Revisiting the Prospects of Plastein: Thermal and Simulated Gastric

Dec 11, 2013 - Truro, Nova Scotia B2N 5E3, Canada. ABSTRACT: Plastein, a product of protease-induced peptide aggregation, is thought to possess unique...
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Revisiting the Prospects of Plastein: Thermal and Simulated Gastric Stability in Relation to the Antioxidative Capacity of Casein Plastein Chibuike C. Udenigwe,* Sihong Wu, Kiesha Drummond, and Min Gong Health and Bioproducts Research Laboratory, Department of Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro, Nova Scotia B2N 5E3, Canada ABSTRACT: Plastein, a product of protease-induced peptide aggregation, is thought to possess unique physical properties and bioactivity, although its formation, stability, and functional mechanisms remain unclear. This study demonstrates that plastein is formed from bovine casein peptides with Alcalase by hydrophobic and electrostatic interactions and less likely by covalent bonding. The peptide aggregation enhanced the Fe(III) reducing potential and decreased the Fe(II) chelating activity (p < 0.05) of casein peptides, but there was no difference in inhibition of Fe-induced linoleic acid peroxidation after plastein reaction. The casein plastein product retained its antioxidative activities after being heated at 100 °C. However, simulated gastric protease treatment with pepsin and pancreatic enzymes resulted in enhanced reducing potential and metal chelation of the casein plastein and reduction of the inhibitory effect on lipid peroxidation. It appears that the plasteins were disintegrated and further hydrolyzed by gastric proteases on the basis of the antioxidative capacity and RP-HPLC profile being similar to those of the casein hydrolysates. Therefore, plastein reaction may not confer metabolic stability or enhance the antioxidative capacity of casein peptides for prospective functional food applications. KEYWORDS: plastein, peptide aggregation, antioxidant, bioactive peptide, stability, gastric protease

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INTRODUCTION Plastein is a product of physical aggregation of peptides catalyzed by proteases at high substrate concentrations. First reported in 1902 by Danilevski and Okuneff, and originally thought to be resynthesized proteins,1 plasteins are held together mostly by hydrophobic interaction of aggregating peptides.2 However, the molecular events catalyzed by the protease during plastein formation remain unclear. Currently, plasteins are yet to be applied in the food system, and decades of research explored utilization of its gel-like physical properties in food.3 Moreover, plastein has prospects for use in delivering desirable amino acids and enhancing the nutritional composition, quality, and health applications of proteins.4 Furthermore, plastein has been explored for masking the bitterness of protein hydrolysates since hydrophobic amino acid residues that contribute to bitterness are buried within the plastein,3 possibly limiting their interactions with taste receptors. Recently, a number of studies reported that plastein formation enhances the bioactivity of peptides, particularly their antioxidative and angiotensin-converting enzyme inhibitory activities.5−7 Consequently, plastein can be explored as a multifunctional peptidebased ingredient for developing functional foods with desirable sensory, physicochemical, and bioactive properties. However, the mechanisms and structural requirements of plastein for enhanced bioactivity are presently not known, although this can be related to the surface property and availability of active functional groups in the peptide aggregates. Moreover, there is a dearth of information on the stability of plastein, which can be susceptible to thermal degradation during food processing and gastric proteolysis during ingestion, potentially leading to loss of structure and bioactivity. The objective of this study was to evaluate the antioxidative property of casein plastein on the basis of the reducing potential, metal chelation, and inhibition © 2013 American Chemical Society

of Fe-induced lipid peroxidation with respect to their structural and functional stability against thermal and gastric protease treatments.

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MATERIALS AND METHODS

Casein Hydrolysis and Plastein Formation. Casein was prepared by isoelectric precipitation by adjusting commercial skim bovine milk to pH 4.6 using 0.5 M HCl followed by centrifugation at 10000g for 15 min and freeze-drying the resulting precipitate. Thereafter, an aqueous suspension of the casein powder (5%, w/v) was heated at 90 °C for 10 min and cooled to 55 °C, followed by addition of protease from Bacillus licheniformis ([Alcalase] ≥ 2.4 U/g) at 0.01 g/g of casein to initiate hydrolysis. After 5 h of reaction maintained at 55 °C and pH 8.5 (adjusted by adding 0.5 M NaOH), the mixture was heated at 90 °C for 15 min to inactivate the enzyme, and the resulting product was freeze-dried. The degree of casein hydrolysis (DH) was determined by the O-phthaldialdehyde (OPA) method8 and the average peptide chain length calculated as previously reported.9 To produce casein plastein, a 40% aqueous suspension of the freeze-dried casein hydrolysates was mixed with 0.01 g of Alcalase/ g of hydrolysates at 43 °C for 6 h at pH 6.5. Sample aliquots were withdrawn from the reaction every 1 h to monitor plastein formation and antioxidative activities. After termination of the plastein reaction by heating, the resulting products were freeze-dried and stored at −20 °C for further analysis. Free Amino Nitrogen Determination. Free amino nitrogen present in the casein hydrolysates and plasteins was analyzed using OPA reagent containing sodium dodecyl sulfate (SDS) and dithiothreitol.8 The OPA reagent (3 mL) was mixed with a 0.4 mL sample (1 mg/mL), and the absorbance of the solution was measured at 340 nm after 2 min. Serine (0.1 mg/mL) was used as the standard, Received: Revised: Accepted: Published: 130

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Figure 1. (A) Turbidity in deionized water, (B) free amino nitrogen concentration, (C) total sulfhydryl group concentration , and (D) surface hydrophobicity of casein hydrolysates (CP0) and plasteins after 1−6 h of reaction (CP1−CP6). Bars with different letters within each graph represent statistically significant means with p < 0.05. using the potassium ferricyanide method.12 The samples and glutathione (positive control) were analyzed at 1 mg/mL final concentration, and the reducing potential was expressed as the absorbance at 700 nm. Thereafter, the Fe(II) chelating assay was conducted as previously reported12 with modifications. Briefly, the 3 mL assay mixture contained 1 mL of peptide solution or EDTA (positive control) at a final concentration of 0.05 mg/mL, 0.05 mL of FeCl2 (2 mM), and 1.85 mL of deionized water. A 0.1 mL solution of 5 mM ferrozine was then added followed by mixing; an equal volume of deionized water was used in the blank assay. The absorbance was then measured at 562 nm and the Fe(II) chelating effect (%) calculated. Last, the assay for inhibition of Fe(II)-induced lipid peroxidation was also conducted as previously reported13 with modifications. A 70 μL solution of the samples or glutathione (final concentration 0.05 mg/mL) was mixed with 3.2 mL of 0.1 M sodium phosphate buffer containing 1% (v/v) Triton X-100 (pH 7), 50 mM linolenic acid (0.72 mL), and 20 mM FeCl2 (10 μL). The mixture was incubated at 80 °C for 1 h, and a 0.1 mL aliquot was mixed with 75% ethanol (4.5 mL), 30% ammonium thiocyanate (0.1 mL), 1 M HCl (0.2 mL), and 20 mM FeCl2 (0.1 mL in 1 M HCl). The absorbance of the solution was then measured at 500 nm followed by calculation of the inhibition (%) of Fe(II)-induced linoleic acid peroxidation. Thermal and Gastric Stability. For thermal stability, aqueous solutions of casein hydrolysates and plastein were heated in a water bath (97−100 °C) for 10 min and cooled to room temperature followed by vortex mixing and measurement of the turbidity and antioxidative properties as described earlier. For gastric stability, the casein hydrolysates and plastein samples (50 mg) were mixed with pepsin solution (1 mg/mL in 0.1 M HCl−KCl buffer, pH 2.0) and incubated at 37 °C for 2 h followed by the addition of 1 mL of NaOH to neutralize the solution and inactivate pepsin.14 Thereafter, 1 mL of pancreatin suspension (3 mg/mL in 0.1 M sodium phosphate buffer, pH 8.0) was added to the mixture, and the resulting mixture was

and the free amino nitrogen was calculated as milliequiv of serine NH2/g of protein as previously reported.8 Surface Hydrophobicity (So). The surface hydrophobicity of the casein hydrolysates and plastein reaction products was determined by the fluorescence method using a hydrophobic probe, 8-anilino-1naphthalenesulfonic acid, as previously reported.10 The samples were assayed at concentrations of 0.0009−0.015% with excitation and emission wavelengths of 390 and 470 nm, respectively. The surface hydrophobicity was determined as the slope of the fluorescence vs concentration (%) plot. Turbidity Measurement. The solution turbidity was measured as reported9 with modifications. The casein hydrolysates and plasteins were dissolved in deionized water at 3 mg/mL and allowed to stand for 15 min at room temperature followed by vortex mixing. The absorbance of the solution was then measured at 460 nm. The same process was repeated with the samples dissolved in 0.2 M SDS or 2 M NaCl instead of distilled water as previously reported9 to evaluate the respective roles of hydrophobic and electrostatic interactions during plastein formation. Sulfhydryl Group Analysis. The total sulfhydryl group concentration of the hydrolysates and plasteins was measured as reported11 with modifications. Samples (0.5 mL, 10 mg/mL) were mixed with 2 mL of 0.1 M Tris−glycine buffer containing 10 M urea (pH 8.0) and incubated at 40 °C for 30 min. Then 62.5 μL of 5,5′dithiobis(2-nitrobenzoic acid) (4 mg/mL 0.1 M Tris−glycine buffer, pH 8) was added to the mixture, the resulting mixture was incubated at room temperature for 10 min, and the absorbance was measured at 412 nm. The total sulfhydryl group concentration (μM SH/g of protein) was calculated as 73.53A412nm(D/C), where D = the dilution factor and C = the sample concentration.11 Antioxidative Assays. Three assays were used to evaluate the antioxidant property of the plasteins. First, the Fe(III) to Fe(II) reducing potential of casein hydrolysates and plasteins was assayed 131

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incubated at 37 °C for 14 h followed by enzyme inactivation at 90 °C for 15 min. The resulting samples were analyzed for free amino group, ferric reducing potential, metal chelation, and inhibition of Fe(II)induced linoleic acid peroxidation. Reversed-Phase (RP) HPLC Analysis. Solutions (20 μL) of 5 mg/mL casein hydrolysates and plastein (dissolved in solvent A, 0.05% trifluoracetic acid (TFA) in water) before and after gastric protease treatment were injected into a Waters 515 HPLC system fitted with a Phenomenex C18 column (250 × 4.6 mm, 5 μm). The samples were eluted at a 1 mL/min flow rate using a linear gradient of 0−66% solvent B (0.05% TFA in acetonitrile) for 45 min and then 66−100% for 5 min and equilibration with 100% solvent A for 10 min. The samples and solvents were filtered through 0.2 μm membrane filters prior to analysis, and peptide elution was monitored at 214 nm with a Waters 2487 dual λ absorbance detector fitted to the HPLC equipment. Statistical Analysis. All data were collected in triplicate and are expressed as the mean ± standard deviation. The statistical significance of difference between treatments was analyzed by one-way analysis of variance and the Holm−Sidak multiple comparison test using SigmaPlot for Windows, version 11.0 (Systat Software, San Jose, CA).

Several studies have demonstrated that hydrophobic interaction plays a major role in peptide aggregation.2,15−17 This was confirmed during casein plastein reaction as the turbidity of the plastein solutions decreased in the presence of a surfactant, SDS (data not shown), which can disrupt hydrophobic interactions in peptide aggregates.2,16,17 This decrease was highest at 2 h of reaction (CP2), corresponding to the highest turbidity value (Figure 1A) and surface hydrophobicity, So (Figure 1D). The peptides’ surface hydrophobicity can impact their physiological biomolecular interactions and influence bioactivity especially if hydrophobic interaction with target molecules (e.g., lipids) is required. Apart from CP2, So did not change with peptide aggregation during casein plastein formation (Figure 1D). Moreover, considering the phosphates and other charged amino acid species in casein, electrostatic interactions may have also played minor roles in plastein formation as demonstrated by the decreased turbidity of the plastein in a high-ionic-strength NaCl solution (data not shown), which disrupted electrostatic interactions in the casein plastein, similar to previous observations during peptide aggregation.15−17 On the basis of these data, it is plausible that most of the casein plasteins are held together by noncovalent bonds, especially for the casein plastein product at 2 h (CP2) under our experimental conditions. Plastein reaction has recently been reported to enhance the bioactivity of peptides including antioxidative properties,5−7 although there is a dearth of clearly defined mechanisms and structural requirements of the aggregates for the enhanced bioactivity. Results from the present study demonstrate that there is no substantial increase in the physiologically relevant antioxidative properties of casein peptides to justify the additional plastein reaction step. However, there were statistically significant (p < 0.05) increases in the ferric reducing potential of the casein plasteins, especially at 2 h (CP2), which enhanced bioactivity by 19% (Figure 2A). In contrast, plastein reaction resulted in a reduced metal (FeII) chelating activity of the casein peptides with up to 42% loss of activity observed in CP3 (Figure 2B). Since the chemical compositions of the casein hydrolysates and plasteins are similar, it appears that molecular rearrangement during aggregation induced the negative effect on bioactivity. Plausibly, the metal chelating residues and phosphates in casein peptides may have participated in stabilizing the plasteins by hydrophobic and electrostatic interactions, respectively, making them unavailable to chelate Fe(II) during the assay. In addition, peptide aggregation during plastein formation did not seem to impact the inhibitory effects of the casein peptides on Fe(II)-induced linoleic acid peroxidation (Figure 2C). It was also observed that the reducing potential of the casein peptides is not related to their total sulfhydryl groups, which is a key electron-donating functionality in peptides. Therefore, the enhanced reducing potential of the plasteins may be due to other antioxidative functionalities of amino acid residues that became proximal and perhaps collectively more reactive within the peptide aggregates. Moreover, the observed inhibitory activities against Fe(II)-induced linoleic acid peroxidation are not exclusively due to their metal chelating effects since there was no correlation between the two antioxidative properties, and a strong metal chelating agent (EDTA) did not inhibit the Fe(II)-catalyzed oxidative process during the assay. Overall, the casein hydrolysates and plasteins competed favorably with equal amounts of the positive controls (EDTA chelated 100% Fe(II) and glutathione inhibited 13% lipid peroxidation) except

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RESULTS AND DISCUSSION Plasteins are believed to be products of entropy-driven physical aggregation of mostly low molecular size peptides induced by proteases at substrate concentrations often higher than 30% (w/v).2,3,15−18 Although proteolytic activity is not required, native enzymes are preferred in inducing peptide aggregation during plastein formation.2 In the present study, Alcalase was used for casein hydrolysis to produce small-chain peptides due to its broad specificity. Extensive proteolysis with the microbial enzyme for 5 h resulted in a 20.4 ± 0.12% degree of casein hydrolysis and a calculated average peptide chain length of 4.9 ± 0.03. The turbidity of the casein peptide solutions was measured at a low concentration (0.3%, w/v) to monitor peptide aggregation induced by the protease during plastein reaction.9,16 In this study, casein peptide aggregation occurred as observed from the increase in the absorbance value at 460 nm (turbidity) by 45% from 0 to 6 h (CP0−CP6), with the highest increase of 54% observed at 2 h of reaction (CP2) (Figure 1A). These results agree with similar studies that reported an increase in solution turbidity during peptide aggregation.9,16 Moreover, the plastein reaction resulted in a decrease in free amino nitrogen of the casein peptide solution by 16% after the 6 h reaction, with CP2 showing the lowest value (Figure 1B). A decrease in free amino nitrogen of protein hydrolysates has been reported to occur during plastein formation.5−7 A possible explanation of the observed changes in free amino nitrogen during peptide aggregation is the enzyme-induced covalent modification of free amino groups, leading to amide bond formation and peptide condensation. However, there is evidence that plastein formation is reversible and that no detectable peptide bonds are formed in casein plasteins.2,17 An earlier study indicated that blocking the amino and carboxyl groups of peptides reduced the plastein yield,2 which supports the possible involvement of free amino groups in peptide aggregation during plastein reaction, although the mechanism remains unclear. It is important to note that peptide aggregation did not occur in the absence of the protease. Another possible contributor to peptide aggregation is covalent disulfide bond formation. However, it appears that disulfide linkages are not involved in casein peptide aggregation in this study since the limited sulfhydryl group concentration in the casein hydrolysates did not decrease during plastein reaction (Figure 1C). 132

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For decades, an important driver of the need to explore plasteins in food applications has included the modified physicochemical functionality of the peptide aggregates that can purportedly confer unique properties such as molecular stability. However, the mechanism of protease-induced plastein formation remains unclear, and the peptide aggregates have yet to find a major place in food product development. Our study has demonstrated that the intramolecular interactions holding the casein peptides together in the plastein appear stable to heat treatment at 100 °C for 10 min on the basis of the solution turbidity (data not shown). Moreover, the antioxidative properties of the hydrolysates and plasteins were conserved after heat treatment, except that the plastein lost about 8% of its ability to inhibit Fe(II)-induced lipid peroxidation (Table 1). There was also a slight but statistically significant (p < 0.05) increase in the ferric reducing potential and metal chelating activity of plastein, indicating possible changes in the noncovalent molecular interactions that exposed functional amino acid residues after heating. That notwithstanding, the same trend was observed for the antioxidative activities of the treated and untreated samples, indicating that the plastein did not have a substantial advantage over the unmodified casein hydrolysates in these model assay systems, which casts doubts on the purported enhanced value and prospective use of casein plasteins in functional food applications. In contrast, gastric treatment resulted in altered antioxidative activities of the hydrolysates and plastein. The gastric enzymes induced a 4-fold increase in the ferric reducing potential of both samples and enhanced the metal chelating effect of the plastein to a value similar to that of the hydrolysates (Table 1). However, there was loss of over 24% of their inhibitory effects on lipid peroxidation after gastric modification. It appears that the casein hydrolysates and plasteins are similar after gastric treatment on the basis of their similar antioxidative activities (Table 1). Moreover, the HPLC profiles of the casein peptides before and after plastein reaction also appeared similar (Figure 3), indicating limited covalent bond formation induced by the microbial protease during aggregation. This suggests that noncovalent interactions played major roles in casein plastein formation in this study, as earlier suggested using other proteases.2 Furthermore, simulated gastric protease treatment resulted in further hydrolysis, especially for highly hydrophobic peptides, yielding similar HPLC profiles for the casein hydrolysates and plastein (Figure 3). These data indicate that the aggregated peptides in the plasteins formed by hydrophobic and ionic interactions (and possibly covalent bonds) are not resistant to further gastric proteolysis, similar to peptides in the original casein hydrolysates. A previous study suggested that plastein reaction can confer stability on peptides against further

Figure 2. Antioxidative properties of casein hydrolysates (CP0) and plasteins after 1−6 h of reaction (CP1−CP6): (A) Fe(III) reducing potential, (B) Fe(II) chelating activity, and (C) inhibition of Fe(II)induced linoleic acid peroxidation. Bars with different letters within each graph represent statistically significant means with p < 0.05.

in the case of the high reducing potential of glutathione (A700nm = 1.91) due to its substantially higher molar amount of reducing sulfhydryl functionality in the assay compared to that of the casein products.

Table 1. Residual Antioxidative Properties of Casein Hydrolysates (CP0) and Plastein (CP2) after Thermal and Gastric Protease Treatmentsa Fe(III) reducing potentialb CP0 CP2

0.098 ± 0.005 d (1.22)c 0.119 ± 0.002 c (1.20)

CP0 CP2

0.346 ± 0.012 a (4.33) 0.320 ± 0.005 b (3.99)

Fe(II) chelation (%)

inhibition of Fe(II)-induced lipid peroxidation (%)

Thermal Treatment (100 °C for 10 min) 97.93 ± 3.42 a (1.00) 82.73 ± 4.23 b (1.15) Gastric Protease (Pepsin and Pancreatin) Treatment 100 ± 0.00 a (1.02) 100 ± 0.00 a (1.39)

28.06 ± 2.79 a (1.02) 27.27 ± 4.36 a (0.92) 21.02 ± 1.10 b (0.76) 21.02 ± 1.10 b (0.71)

a

Data with different letters within a column are statistically significant (p < 0.05). bAbsorbance values of assay mixtures at 700 nm. cFold change in activity in parentheses. 133

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Figure 3. Reversed-phase HPLC profiles of casein hydrolysates (blue) and plastein (red) before and after gastric protease treatment.

Funding

proteolysis after short-term (up to 30 min) exposure to proteases, including gastric trypsin and pepsin.7 However, plastein stability may not be guaranteed since the authors’ conclusion was based on short-term exposure to proteases and retention of bioactivity and did not consider the peptide aggregate structure or fingerprints, although the authors observed an increase in free amino group postprotease treatment,7 suggesting susceptibility of the plastein to proteolysis. Gastric metabolic instability of peptides has been a major concern in translating food peptide research due to potential loss of structure and function. However, the present study demonstrated that the overall antioxidative potential of the casein plastein was enhanced as a result of gastric proteolysis, possibly due to further release of cryptic antioxidative peptides. Despite the enhanced bioactivity, the lack of clear distinction in the molecular profile and antioxidative activities of the casein hydrolysates and plastein postmodification by gastric proteases indicates that plastein reaction did not enhance gastric metabolic stability and overall prospects of the casein peptide aggregates. In conclusion, this study has demonstrated that plastein reaction catalyzed by microbial proteases resulted in the formation of peptide aggregates that are held together mostly by hydrophobic and electrostatic interactions and less likely by covalent bonding. Although plastein reaction enhanced the reducing potential and decreased the metal chelating ability, the overall activity of the casein plastein did not differ substantially from that of the hydrolysates, especially in their effects in inhibiting linoleic acid peroxidation. Despite the thermal stability and retention of antioxidative activities after heating, the structure and bioactivity of casein plastein were modified substantially after simulated gastric treatment. These findings suggest that the aggregates in the plastein product may not improve the metabolic stability of casein peptides in functional food applications.

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The research program of CCU is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) through a Discovery Grant. Notes

The authors declare no competing financial interest.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (902) 893-6625. Fax: (902) 893-1404. 134

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