Article pubs.acs.org/JAFC
Dietary Egg White Protein Inhibits Lymphatic Lipid Transport in Thoracic Lymph Duct-Cannulated Rats Ryosuke Matsuoka,†,§,∥ Bungo Shirouchi,†,∥ Sayaka Kawamura,† Sanae Baba,† Sawako Shiratake,† Kazuko Nagata,† Katsumi Imaizumi,† and Masao Sato*,† †
Laboratory of Nutrition Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School of Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan § R&D Division, Kewpie Corporation, Sengawa Kewport, 2-5-7 Sengawa-cho, Chofu-shi, Tokyo 182-0002, Japan ABSTRACT: Dietary egg white protein (EWP) decreases serum cholesterol levels. We previously showed that EWP decreased cholesterol absorption in the intestine. Rats subjected to permanent lymph duct cannulation were used to investigate the effects of dietary EWP on lipid transport. They were fed diets with 20% EWP and casein, and their lymph was collected to quantify lymphatic lipid levels. Dietary EWP decreased lymphatic cholesterol transport compared with casein. It was previously shown that EWP excluded cholesterol from bile acid micelles. Therefore, pepsin-hydrolyzed EWP and casein were prepared. EWP was not completely digested. Ovalbumin, which is the most abundant protein in EWP, showed resistance to digestion by pepsin. This study investigated the effects of EWP pepsin hydrolysate (EWP-ph) on cholesterol micellar solubility, cholesterol transfer from the micellar to the oil phase, water-holding capacity (WHC), settling volume in water (SV), and relative viscosity and compared them with the effects of casein pepsin hydrolysate (C-ph). EWP-ph significantly decreased the micellar solubility and transfer rate and increased the WHC, SV, and relative viscosity compared with C-ph. Moreover, the pepsin hydrolysate of ovalbumin, a major protein in EWP, played a role in decreasing cholesterol micellar solubility, leading to the inhibition of cholesterol absorption. In conclusion, dietary EWP decreased cholesterol intestinal absorption by exerting combined effects of these physicochemical properties in the gut. KEYWORDS: egg white protein, ovalbumin, permanent thoracic lymph duct cannulation, cholesterol absorption
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INTRODUCTION Hen eggs are a cholesterol-containing food; therefore, their consumption can increase serum cholesterol levels.1 Worldwide, physicians recommend that patients with lifestyle-related diseases should avoid eggs. However, several papers have shown no correlation between egg consumption and serum cholesterol levels.2−5 Although eggs can provide cholesterol in the diet, the dietary cholesterol contained in eggs may not increase serum cholesterol levels.2−5 These reports suggested that eggs may contain components with serum cholesterollowering activity. Phosphatidylcholine (PC) is a major component of egg yolk and has a serum cholesterol-lowering effect.6,7 Egg yolk PC inhibited cholesterol absorption in the gut.8 In this mechanism, excess dietary PC, a component of micelles, shows an increased affinity to cholesterol; the PC undergoes digestion to become lyso-PC, which has a cholesterol affinity lower than that of PC.8 In addition, dietary phosphatidylethanolamine exerts a stronger hypocholesterolemic effect in rats.9 In addition, dietary egg white protein (EWP) decreases serum cholesterol levels in humans10 and rats.11 Our previous study showed that mechanisms underlying the cholesterol-lowering activity of EWP participated directly in micellar formation in the gut through the physicochemical properties of EWP.11 The cholesterol in the gut moves in and out of the micelles. Several food components such as plant sterols12 and catechins13 inhibit this behavior. EWP or digestive EWP may also inhibit cholesterol absorption, depending on its physicochemical properties.11 However, there is no existing report evaluating © 2014 American Chemical Society
the effect of EWP on lymphatic dietary lipid transport. In this study, we permanently cannulated the thoracic lymph duct to measure the lymphatic transport of dietary lipids in rats under near-physiological conditions.14,15 This method is superior because it evaluates the lymphatic lipid transport during actual dietary lipid absorption from a normal diet without restraint stress.14,15 In this study, we demonstrated EWP inhibition of intestinal cholesterol absorption by measuring the lipid transport in thoracic lymph duct-cannulated rats. Moreover, we determined the physicochemical properties, including micellar formation, of pepsin-digested EWP and the constituent proteins of EWP.
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MATERIALS AND METHODS
Materials. EWP was prepared from unsterilized egg white (Q. P. Egg Corp., Tokyo, Japan), which was freeze-dried and uniformly crushed. Casein was purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). The protein contents of EWP and casein were estimated by determining the total nitrogen content according to the Dumas method (AOAC 968.06).16 The nitrogen-to-protein conversion factor used for the calculation of protein content was 6.25. The measured protein content was 80.2% for EWP and 84.8% for casein. Ovalbumin (OA), ovotransferrin (OT), and ovomucoid (OM) were purchased from Sigma-Aldrich (Tokyo, Japan). Cholesterol, oleic acid, 1monooleoyl-rac-glycerol (monoolein), sodium taurocholate, pepsin, Received: Revised: Accepted: Published: 10694
June 14, 2014 September 1, 2014 October 9, 2014 October 10, 2014 dx.doi.org/10.1021/jf502741b | J. Agric. Food Chem. 2014, 62, 10694−10700
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Micellar Solubility of Cholesterol in Vitro. The micellar solubility of cholesterol was measured according to the method of Ikeda et al.20 A solution containing C-ph, EWP-ph, ovalbumin, ovotransferrin, ovomucoid, or lysozyme was added to the micelles. In the experiments shown in Figure 3A,C, the samples were added at a final concentration of 10 mg/mL to the micelles. In the experiment shown in Figure 4A, the samples were added at a final concentration of 5 mg/mL to the micelles. In the experiment shown in Figure 4B, EWP-ph was added at a final concentration of 10 mg/mL to the micelles, whereas the reconstituted EWP-ph was added at a final concentration of 8 mg/mL to the micelles. In accordance with a previous study,21 the reconstituted EWP-ph included 54% OA-ph, 13% OT-ph, 11% OM-ph, and 3.5% Ly-ph. The samples were incubated at 37 °C for 1 h and passed through the 0.2 μm syringe filter. In the experiment shown in Figure 3A, pancreatin was added at a final concentration of 0.15 mg/mL during the incubation time. After lipid extraction from the filtrate, cholesterol levels were measured by gas chromatography (GC).20 Transfer of Cholesterol from the Bile Acid Micelle to Triolein. The transfer rate of triolein to cholesterol was measured according to the method of Hamada et al.22,23 One and a half milliliters of the micellar solution, 0.5 mL of triolein, and C-ph or EWP-ph (final concentration, 10 mg/mL) were placed in a plastic tube, which was flushed with N2 and sealed. The tubes were incubated at 37 °C for 1 h with shaking. After incubation, the contents of each tube were transferred to a sealed tube, and the oil and aqueous phases were separated by centrifugation at 100000g for 1 h at 37 °C. The oil and micellar phases were collected, and the cholesterol levels in each phase were analyzed by GC using 5α-cholestane as an internal standard. Water-Holding Capacities (WHCs) of Casein, EWP, and Its Pepsin Hydrolysate. The WHCs of casein, EWP, C-ph, and EWPph were measured as described previously.24 Samples (0.1 g) were placed on two filter papers fitted in a stainless steel container (hole diameter, 2.5 cm; diameter, 5 cm; height, 1.5 cm) with a polypropylene net. This weight was measured as dry weight. After being placed in a case with distilled water for 24 h at room temperature, the container was removed and weighed. The WHCs of casein, EWP, C-ph, and EWP-ph were evaluated by measuring the difference between the wet and dry weights. Each measurement was repeated three times. Settling Volumes (SVs) in Water of Casein, EWP, and Its Pepsin Hydrolysate. The SVs of casein, EWP, C-ph, and EWP-ph were measured as described previously.24 Samples (7 mg) were blended with 7 mL of distilled water in 10 mL graduated cylinders. The cylinders were left to stand for 24 h at room temperature. The SVs of casein, EWP, C-ph, and EWP-ph were evaluated on the basis of precipitation volume. Each measurement was repeated three times. Viscosities of Casein, EWP, and Its Pepsin Hydrolysate. The viscosities of casein, EWP, C-ph, and EWP-ph were measured using an Ostwald viscometer (Sansyo Co., Ltd., Tokyo, Japan) with a 0.75 mm capillary diameter, as described previously.24 Samples (1 g) were blended with distilled water. After being stored for 24 h at room temperature, 1% (v/v) suspending solutions were prepared in 100 mL volumetric flasks. The solutions were preheated at 37 °C for 15 min and the flow times determined. Each measurement was repeated three times. The viscosities of casein, EWP, C-ph, and EWP-ph were calculated relative to the viscosity of distilled water. Statistical Analysis. All values are expressed as the mean ± standard error of mean (SEM). Comparisons between two groups were performed by Student’s t test, whereas those among more than three groups were performed by using one-way ANOVA followed by the Tukey−Kramer multiple-comparison post hoc test. Differences were considered to be significant at P < 0.05. Statistical analysis was performed using StatView version 4.5 (Abacus Concept Inc., Berkeley, CA, USA).
and pancreatin were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Phosphatidylcholine and lysozyme (Ly) were obtained from Kewpie Corp. (Tokyo, Japan). Animals, Diets, and Permanent Thoracic Lymph Duct Cannulation. Seven-week-old male Sprague−Dawley rats (Kud:SD) were obtained from Kyudo (Kumamoto, Japan). The rats were housed individually in metal cages in a temperature-controlled room (21−23 °C) under a 12 h light/dark cycle. Experimental diets were prepared according to the recommendations of the AIN-93G17 and contained the following ingredients (in weight, %): casein or EWP (20), αcornstarch (13.2), sucrose (10), cellulose (5), mineral mixture (3.5), vitamin mixture (1), choline bitartrate (0.25), soybean oil (7), cholesterol (0.5), cholic acid (0.125), tert-butylhydroquinone (0.0014), and β-cornstarch (39.4236). The rats were trained to consume the casein diet as a basal diet twice a day from 10:00 to 11:00 a.m. and from 4:00 to 5:00 p.m., respectively, for 5 days. On day 6, the rats were anesthetized using nembutal before permanent cannulation of the thoracic duct lymph, as described previously.14,15 In brief, a cannula [SH silicon tube, 0.5 mm inner diameter (i.d.) and 1.0 mm outer diameter (o.d.), Kaneka Medics, Osaka, Japan] filled with heparinized saline was inserted into the thoracic duct and secured within the abdominal cavity. After surgery, the rats were returned to their cages and provided free access to isotonic glucose solution (139 mM glucose and 85 mM NaCl in distilled water). On days 7 and 8, the rats were provided with the casein diet twice a day, as described. On day 9, the rats were attached to a long polyethylene cannula (0.58 mm i.d. and 0.97 mm o.d.; Becton, Dickinson, and Co., Franklin Lakes, NJ, USA). The end of the cannula was 5−10 cm below the bottom of the cage to allow the lymph to drain. After a 20 min collection period, the rats were provided free access to the casein diet or the EWP diet for 30 min. Then, the lymph was collected every hour for 7 h. The rats had free access to deionized water throughout the feeding periods and during lymph collection. The collected lymph was maintained at 4 °C overnight before the fibrin was removed, and the samples were stored at −30 °C until lipid analysis. Following lymph collection, the rats were anesthetized with nembutal and sacrificed by exsanguination. This experiment was conducted according to the Guidelines for Animal Experiments of Kyushu University (Fukuoka, Japan) and the law (no. 105) and notification (no. 6) of the government of Japan. The authorization number was A19-195-0. Lymphatic Lipid Transport Measurement. Total cholesterol, triacylglycerol (TAG), and phospholipid levels in the lymph were measured using commercial enzyme assay kits (T-CHO Kainos from Kainos Laboratories, Inc., Tokyo, Japan; Triglyceride E-Test and Phospholipid C-Test from Wako Pure Chemicals, Tokyo, Japan). If the lymphatic lipid levels exceeded the range of a standard curve, the lymph was diluted with physiological saline and retested. Preparation of Micellar Solutions. Micellar solutions containing 6.6 mM sodium taurocholate, 0.6 mM phosphatidylcholine, 50 μM cholesterol, 1.0 mM oleic acid, 0.5 mM monoolein, and 132 mM NaCl in 15 mM sodium phosphate buffer (pH 6.8) were prepared by sonication. The micelles were passed through a 0.2 μm syringe filter (DISMIC-25CS, Toyo Roshi Kaisha Ltd., Tokyo, Japan) and maintained at 37 °C for 24 h for stabilization of the micelles. Preparation of Casein, EWP, and EWP Constituent Protein Hydrolysates. Ten grams of casein, EWP, OA, OT, OM, and Ly was hydrolyzed by 400 mL of experimental gastric juice [0.1% (w/v) pepsin in 100 mM KCl] at pH 2.0 with HCl and 37 °C for 24 h.18 The reaction mixtures were adjusted to pH 7.0 with 1 M KOH, freezedried, and powdered to obtain casein pepsin hydrolysate (C-ph), EWP pepsin hydrolysate (EWP-ph), OA pepsin hydrolysate (OA-ph), OT pepsin hydrolysate (OT-ph), OM pepsin hydrolysate (OM-ph), and Ly pepsin hydrolysate (Ly-ph). The degree of hydrolysis of casein and EWP was determined by SDS-PAGE. The protein content of casein, C-ph, EWP, and EWP-ph was determined according to the method discussed by Lowry et al.19 The samples in the SDS-PAGE sample buffer were incubated at 95 °C for 5 min using a thermal cycler, and 5 μg of protein from each sample was then electrophoresed on a 5−20% gradient polyacrylamide gel. The gel was stained using a silver staining kit (Wako Pure Chemical Industries Ltd.). 10695
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Figure 1. Cumulative lymph flow (A), lymphatic transport of cholesterol (B), triacylglycerol (C), and phospholipids (D) in rats fed diets containing casein (black circles) or EWP (red circles). Values are expressed as the mean ± SEM for six rats. (∗) P < 0.05 and (∗∗) P < 0.01 versus casein group by Student’s t test.
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RESULTS Effects of Dietary EWP on Lymphatic Lipid Transport. The two groups of rats did not differ in final body weight (casein, 232.3 ± 3.7; EWP, 228.4 ± 6.5 g). The rats consumed the casein and EWP diets for 30 min (4.6 ± 0.3 and 4.4 ± 0.4 g, respectively). Figure 1 shows the cumulative lymph flow and lipid transport. There was no significant difference in lymph flow (Figure 1A). The lymphatic transport of total cholesterol at 7 h (Figure 1B) and TAG at 6 and 7 h (Figure 1C) after feeding was significantly decreased in the EWP group compared with that in the casein group. In addition, the lymphatic transport of phospholipids at 1, 2, and 3 h after feeding was significantly increased in the EWP group compared with that in the casein group (Figure 1D). Degree of Hydrolysis of Casein and EWP by Pepsin. Figure 2 shows the degree of hydrolysis of casein and EWP by pepsin. Two bands were recognized in the gel. These bands were identified as ovalbumin (45 kDa) and lysozyme (14.3 kDa) in EWP-ph.25 The casein was almost digested with pepsin. Effects of EWP Pepsin Hydrolysate and EWP Constituent Protein Pepsin Hydrolysates on the Micellar Solubility of Cholesterol. EWP-ph significantly inhibited the micellar solubility of cholesterol to a greater extent than C-ph did (Figure 3A). There was no effect of pancreatin on micellar solubility (Figure 3A). The inhibitory effect of EWP-ph on micellar solubility was dose-dependent and reached a plateau at a final concentration of 10 mg/mL (Figure 3B). There was no effect of the presence of phosphatidylcholine in bile acid micelles on micellar solubility (Figure 3C). Compared with Cph, EWP-ph, OA-ph, and OT-ph significantly inhibited micellar solubility (Figure 4A). Furthermore, there was no significant difference in micellar solubility between EWP-ph and reconstructed EWP-ph (Figure 4B). Effects of EWP on Transfer of Cholesterol from Micelles to Triolein. EWP-ph significantly inhibited the transfer of cholesterol from micelles to triolein (Figure 5). That
Figure 2. Results of electrophoresis of casein, EWP, and its pepsin hydrolysate.
is, EWP-ph inhibited cholesterol monomer release from micelles. Effects of EWP and Its Pepsin Hydrolysate on WaterHolding Capacity, Settling Volume in Water, and Relative Viscosity. Table 1 summarizes the effects of EWP and its pepsin hydrolysate on WHC, SV, and relative viscosity. There was no significant difference in WHC between casein and EWP. However, the WHC of EWP-ph was approximately 10696
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Figure 3. Effects of EWP hydrolysate on the micellar solubility of cholesterol in vitro: (A) effects of EWP pepsin hydrolysate or EWP pepsin + pancreatin hydrolysate; (B) dose-dependent effects of EWP pepsin hydrolysate; (C) effects of phosphatidylcholine containing the micelles. C-ph, casein pepsin hydrolysate; EWP-ph, egg white protein pepsin hydrolysate; PC, phosphatidylcholine. Data were calculated using the following formula: micellar cholesterol contents incubated with protein/micellar cholesterol contents incubated without protein × 100. Values are expressed as the mean ± SEM for three samples. Different letters (a−c) show a significant difference by the Tukey−Kramer test (P < 0.05).
Figure 4. Effects of EWP constituent protein pepsin hydrolysates and reconstructed EWP-ph on the micellar solubility of cholesterol in vitro: (A) effects of EWP-ph and EWP constituent protein pepsin hydrolysates (each protein was added at a final concentration of 5 mg/mL); (B) effects of EWP-ph and reconstruction EWP-ph (reconstructed EWP-ph included 54% OA-ph, 13% OT-ph, 11% OM-ph, and 3.5% Ly-ph; EWP-ph was added at a final concentration of 10 mg/mL; reconstruction EWP-ph was added at a final concentration of 8 mg/mL). C-ph, casein pepsin hydrolysate; EWP-ph, egg white protein pepsin hydrolysate; OA-ph, ovalbumin pepsin hydrolysate; OT-ph, ovotransferrin pepsin hydrolysate; OM-ph, ovomucoid pepsin hydrolysate; Ly, lysozyme pepsin hydrolysate. Data were calculated using the following formula: micellar cholesterol contents incubated with protein/micellar cholesterol contents incubated without protein × 100. Values are expressed as the mean ± SE for three samples. Different letters(a−c) show a significant difference by the Tukey−Kramer test (P < 0.05).
casein was higher than that of EWP, whereas that of EWP-ph was significantly higher than that of C-ph.
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DISCUSSION We confirmed that dietary EWP inhibited intestinal cholesterol absorption in thoracic lymph duct-cannulated rats (Figure 1B). These data supported the serum and hepatic cholesterollowering action of EWP in our previous paper.11 In that previous paper, evidence for the inhibition of cholesterol absorption was the fecal excretion of neutral steroids in rats fed the 20% EWP diet compared with that in rats fed the 20% casein diet. Most of the dietary lipids are absorbed in the proximal small intestine and then transported by the lymphatic system. We hypothesized that EWP hydrolysate by pepsin in the stomach may play a major contributing role to the inhibition of cholesterol absorption by EWP. To test the hypothesis, we prepared EWP-ph and then conducted the in vitro experiments. The results showed that EWP-ph inhibited the micellar solubility of cholesterol (Figures 3A−C and 4A) and cholesterol monomer release from micelles (Figure 5). In addition, there was no effect of pancreatin on the micellar solubility of cholesterol (Figure 3A).
Figure 5. Effects of EWP-ph on the transfer of micellar cholesterol to triolein in vitro. Data were calculated using the following formula: cholesterol contents in the triolein phase/original micellar cholesterol contents × 100. Values are expressed as the mean ± SEM for three samples. (∗) P < 0.05 versus casein group by Student’s t test.
7.6 times higher than that of C-ph. In addition, the SV of casein was higher than that of EWP, whereas the SV of EWP-ph was approximately 4.5 times higher than that of C-ph. In accordance with the behavior of SV, the relative viscosity of 10697
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Table 1. Water-Holding Capacities, Settling Volumes in Water, and Relative Viscosities of Casein, EWP, and Its Pepsin Hydrolysatea water-holding capacity [wet (g)/dry (g)] settling volume (mL/g) relative viscosity (η) a
casein
EWP
C-ph
EWP-ph
2.87 ± 0.03a 4.14 ± 0.22a 1.115 ± 0.000a
2.87 ± 0.13a 0.95 ± 0.13b 1.096 ± 0.001b
0.32 ± 0.09b 1.80 ± 0.17b 1.054 ± 0.001c
2.11 ± 0.09c 8.09 ± 0.34c 1.059 ± 0.001d
Different letters (a−d) show a significant difference by the Tukey−Kramer test. Mean ± SE of three samples.
concluded that the primary mechanism underlying the hypocholesterolemic effect of dietary EWP was the inhibition of dietary cholesterol absorption.11 In addition, in this study, lymphatic cholesterol transport was decreased by dietary EWP (Figure 1B). As shown in Figure 2, there were proteins that were resistant to pepsin in EWP. On the other hand, there was no band in the C-ph lane. Two major bands were recognized in the EWP lane after pepsin digestion (EWP-ph lane). These bands were individually identified as ovalbumin (MW 45 kDa) and lysozyme (MW 14.3 kDa). EWP contains 54% ovalbumin and 3.5% lysozyme.21 Ovalbumin is the most abundant protein in EWP. Ovalbumin almost remained in EWP digestion by pepsin. We attempted to do a reverse experiment. A reconstructed EWP-ph was used; four pepsin hydrolysates of the EWP protein components were mixed with the EWP-ph while considering an intact ratio for the EWP composition, as described in Figure 4. The cholesterol micellar solubility was found to be similar for reconstructed EWP-ph and EWP-ph (Figure 4B). There was no ingredient in EWP that altered the solubility. According to the results of inhibition of cholesterolmicellar solubility, ovalbumin is responsible for the cholesterollowering activity. The solubility of EWP-ph was as low as that of OA-ph (Figure 4A). Ovalbumin may pass through the intestine, as evidenced by difficulty in its digestion according to the results of the in vitro protease digestion experiment (Figure 2). When EWP was the only dietary protein source in the rats fed the EWP diet, approximately half the dietary protein could not be absorbed. However, in the previous study, body weight gain in rats fed the EWP diet was not different from that in rats fed the casein diet.11 From the perspective of nutrition, the EWP diet supplemented with biotin did not cause growth inhibition in animal models, similar to that observed with egg white injury. It was reported that nitrogen excretion in the feces of rats fed EWP was not high.32 Furthermore, EWP exhibits greater net protein utilization compared with other protein sources.32 It has also been reported that EWP includes protease inhibitors.33 Protease inhibition by EWP components or ovalbumin may be selective for proteases during gastric digestion. In this study, ovalbumin was resistant to stomach pepsin, but it may not be resistant to the other proteases in the intestine. We newly observed that EWP inhibited lymphatic triacylglycerol (TAG) transport (Figure 1C). This observation reflected on serum and hepatic TAG levels from the previous study, where the efficiency of inhibition in rats fed the EWP diet for 3 weeks was 26 and 37%, respectively.11 However, there were no significant differences in serum and hepatic TAG levels between the casein and EWP diets. These TAG-lowering effects were not observed in rats fed the EWP diet for 1 week; however, the hypocholesterolemic effect of EWP was evident. Ovalbumin has a pancreatic lipase inhibitory activity.34 It is not digested by pepsin in the stomach because of its protease inhibitory activity as described above; it reaches the intestine to
The hypocholesterolemic manners of EWP resemble those of plant sterols, which have hypocholesterolemic activity.12,23 The efficiency of lymphatic cholesterol transport inhibition by βsitosterol, a major plant sterol, was 57% in thoracic lymph ductcannulated rats administered lipid emulsions in the stomach.12 The efficiency of a hypocholesterolemic effect of β-sitosterol in rats fed a 0.5% cholesterol-containing diet for 2 weeks was 21%.26 According to a comparison of these calculated data, it was estimated that dietary β-sitosterol inhibited intestinal cholesterol absorption more strongly than dietary EWP did. However, the hypocholesterolemic effect of dietary plant sterol was similar to that of dietary EWP. To assess intestinal lipid absorption, the administration of two types of diets containing lipids and infusion lipid emulsions into the stomach or duodenum was used. The method that used lipid emulsions failed to consider the interaction of the lipid emulsion with other dietary components, particularly proteins and carbohydrates.27 Therefore, differences in the methods of administration may contribute to the fact that the hypocholesterolemic effects of dietary plant sterols and EWP were similar. Otherwise, EWP may have other functions for the hypocholesterolemic effect. It has been reported that a commercially available EWP hydrolysate produced by microbial proteases, with an average molecular weight of approximately 1100, as well as EWP, have hypocholesterolemic effects in rats and mice.28 In this study, the hypocholesterolemic effect of EWP through its physicochemical properties depended on its protein structure, which resisted protease digestion in the gut and inhibited intestinal cholesterol absorption. The commercial EWP hydrolysate primarily comprises peptides and amino acids. The hypocholesterolemic effect of the hydrolysate may be exerted on the intestinal mucosa after absorption as smaller size peptides except for the physiological properties of the hydrolysate described above. Many studies have reported that other dietary proteins such as soy protein isolate29 and whey protein30 can provide several peptides that can exert a hypocholesterolemic effect. Therefore, some peptides derived from EWP may have a hypocholesterolemic effect inside animal bodies. In this study, the efficiency of the inhibition of lymphatic cholesterol transport was approximately 14%. In the previous study, the decrease in serum cholesterol levels in rats fed the EWP diet was approximately 17%.11 The serum cholesterol-lowering activities of several dietary proteins compared with those of casein were found to depend on their amino acid compositions.31 Serum cholesterol levels were found to be similar in rats fed a casein diet supplemented with cystine as an amino acid rich in EWP and those fed a casein diet, as described in the previous paper.11 To exert the serum cholesterol-lowering action of EWP, protein structures, including the primary structure, may be important; however, the amino acid composition does not obviously. Further studies are needed to investigate whether a reconstructed amino acid mixture of the EWP amino acid composition has a hypocholesterolemic effect. Therefore, we 10698
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and may possess properties capable of exerting a hypocholesterolemic effect.
act as a lipase inhibitor. Therefore, the intestinal absorption and lymphatic transport of TAG decreased and serum and hepatic TAG levels decreased. The difference in sensitivities for the inhibition of intestinal absorption of dietary cholesterol and TAG may rely on the feeding periods. The inhibition of cholesterol absorption may be more sensitive to its accumulation in the animal body compared with the inhibition of TAG. As shown in Figure 1D, the mechanisms underlying temporal promotion of lymphatic phospholipid transport are not clear for the EWP diet without phospholipids. The rats may have temporarily experienced a condition of less intestinal phospholipid from 1 to 4 h after consuming the EWP diet. As shown in Figure 3C, there was no effect of the presence of PC in the bile acid micelles on the micellar solubility of cholesterol. Also, EWP-ph has better physicochemical properties of WHC, SV, and relative viscosity compared with casein and C-ph. These characteristics of EWP-ph are similar to those of water-soluble fibers such as guar gum.24 Under alkaline conditions, fatty acids, water, and EWP combine in the intestine after ingestion, and EWP turns into a gel because of its physicochemical properties.35−39 According to these results and information, the gel form of EWP may hold the micelles. Moreover, the gel mixture that contained EWP, fatty acids, and water may lead to inhibited fatty acid absorption. The timedependent effect of dietary EWP on lymphatic transport of lipids was totally exerted after 3 h of feeding. In research on the effects of dietary protein on gastric emptying, compared to casein, gluten, and fish protein, whey protein delays gastric emptying time to suppress plasma FFA levels because of their controlling gastric hormone secretion and/or their physicochemical properties such as solubility of acidic solution.40 Physicochemical properties and stimulation of gastric hormone secretion of dietary protein influence gastric emptying time, and then the influences reach intestinal absorption of nutrients. In fact, the protein form of EWP partly remained after the digestion (Figure 2). In the early phase of lipid transfer, EWP may stay in the stomach and the TAG and cholesterol may reach the duodenum more rapidly than EWP-ph. In the late phase, EWP-ph may exert effects of inhibition of intestinal lipid absorption. In this study, the lymphatic phospholipids may be derived from bile, intestinal mucosa exfoliation, bacterial flora, etc., but not from the diets and stomach. The lymphatic phospholipid transport may primarily suffer from the influence of phase shifting of gastric emptying. Taken together, the differences of the time course of lymphatic lipid transport may depend on the differences of gastric emptying time of protein. These mechanisms remain a hypothesis. Therefore, we have to organize acceptable plans for each dietary lipid in future studies. The efficiency of intestinal cholesterol absorption is approximately 50%.17,41 It assumes a one-to-one relationship between a passive diffusion process and a process through the Niemann−Pick C1-like 1 (NPC1L1) protein, a cholesterol transporter.17,41 Possible findings about the effect of dietary EWP on NPC1L1 would be of great interest for future study, although none of the studies have shown that dietary components inhibit NPC1L1 protein. Further studies are also needed to investigate whether dietary EWP affects the assembly and secretion of chylomicrons. In summary, dietary EWP inhibited dietary cholesterol absorption via decreased cholesterol passage in and out of the micelles and increased WHC, SV, and relative viscosity. Ovalbumin, a major protein in EWP, resisted protease digestion
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AUTHOR INFORMATION
Corresponding Author
*(M.S.) Phone/fax: +81-92-642-3004. E-mail: masaos@agr. kyushu-u.ac.jp. Author Contributions ∥
R.M. and B.S. contributed equally to the study.
Author Contributions
R.M. and B.S. wrote the manuscript and contributed equally to the study. R.M., B.S., S.K., S.B., S.S., and K.N. participated in the experimental work and collected and analyzed data. K.I. and M.S. supervised the study design and commented on the manuscript. All authors read and approved the final manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Enago (www.enago.jp) for the English language review.
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REFERENCES
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