c/p\u003e \u003cdiv class=\"Credit\"\u003e\u003cp\u003e(adapted from Marques, et.al. 2015a).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePeptide\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eObserved molecular weight /theoretical molecular mass (Da) \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePossible biological activities\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTotal hydrophobic ratio (%)\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIso-electric point \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNet charge at pH 7 \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCowpea protein source\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFFGQDGAVVAGSC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1256.5/1257.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eACE inhibitor; neuropeptideinhibitor; dipeptidyl-aminopeptidase IV inhibitor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e53.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003epH 3.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eZeaxanthinepoxidase; Glutathione reductase\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLLNPDDEQL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1055.5/1056.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eACE inhibitor; dipeptidyl-aminopeptidase IV inhibitor; Glucose uptake stimulating peptide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e30.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003epH 2.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAcetyl-CoA carboxylasecarboxyltransferase;Phospholipase D alpha 1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLNL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e358.2/358.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eACE inhibitor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e66.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003epH 6.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo significant similarity found\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u003c/sup\u003ePeptides with PEAKS ALC score of 50% or greater\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003eb\u003c/sup\u003eDetermined using BIOPEP\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003ec\u003c/sup\u003eCalculated the percentage of hydrophobic residues (I, V, L, F, C, M, A, W) in the peptide sequence.\u003c/p\u003e\u003cp\u003e\u003csup\u003ed\u003c/sup\u003eDetermined using BLAST tool\u003c/p\u003e\u003cp\u003e\u003csup\u003ee\u003c/sup\u003e Determined using Innovagen\u0026acute;s peptide property calculator\u003c/p\u003e\u003cp\u003eNext, the impact was tested of the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction and the three synthesized peptides on cholesterol solubilisation, micelle size, bile acid binding, phosphatidylcholine binding and \u003cem\u003ein vitro\u003c/em\u003e cholesterol crystal formation.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Preparation of in vitro cholesterol micelles\u003c/h2\u003e\u003cp\u003eMicelles were prepared according to the method of Zhang et al.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The lipids (final concentrations in the aqueous buffer: 0.5 mM cholesterol, 1 mM 9-\u003cem\u003ecis\u003c/em\u003e,12-\u003cem\u003ecis\u003c/em\u003e-linoleic acid and 2.4 mM phosphatidylcholine from egg: 1,2-Diacyl-\u003cem\u003esn\u003c/em\u003e-glycero-3-phosphocholine) were dissolved in methanol and added to tubes of 2 mL and dried under N\u003csub\u003e2\u003c/sub\u003e flow. The dried lipid mixture was combined with 500 \u0026micro;L of 15 mM sodium phosphate monobasic buffer containing 6.6 mM taurocholic acid sodium salt and 132 mM NaCl at pH 7.4. Thus, the [phosphatidylcholine]/([ phosphatidylcholine] + [bile acids]) ratio was 0.26. This ratio is considered the Cholesterol Saturation Index (CSI) and it was calculated based on Carey\u0026rsquo;s critical tables[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This index represents the maxima amount of cholesterol dissolved in bile acids in an equilibrium of solubility. All the chemicals were purchased from Sigma-Aldrich\u0026reg;. The suspension was submitted to sonication in an ice bath for 15 min and incubated for a further 30 min at 37\u0026deg;C to form the micelles.\u003c/p\u003e\u003cp\u003eWhen required, \u0026le; 3 kDa hydrolysate fraction (ranging from 1 to 200 mg/mL in protein) and peptides (0.3 mg/mL to 1 mg/mL) were added and the mixture was submitted to sonication for 1 min and incubated for 1 h at 37\u0026deg;C. The solution was then centrifuged at 1,000 x \u003cem\u003eg\u003c/em\u003e for 10 min and filtered through a 0.20 \u0026micro;m Millex-GP filter (Millipore, Bedford, MA, USA). All analyses were performed after 2 days to ensure the system stabilization. The micelle formation was verified by fluorescence spectroscopy described below.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Fluorescence spectroscopy\u003c/h2\u003e\u003cp\u003eFollowing the work of Greenspan and Fowler[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], we used the solvatochromic dye Nile Red as a reporter of micelle formation and changes in the micelles upon addition of the \u0026le;\u0026thinsp;3 kDa hydrolysate and peptides. To this end, samples were prepared by adding 0.83 \u0026micro;L of a 0.1 mg/mL Nile Red solution in acetone to 0.5 mL of a micellar solution. Fluorescence emission spectra were recorded at 25\u0026deg;C in a wavelength range from 575 to 700 nm upon excitation at 549 nm on a Varian\u0026reg; Cary Eclypse fluorimeter equipped with a xenon lamp. The normalized fluorescence intensity of the samples was obtained after subtraction of the buffer solution spectrum (blank). For that, this experiment monitored the micelle formation with 0.5 mM cholesterol (standard micelle as shown in above section) and without cholesterol (buffer plus taurocholate without cholesterol) used as a control of spontaneous micelle formation. The baseline containing only phosphate buffer (without taurocholate and without cholesterol) was also controlled. In the same micelle with cholesterol (0.5 mM cholesterol), it was added hydrolysate and peptides to test the variation on Nile Red spectra, searching for blue shift. All samples tested were containing the same amount of Nile Red.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Cholesterol solubilisation in micelles\u003c/h2\u003e\u003cp\u003eThe impact of the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction and peptides on cholesterol solubilisation into micelles was examined spectroscopically. The compounds were separately added to a micelle solution, which was sonicated in a bench top ultrasound bath (Transsonic Digital S Elma) during 1 min, and subsequently incubated for 1 h at 37\u0026deg;C. The solution was then centrifuged at 1,000 x \u003cem\u003eg\u003c/em\u003e for 10 min and the supernatant filtered through 0.20 \u0026micro;m Millex-GP filters (Millipore, Bedford, MA, USA) to exclude other non-micelles fractions. After filtration, 50 \u0026micro;L of this solution was collected, and its cholesterol concentration was determined using Amplex\u0026reg; Red Cholesterol Assay Kit (Invitrogen, Paisley, UK) by fluorescence according to the manufacturer instructions. Excitation wavelength was 555 nm and emission detection was at 590 nm, measured with SpectraMax M5 equipment (Molecular Devices, Sunnyvale, CA, USA). Cholesterol standard curves were obtained using calibration standards.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Micelle sizing by dynamic light scattering\u003c/h2\u003e\u003cp\u003eThe apparent hydrodynamic radii of the micelles were measured by dynamic light scattering (DLS). The experiments were performed on an ALV/CGS-3 MD-4 compact goniometer system equipped with a Multiple Tau digital real time correlator (ALV-7004) and a solid-state laser (λ\u0026thinsp;=\u0026thinsp;532 nm; 40 mW). Typical experiments cover scattering angles from 60 to 120\u0026deg;, averaging over 3 x 20 sec runs at 20\u0026deg;C. As a control, the sodium phosphate monobasic buffer containing 6.6 mM sodium taurocholate salt and 132 mM NaCl at pH 7.4 was also measured; here, spontaneous micelle formation was not detected.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Phosphatidylcholine solubilisation in micelles\u003c/h2\u003e\u003cp\u003eTo evaluate the interaction between micellar phosphatidylcholine and the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction and peptides, the micelle solution was prepared and stored at 37\u0026deg;C. Two hundred microliters of two different concentrations of each peptide (0.3 and 1 mg/mL) were added to the 6 mL micellar solutions. The mixture was incubated for 1 h at 37\u0026deg;C and then centrifuged at 25,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 1 h. In order to exclude other non-micelles fractions, the supernatant was passed through a 0.20 \u0026micro;m Millex-GP filter (Millipore, Bedford, MA, USA) syringe filter, and the phosphatidylcholine solubilisation in the presence and absence of peptides was quantified according to a phospholipids assay kit (n\u0026deg; cat. MAK122, Sigma-Aldrich\u003csup\u003e\u0026reg;\u003c/sup\u003e) in the filtrate, according to the manufacturer instructions[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This method is based on the enzymatic hydrolysis of the choline fraction present in the phospholipids and their subsequent spectrophotometric detection at 570 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Bile acid binding assay\u003c/h2\u003e\u003cp\u003eThe interaction between the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction and peptides with micellar bile acids was assessed according to Yoshie-Stark and Wasche[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Different concentrations of the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction and peptides were added in phosphate buffer solution (0.1 M) containing sodium taurocholate 2 mM at pH 7. After incubation at 37\u0026deg;C for 2 h, each sample was centrifuged, and the supernatant was transferred to a volumetric flask. A further 1 mL of 0.1 M sodium phosphate buffer at pH 7.0 was added to the sediment, thoroughly mixed and centrifuged. The supernatant was removed and combined with the earlier supernatants. This procedure was repeated, and the supernatants were collected to the existing supernatants in a volumetric flask. The concentration of bile acids was measured spectrophotometrically at 405 nm according to the manufacturer instructions of the Total Bile Acids Assay kit (Diazyme, Poway, CA, USA).\u003c/p\u003e\u003cp\u003eThe experimental data was obtained from a calibration curve obtained using calibrated cholic acid standard solutions. Cholestyramine resin, a drug that binds bile acid and lowers cholesterol, was also evaluated for its ability to bind bile acid. All analyses were performed in triplicate. The capacity of bile acid binding was expressed as \u0026micro;mol of bile acids per gram of sample (precipitate after centrifugation).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Separation of micelles and cholesterol crystals\u003c/h2\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.8.1. The intermixed micellar bile salt concentration (IMC) measurements\u003c/h2\u003e\u003cp\u003eThe intermixed micellar/vesicular (non-phospholipid associated) bile salt concentration (IMC) is the sum of the concentrations of monomeric and simple micellar bile salt. This parameter was measured firstly to determine the minimum amount of bile salt (deoxycholate) required to prepare the cholesterol crystals for quantitative analysis (\u003cem\u003evide supra\u003c/em\u003e). Briefly, a 10 kDa Centrisart ultrafilter was rinsed with ultrapure water and submitted to centrifugation for 5 min at 500 \u0026times;\u003cem\u003eg\u003c/em\u003e. The remnant water after centrifugation was totally removed by a syringe. The filter was then pre-incubated during 1 h at 37\u0026deg;C. After the incubation, 2 mL of micellar solution without \u0026le;\u0026thinsp;3 kDa hydrolysate fraction or peptides were added, centrifuged in a 10 kDa Centrisart ultrafilter for 5 min at 500 \u0026times; \u003cem\u003eg\u003c/em\u003e for three times, and the filtrates pooled. In the filtrates, the amount of bile acids was quantified to determine the IMC value.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.8.2. Separation and quantification of the amount of cholesterol crystals and micelles\u003c/h2\u003e\u003cp\u003eAiming to evaluate whether cholesterol crystals form and if so, separate these from the micellar phase, the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction and peptides were first added to a solution of cholesterol micelles, after which it was submitted to centrifugation for 10 minutes. After then, it was added sodium deoxycholate in a sufficient amount to desaturate the model system (cholesterol saturation index\u0026thinsp;\u0026lt;\u0026thinsp;1)[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Subsequently 2 mL of taurocholate solution 197 mM were then added to a 300 kDa Centrisart ultrafilter and centrifuged for 5 minutes at 500 \u0026times; \u003cem\u003eg\u003c/em\u003e. The residues were carefully withdrawn by a syringe and discarded.\u003c/p\u003e\u003cp\u003eThe minimum concentration of deoxycholate was determined previously (197 mM), based on IMC[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and it was checked against tabulated values to determine the cholesterol saturation index[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNext, each micellar solution was centrifuged at 50,000 \u0026times; \u003cem\u003eg\u003c/em\u003e (37\u0026deg;C for 30 minutes). The supernatant was collected and applied on a disposable filter and submitted to gravitational filtration during 1 h. The mass of cholesterol in crystals was determined in the filtered fraction using the assay \u003cem\u003eAmplex \u0026reg; red\u003c/em\u003e (Invitrogen, Paisley, UK).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using analysis of variance (ANOVA) and when necessary, a Tukey multiple comparison \u003cem\u003epost hoc\u003c/em\u003e test was run, with in both cases \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 signalling significant differences between the studied conditions. To compare the systems where the proteins or peptides were added to the control micellar solution, the t-Student paired test with also signalling significant differences in \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 was used. The statistical analyses were performed using software GraphPad Prism version 5 for Windows.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eAiming to advance the current understanding of the mechanisms through which hydrolysed cowpea protein, and its peptides reduce the solubility of cholesterol in cholesterol mixed micelles and thereby cholesterol absorption by the gut and cholesterol plasma concentrations, we herein study the effect of cowpea hydrolysate and peptides on the structure and composition of artificial cholesterol micelles. There are different pathways which may contribute to the way in which cowpea hydrolysate and peptides may disturb micelle formation and impair recognition of the micelles by the border-brush intestinal cell receptors. These include changes in micellar dimensions and composition, e.g., variations in the micellar content of cholesterol, phosphatidylcholine, and bile acids, as well as cholesterol crystallisation.\u003c/p\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Impact of cowpea derived peptides on micelle formation\u003c/h2\u003e\u003cp\u003eThe formation of cholesterol micelles and changes induced by hydrolysates and peptides were monitored using Nile Red fluorescence emission spectra with and without added hydrolysate and peptides (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). According to Greenspan and Fowler[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], Nile Red exhibits a blue shift in emission when bound within hydrophobic environments, such as micelles, but shows negligible fluorescence in water (λ\u003csub\u003eexc\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;549). It shifts increasingly towards the blue if the local environment becomes more hydrophobic. In this situation, when binding to cholesterol which is internalised within the hydrophobic environment of a micelle. As expected, we observe no discernible signal in the phosphate buffer (control sample), since this does not contain any micelles. A modest signal peaked around an emission maximum at λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;641 nm appears when bile salt (taurocholate) but no cholesterol is added, signalling the presence of more hydrophobic domains. A marked increase in emission and blue shift of the emission maximum to λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;629 nm is observed for the micellar solution containing 0.5 mM cholesterol. Interestingly, the emission maximum remains fairly unaffected at λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;629 \u0026plusmn; 1 nm, whilst the fluorescence intensity is noticeably reduced upon addition of 50 mg/mL and 100 mg/mL hydrolysate. Similarly, addition of either LLNPDDEQL, FFFGQDGGSKGEE or LNL also reduces the fluorescence intensity (emission maximum respectively λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;625 nm, λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;628.9 nm and λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;631 nm). The decrease in Nile Red fluorescence in all samples with hydrolysate and peptides signals a reduction in cholesterol solubilisation, which is presumably due to displacement by the hydrolysate and peptides of cholesterol from the micelle.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Impact of cowpea derived peptides on micellar dimensions\u003c/h2\u003e\u003cp\u003eThe literature reports conflicting \u003cem\u003ein vitro\u003c/em\u003e results regarding experiments about variations in micellar dimensions. It seems possible that larger and smaller mixed micelles could negatively impact the intestinal absorption of cholesterol[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To examine whether cowpea hydrolysate and peptides affect micellar dimensions to such a large extent, dynamic light scattering experiments were performed to determine the hydrodynamic radius \u003cem\u003eR\u003c/em\u003e\u003csub\u003eh\u003c/sub\u003e of the micelles as a function of the added amount of hydrolysate and peptides (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The size of the cholesterol micelles varied little up to \u0026le;\u0026thinsp;3 kDa hydrolysate concentrations of 10 mg/mL, whilst the micelles grew larger in a dose-dependent manner for hydrolysate concentrations above 100 mg/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Surprisingly, the addition of the analogous synthetic peptides, led to a reduction in micellar size for much lower peptide concentrations up to 1 mg/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Addition of 1 mg/mL of the FFFGQDGGSKGEE and LNL peptides reduced micellar dimensions from about 65.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 nm in the absence of either to 58.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 nm (FFFGQDGGSKGEE) and 59.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 nm (LNL), for respectively. Nonetheless, according to \u003cem\u003ein vitro\u003c/em\u003e experiments on the cholesterol absorption, neither the up to 3-fold increase induced by the hydrolysate, nor the slight decrease due to the synthetic peptides is sufficiently large to significantly affect the cholesterol absorption through the gut[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Cholesterol micellar solubilisation \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eSince changes in micelle size alone did not account for the reduced cholesterol absorption previously reported at 1 mg/mL compound concentration[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], we next examined cholesterol solubilisation directly. Both the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction and the synthetic peptides reduced micellar cholesterol content in a dose-dependent manner. The \u0026le;\u0026thinsp;3 kDa hydrolysate fraction decreased cholesterol solubility by 50\u0026ndash;60% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), whereas peptides reduced solubility by 20\u0026ndash;30%. Although the peptide concentrations required were relatively high, such levels are physiologically achievable in the intestinal lumen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Peptide-phosphatidylcholine and peptide-bile acids interaction\u003c/h2\u003e\u003cp\u003eTo examine whether cowpea peptides interfere with other components in the micelles, the phosphatidylcholine and bile acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) solubilized in the presence of the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction and the analogue synthetic peptides were quantified. Absorbance measurements revealed an increase in phosphatidylcholine dispersion compared with controls. The \u0026le;\u0026thinsp;3 kDa hydrolysate fraction increased solubilised phosphatidylcholine nearly threefold (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), while synthetic peptides produced a similar, though less pronounced, effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eThe Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed depicts the peptide-bile acids interaction with cholestyramine being used as positive control. Cholestyramine is a drug that has been used in hypercholesterolemia treatments capable of binding intestinal bile acids. The bile acids COO\u003csup\u003e-\u003c/sup\u003e tail is the main structural target for anionic swap, promoted by cationic resins, clinically used as bile acids abductors. Food proteins from soy, white rice, fishes, milk goat and others were described as bile acids abductors. They reduce the amount of bile acids recycled in the liver, and increase the bile acids synthesis by cholesterol clearance from plasma[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It is possible to observe that the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction was able to bind the bile acids at 200 mg/mL.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed shows that there were not significant differences in bile acids binding by the synthetic analog cowpea peptides, except by \u0026le;\u0026thinsp;3 kDa hydrolysate fraction at 200 mg/mL and the LNL peptide.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Cholesterol crystals formation\u003c/h2\u003e\u003cp\u003eFinally, the potential for cholesterol crystallisation was examined. Micelles phase containing peptides or hydrolysates were centrifuged, and crystallised cholesterol was quantified \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e. Only cowpea analogue synthetic peptides induced cholesterol crystal formation, whereas the hydrolysate fraction did not. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe maximum emission spectra of Nile Red depend on the environment polarity. Variations in fluorescence spectra of the solubilised Nile Red indicate the presence of different association complexes in solution (i.e. micelles, bilayers membranes, vesicles)[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This study corroborates previous findings [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] that is possible simulate a stable human micelle \u003cem\u003ein vitro\u003c/em\u003e with a hydrophobic internal core filled with cholesterol. A direct relationship was also observed between the cholesterol content of micelles and the fluorescence signal intensity.\u003c/p\u003e\u003cp\u003eIt is known that enzymatic digestion of proteins leads to extensive chemical modifications in the final products and may alter micellar dimensions. Reynier and co-workers [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] attested that the diffusion rate of the micelles through the unstirred water, layer decreases as micellar size increases, suggesting a decrease in intestinal absorption of huge cholesterol micelles. Our earlier study showed that 1 mg/mL of the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction sufficed to impair cholesterol solubilisation, though micellar size was not assessed at that time[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs a result, clearly the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction could not alter the micelle radius at lower concentrations tested (less than 100 mg/mL) as compared, for example, with other compounds such as phytosterols[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Significant increases in micelle size were observed only at hydrolysate concentrations above 100 mg/mL of \u0026le;\u0026thinsp;3 kDa hydrolysate fraction, far above that where significant reduction in cholesterol solubilisation was observed. This suggests that the reduced cholesterol solubilisation at hydrolysate concentrations below 100 mg/mL was mediated by mechanisms other than micellar size modification.\u003c/p\u003e\u003cp\u003eConsidering Khoshakhlagh [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] report on a drug-delivery system based on cholesterol micelles, these results raised the possibility of a non-linear relationship between solubilisation of cholesterol and the increase or decrease in the micelle size in the presence of protein hydrolysate as it is common with drugs in a micellar system. Similarly, Hu and colleagues [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] did not verify apparent change in the size distribution data at different dosages of \u003cem\u003eβ\u003c/em\u003e-sitosterol glycosyl derivatives in presence of cholesterol micelles. They concluded that \u003cem\u003eβ\u003c/em\u003e-sitosterol glycosyl derivatives preferred to interact with bile salts at the interface of micelles rather than being incorporated into the hydrophobic cores of micelles. The reasons are due to their amphiphilic structure, comprising hydrophilic polar groups and a hydrophobic \u003cem\u003eβ\u003c/em\u003e-sitosterol tail. This decrease in size supported the interaction of \u003cem\u003eβ\u003c/em\u003e-sitosterol glycosyl derivatives with cholesterol-loaded bile salt micelles\u003c/p\u003e\u003cp\u003eNevertheless, the cholesterol solubility was found reduced by about 50\u0026ndash;60% after adding the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction in a dose-dependent manner. Although relatively high concentrations of cowpea peptides were required, such levels are physiologically attainable following legume consumption and digestion. Comparable reductions in micellar cholesterol solubility have been reported for milk casein (four peptides at 3 mg/mL decreased 32\u0026ndash;53%) using the same protocol[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and gluten hydrolysates\u0026thinsp;\u0026lt;\u0026thinsp;1kDa from wera able to inhibit 47.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71% of micellar cholesterol solubilisation at 2 mg/mL[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlthough the displacement of cholesterol by peptides was evident, the precise mechanism remains unclear. A possible explanation would be that hydrophobic peptides interfere with the structural formation of the micelles, competing then with cholesterol as phytosterols do [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, when using peptides with distinct hydrophobicity in our study, the absolute effects of reduction of cholesterol solubility among peptides are comparable. In line with this, the pentapeptide IIAEK, derived from β-lactoglobulin, was shown to reduce cholesterol levels in an \u003cem\u003ein vivo\u003c/em\u003e model, although it did not alter cholesterol or bile acid solubility \u003cem\u003ein vitro\u003c/em\u003e. The authors suggested that the peptide may act as a surfactant or interfere with gene expression [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt is plausible that peptides act via multiple mechanisms simultaneously. Therefore, the hydrophobicity does not fully explain the peptide action. Isolated proteins and phospholipids can present synergism or antagonism in the stability of colloidal systems. Interaction between proteins and phospholipids can lead to changes in their superficial activity, in their structural conformation and in their incorporation in surfactant-based structures, such as micelles and vesicles [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In Brown and co-worker\u0026acute;s [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] study, phytosterols were also able to reduce cholesterol solubility without interfering with micelle size. Intending to clarify this behaviour, we evaluated whether cowpea peptides interact with phosphatidylcholine or bile acids.\u003c/p\u003e\u003cp\u003eFood-derived components have previously been shown to bind phosphatidylcholine. For instance, the spectroscopic study done with curcumin to assess the peptide interaction with phosphatidylcholine suggests that an absorbance increase, is a consequence of an increase of the hydrophobic environment in the micelles [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The α-lactalbumin from milk was also described as being able to penetrate in phosphatidylcholine vesicles through the most hydrophobic regions of the protein[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHence, the cowpea protein seems to reorganize the structure; changing the micellar composition due to effects sum between fluctuation of cholesterol and phosphatidylcholine concentrations. Thus, the cholesterol is not satisfactory solubilised, or it is inefficiently incorporated into the mixed micelle formed. Again, the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction of cowpea was more effective than the isolated peptides from cowpea bean, which exhibit a tiny action featuring the LNL sequence; and this effect does not seem to be dose-dependent. Similarly to α-lactalbumin and hydrolysed dry beans (\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e L.) protein, the hydrophobicity of the LNL peptide can explain the featured effect[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Despite of being a hydrophobic peptide, the mechanism of protein action on cholesterol micelles (prepared with phosphatidylcholine) is not the same found for other polyphenols that consider the hydrophobicity as an important chemical characteristic condition[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBile acid binding represents another potential mechanism. Peptides, including those from soy and amaranth, can sequester bile acids at levels comparable to cholestyramine, reducing cholesterol solubility[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. However, this action seems to be sensitive to many parameters such as the hydrolysis conditions (enzymes used, time, temperatures) and bile acids type.\u003c/p\u003e\u003cp\u003eThe information about the structure-function of peptides/proteins and the intestinal bile acid sequestration is scarce. A successful bile acids binding is explained for their action points towards a low digestibility and a higher amount of hydrophobic amino acids in the structure[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The study of Johns and Bates [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] proved that the cholestyramine has affinity dependent in part on the extent of the hydrophobic character of bile salt anions to the possibility the anionic swap. In our system the LNL (hydrophobic), in fact, could not perform this swap due to its net neutral charge at pH\u0026thinsp;=\u0026thinsp;7, leading to insolubility. Moreover, the peptide was also engaged with phosphatidylcholine.\u003c/p\u003e\u003cp\u003eThe increase in bile acid solubilisation observed with LNL is consistent with models of cholesterol\u0026ndash;bile acid crystallisation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and more recently corroborate by Matsuoka [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] about the crystallization mechanisms of cholesterol and bile acids. A relationship between the micellar solubilisation and the peptide-bile acid binding can be established; being this peptide responsible for the composition change of the micelles. The increase of the bile acids solubilisation in the system causes the reduction of ([phospholipid]/([bile acids\u0026thinsp;+\u0026thinsp;phospholipid]) ratio. Interestingly, the alteration in this micellar equilibrium (with bile acids concentration changing) is associated with a higher precipitation of cholesterol crystals.\u003c/p\u003e\u003cp\u003eAlthough the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction reduces cholesterol solubilisation, it has no effect in promoting the cholesterol crystallization. This is directly correlated with the increase of phosphatidylcholine that is solubilised in the presence of \u0026le;\u0026thinsp;3 kDa hydrolysate fraction. The excess of phosphatidylcholine liberated from the micelles can act as a cholesterol binder, not necessarily forming crystals. This binding capacity was demonstrated by Yang et.al.[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], who observed that egg phosphatidylcholine attenuated cholesterol solubilisation and its absorption. The strong cholesterol inhibitory effect observed is associated with the higher degree of saturation and the long chains of its acyl groups in the phosphatidylcholine molecule, able to reorganize the micellar structure.\u003c/p\u003e\u003cp\u003eCholesterol is critical for micelle stability, and its removal disrupts micelle self-organisation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Summarizing the events, the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction binds phosphatidylcholine and then cholesterol, forming a large complex. This rearrangement is responsible for micelle size increasing in the presence of \u0026le;\u0026thinsp;3 kDa hydrolysate fraction. On the other hand, in the presence of the isolated peptides, a significant precipitation of cholesterol crystals is observed. Even though this is not a dose-dependent phenomenon, these results show that the solubilisation of cholesterol is due to the alteration of ([phospholipid]/([bile acids\u0026thinsp;+\u0026thinsp;phospholipid]) ratio, and the crystal formation are caused by the peptides tested. Several reports with food proteins describe the various possible mechanisms of protein/peptide \u0026ndash; micelle interaction; however, none of them details which is the main target of this interaction: cholesterol, phosphatidylcholine or bile acids[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The present study shows that the mechanism by which less cholesterol is incorporated into the micelles is through the competition between the micellar constituents (phosphatidylcholine or bile acids) and the cowpea derivatives, rather than specifically with cholesterol. This raises multiple possibilities for the inhibition of cholesterol absorption by the hydrolysates and peptides.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study extends our knowledge about the physical-chemical interaction between cholesterol micelle components and peptides generated from diet. Our experiments have shown that the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction, and all tested peptides could reduce the cholesterol internalisation of \u003cem\u003ein vitro\u003c/em\u003e micelles. However, the changes in micelle size are not related only to this lack of cholesterol inside the structure. The mechanism of lower cholesterol internalization in micelles by the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction and LNL peptide is related to peptides\u0026rsquo; binding to phosphatidylcholine and then to cholesterol, forming a large complex. The hydrolysate binding with bile acids was less effective.\u003c/p\u003e\u003cp\u003eThe mechanisms of cholesterol insolubilisation achieved by LLNPDDEQL, FFFGQDGGSKGEE and LNL peptides are a consequence of a combination between bile acids and phosphatidylcholine interaction with the peptides tested. However, the mechanism of the peptides additionally showed some kind of direct interaction with cholesterol due to the formation of cholesterol crystals. In all cases, the peptides are less effective than the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction.\u003c/p\u003e\u003cp\u003eA key contribution of the present study was to verify that, unlike polyphenols and phytosterols, the mechanism of competition for the intra-micelle space with cholesterol is better explained by an interaction of peptides with other micelle constituents rather than a change in their size or a direct interaction with the cholesterol in the micelles.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interest\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Marcelo Rodrigues Marques , Neus Vilanova Garcia and Gustavo Guadagnucci Fontanari. The first draft of the manuscript was written by Marcelo Rodrigues Marques with contribution of Ilja Karina Voets and Jose Alfredo Gomes Ar\u0026ecirc;as. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe author MR Marques is grateful to FAPESP (Foundation for research support of the State of S\u0026atilde;o Paulo, Brazil) [grant 2013/09304-2], [grant 2012/15900-4] and CNPq (National Council for Scientific and Technological Development) [PhD grant 202992/2015-2]. This research is also part of the research program of the DPI (Dutch Polymer Institute), project #772ap. The authors wish to thank Rosana A.M. Soares-Freitas and Cintia P. Silva for technical support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSchade, D.S., et al., \u003cem\u003eResolving the Egg and Cholesterol Intake Controversy: New Clinical Insights Into Cholesterol Regulation by the Liver and Intestine.\u003c/em\u003e Endocrine Practice, 2022. \u003cstrong\u003e28\u003c/strong\u003e(1): p. 102-109.\u003c/li\u003e\n\u003cli\u003eFogacci, F., et al., \u003cem\u003eCholesterol-Lowering Bioactive Foods and Nutraceuticals in Pediatrics: Clinical Evidence of Efficacy and Safety.\u003c/em\u003e Nutrients, 2024. \u003cstrong\u003e16\u003c/strong\u003e(10): p. 1526.\u003c/li\u003e\n\u003cli\u003eMarrink, S.J. and A.E. 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This study explored the mechanistic effects of hydrolysed cowpea proteins and sequence-defined synthetic peptides on cholesterol micelle structure and solubilisation in vitro. Synthetic peptides consistently reduced micelle size by at least 6 nm, whereas the \u0026le;\u0026thinsp;3 kDa hydrolysate fraction induced a threefold increase in micelle radius. This hydrolysate fraction also decreased cholesterol solubility and phosphatidylcholine content in a dose-dependent manner, forming insoluble aggregates through 50\u0026ndash;60% complexation with phosphatidylcholine and thereby disrupting micellar organisation. The three synthetic peptides reduced cholesterol solubility by 10\u0026ndash;20% relative to untreated micelles, effects attributed to altered bile acid and phosphatidylcholine composition, as well as potential direct peptide\u0026ndash;cholesterol interactions. These findings indicate that micellar competition between peptides and non-cholesterol constituents is a central mechanism modulating cholesterol solubilisation. By examining both complex hydrolysate fractions and precise peptide sequences, this study provides detailed mechanistic insight into cowpea protein activity. These results not only enhance our understanding of peptide\u0026ndash;micelle interactions but also establish an \u003cem\u003ein vitro\u003c/em\u003e foundation for future validation studies, informing potential dietary or therapeutic strategies aimed at modulating cholesterol absorption.\u003c/p\u003e","manuscriptTitle":"Mechanistic insights into cholesterol and micellar components insolubilisation by cowpea bioactive peptides in a model system","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-06 06:07:25","doi":"10.21203/rs.3.rs-7612609/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"301cf8f9-97e4-413b-a4b4-610fc26b573a","owner":[],"postedDate":"October 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-23T17:53:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-06 06:07:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7612609","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7612609","identity":"rs-7612609","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}