As shown in Figure 2, all the hydrolysates resulting from the five enzymes were capable of binding sodium glycocholate. Sodium glycocholate was bound by hydrolysate from Alcalase, Trypsin, Neutrase, Protamex, and Flavourzyme to the degree of 9.53, 11.16, 16.80, 12.15 and 19.01%, respectively. Cholestyramine was used as a positive control and displayed 72.77% glycocholate binding capacity. Hydrolysate from Alcalase showed a significantly lower glycocholate binding capacity while the value for the hydrolysate from Flavourzyme was significantly higher than those of the other hydrolysates. Cholestyramine bound sodium glycocholate and taurocholate by 87% and 93%, respectively [26]. Story and Krichevsky [27] reported that cholestyramine bound glycocholate under various conditions to a degree of 74.2%. In our study, cholestyramine binding by glycocholate was similar to that observed by Story and Krichevsky [27]. Alfalfa and wheat bran have been shown to have a glycocholate binding capacity of 11.5% [27] and 100% [28], respectively; and the defatted soybean hydrolysate 0.2 to 8.5% [19]. Hydrolysate from Flavourzyme showed higher glycocholate binding capacity than alfalfa and defatted soybean hydrolysate.

Sodium cholate was bound by hydrolysate from Alcalase, Trypsin, Neutrase, Protamex, and Flavourzyme to the degree of 1.93, 5.52, 6.26, 8.82 and 9.99%, respectively, while cholestyramine, used as a positive control, had 85.78% cholate binding capacity (Figure 3). These values are much lower than those recorded with sodium glycocholate.

Hydrolysate from Alcalase showed significantly (p < 0.05) lower cholate binding capacity than cholestyramine, which has been reported to bind sodium cholate to the degree of 80% [13] and 60.7% [27]. Camire and Camire et al. [13,28] reported cholate binding by cholestyramine, three types of raisins, wheat bran and various types of potato peels at a cholate concentration of 12.5 mM. Their work indicated cholate binding of 75%, 15–20%, 10% and 1.9–8.1%, respectively. Hydrolysate from Flavourzyme exhibited higher cholate binding capacity than potato peels.

Under similar condition, all hydrolysates from defatted corn protein hydrolyzed by proteases showed higher percentage of bile acid binding with sodium deoxycholate than sodium glycocholate and sodium cholate (Figure 4). Sodium deoxycholate was bound by hydrolysate from alcalase to a degree of 55.76%, 78.68% by hydrolysate from Trypsin, 50.77% by hydrolysate from Neutrase, 43.40% by hydrolysate from Protamex, 86.90% by hydrolysate from Flavourzyme and 99.15% by cholestyramine. The hydrolysate from Flavourzyme had the highest binding capacity as compared to the other hydrolysates. Cholestyramine has been shown by other researchers to bind sodium deoxycholate by 99% [13], 92.5% [27] and 85% [28]. Our result was similar to that observed by Camire et al. [13]. Protein isolates F and its hydrolysate [19], Alfalfa [27], raisins, wheat bran [28], and potato peels [13] showed 58.4–69.5%, 10.8%, 5–10%, 15%, and 10.6–18.9%, respectively. Compared to these samples, our hydrolysates from Flavourzyme showed a much higher deoxycholate binding capacity.

As demonstrated by previous works [14,29–32], our investigations confirmed that bile acid binding degree decreases with hydroxyl group increment in steroid ring structure. In addition, we found that sodium cholate primary bile acid (three hydroxyl) was the less bound with hydrolysate from Alcalase than sodium glycocholate, a conjugation of sodium cholate with glycine; however, sodium deoxycholate secondary bile acid (two hydroxyl) was the best bound. Numerous authors [18,33,27] have reported a greater binding capacity of sodium deoxycholate than sodium cholate. Kern et al. [34] proposed that less sodium cholate, a trihydroxy bile acid, was bound than dihydroxy bile acid because hydrophobic interactions are involved with binding.

The method reported here appears to be a satisfactory technique for the measurement of the bile acid binding capacity of peptides. Cholestyramine, as expected, bound all the bile acids selected under the same treatment. The concentration of bile acids used and the percentage bound in the present study are in accordance with findings from previous studies. However, there is no obvious correlation between bile acid binding and the degree of hydrolysis. As the results showed, hydrolysate obtained with Flavourzyme contained effective peptides capable to bind bile acids. This could be explained by the presence of Flavourzyme acting as an endoprotease and exopeptidase, providing a broader specificity to release peptides rich in hydrophobic amino acid residues. These hydrophobic amino acids can bind bile acids strongly via hydrophobic reactions, since the hydrophobic amino acids present a strong interaction with lipids (cholesterol, bile acids, others sterols and others lipids). Moreover, higher bile acid binding by Flavourzyme hydrolysate in our studies may be due to the use of physiological pH of 6.5, which is closer to the pHi of the hydrophobic amino acids. The results given in the literature have shown that the binding of bile acid mixture, simulating the condition in the human body, occurred in a noncompetitive manner. Despite the need for bile acid mixture analysis, Flavourzyme hydrolysate could be considered as a suitable natural compound for bile acid binding.

Bile acids, especially deoxycholate, are thought to be involved in the etiology and development of colorectal cancer [35]. The synthesis of bile acids from cholesterol is adjusted by their concentration in the liver [36]. The presence of Flavourzyme hydrolysate in the intestine can decrease not only enterohepatic circulation of bile acids, but also their contact with colorectal mucosa through the binding effect. Therefore, it is possible for humans to ingest some Flavourzyme hydrolysate to prevent hypercholesterolemia and colorectal cancer at the same time.

Defatted corn proteins isolates were prepared by alkaline extraction at pH 11.50, followed by precipitation at its isoelectric point of pH 4.50, and freeze-dried. The freeze-dried defatted corn protein was dissolved in distilled water at a concentration of 5% (w/v) before hydrolysis with five different enzymes independently. Homogenization was carried out for each enzyme for 30 min in order to adjust the pH (through addition of 0.5 M NaOH) and temperature to the appropriate values. After the optimum condition was reached, the reaction was initiated by adding each of the five different enzymes with continuous stirring. Hydrolysis was carried out for 90 min and the pH of the mixture was kept constant by adding 0.5 M NaOH solution continuously to the reaction mixture. The amount of alkali added to keep the pH constant was recorded and used to calculate the degree of hydrolysis (DH). Then, the mixture was heated at 85 °C for 10 min to inactivate the enzyme and centrifuged by freezing centrifugation (ZOPR-52D, Hitachi Koki Co., Japan) at 10,000 rpm for 25 min. The supernatant was freeze-dried and stored in a desiccator for further use.

Degree of hydrolysis (DH) was defined as the percentage ratio between the number of peptide bonds cleaved (h) and the total number of peptide bonds in the substrate studied (h_tot). DH was evaluated by pH-stat method which allowed the estimation of DH based on the consumption of alkali to maintain a constant pH at the desired value. The degree of hydrolysis was calculated using the following equation (1) [24]: (1) DH ( % ) = V b × N b × 1 α × 1 M p × 1 h tot × 100where V_b is the amount of alkali consumed in mL; N_b is the normality of the alkali; M_p is the mass of protein (N × 6.25) in g; h_tot is the total number of peptide bonds in the protein substrate (9.2 meqv/g defatted corn protein) and, α is the average degree of dissociation of the α-NH₂ groups released during hydrolysis. The DH of the five defatted corn protein hydrolysates can be seen in Table 5.

The in vitro bile acid binding was executed through modification of the procedure previously by Hu et al. [12]. Each bile acid (as substrate) was dissolved in 50 mmol/L phosphate buffer (pH 6.5) to make a 2 mM bile acid solution, which is in the same range of bile acid concentration in the human body (1.5–7 mM), and the pH was simulated to the physiological pH of the duodenum. Ten milligrams of the hydrolysate sample was added to each tube containing one milliliter of bile acid solution, and the individual substrate solution without samples was used as blank. Then tubes were incubated for one hour at 37 °C in a shaking water bath. Mixtures were centrifuged at 10 °C with 10,000 rpm for 30 min in an ultracentrifuge (Model J-26XPI, Beckman, USA). The supernatant was removed into a second set of tubes and frozen at −20 °C for bile acid analysis. Bile acids were analyzed using HPLC (Model 1525, Waters, USA) on a symmetry C18 column (3.9 × 150 mm i.d., 5 μm particle size, Waters, USA), maintained at 30 °C. The injected sample volume was 10 μL for each bile acid analysis. The bile acid was eluted with 40% acetonile + 60% of 0.05% H₃PO₄ at a flow rate of 1 mL/min for 24 min. The absorbance of the eluate was monitored continuously at 210 nm (Model 2996 PDA detector, Waters, USA) and quantitated using standard calibration curves generated from the peak area responses of the standard solutions. Duplicate assays were conducted for each bile acid essay.

The percentage of bound bile acid was calculated as bound (%) = [(C_c − C_s)/C_c] × 100, where C_c and C_s represent bile acid concentrations in the control and in samples, respectively.

DH and bile acid binding capacity obtained from Flavourzyme hydrolysate were significantly higher in comparison to hydrolysate obtained from Alcalase, Trypsin, Neutrase and Protamex. Therefore, further experiments were focused on Flavourzyme hydrolysate.

The stability against in vitro gastric proteases was assessed based on the method described by Wu and Ding [49]. A 1% (w/v) hydrolysate solution in 0.1 M KCl–HCl buffer (pH 2.0) was treated with pepsin for 4 h in a rotary water bath at 37 °C. The pepsin-treated peptide was heated to boiling in a water bath for 15 min then adjusted to pH 7.0 with addition of 2 N NaOH. A 1 mL aliquot of the neutralized suspension was centrifuged at 10,000 g for 40 min and the supernatant portion was used to determine bile acid binding. The remaining portion of the suspension was further digested by 2% (w/w) pancreatin at 37 °C for 4 h, followed by enzyme inactivation by boiling for 15 min. The reaction solution was centrifuged at 10,000 g for 40 min. The supernatant portion was used for additional testing on bile acid binding capacity.

The dried Flavourzyme hydrolysate (100 mg) was subjected to acid hydrolysis with 5 mL of 6 M HCl under nitrogen atmosphere for 24 h at 110 °C. The hydrolysate was washed into a 50 mL volumetric flask and made up to the mark with distilled water. The amino acids were subjected to RP-HPLC analysis (Agilent 1100, USA) after precolumn derivatization with o-phthaldialdehyde (OPA). Amino acid composition was expressed as g amino acid per 100 g protein.

The dried Flavourzyme hydrolysate (1 g) was dissolved in 5% (w/v) TCA (25 mL) and centrifuged at 10,000 g for 10 min. The clear supernatant containing mainly free amino acids and some short peptides were derivatized by o-phthaldialdehyde (OPA) followed by reversed phase high performance liquid chromatography (RP-HPLC) analysis, carried out in an Agilent 1100 (Agilent Technologies, Palo Alto, CA, USA). Sample (1 mL) was injected on a Zorbax 80A C18 column (4.6 i.d. × 180 mm, Agilent Technologies, Palo Alto, CA, USA) at 40 °C with detection at 338 and 262 nm. Mobile phase A was 7.35 mmol/L sodium acetate/triethylamine/tetrahydrofuran (500:0.12:2.5, v/v/v), adjusted to pH 7.2 with acetic acid, while mobile phase B (pH 7.2) was 7.35 mmol/L sodium acetate/methanol/acetonitrile (1:2:2, v/v/v). The amino acid composition was expressed as g of amino acid per 100 g of protein. The results were processed with the aid of ChemStation for LC 3D software (Agilent Technologies, Palo Alto, CA, USA).

Molecular weight distribution was determined using a Waters^TM 600E Advanced Protein Purification System (Waters Corporation, Milford, MA, USA). A TSK gel 2000SWXL (7.8 × 300 mm) column with 10% acetonitrile + 0.1% TFA in HPLC grade water as the mobile phase.

The calibration curve was obtained by running horse heart cytochrome C (12,400 Da), bacitracin (1450 Da), gly-gly-tyr-Arg (451 kDa) and gly-gly-gly (189 Da). The results obtained were processed with the aid of Millennium³² Version 3.05 software (Waters Corporation, Milford, MA 01757, USA).

All analyses were carried out in duplicate and data were presented as mean ± SD. Analysis of variance (ANOVA) was carried out using SAS package (SAS Institute, Cary, NC). Comparisons between means were done using a Duncan’s multiple-range test with a probability of p < 0.05.