Characterization and bioaccessibility of β-carotene in re-assembled casein

Abstract

The characterization and bioaccessibility of β-carotene (β-CE) in re-assembled casein (CN) were investigated in this study. At pH 4.6, β-CE in re-assembled CN significantly lowered the zeta-potential to a negative value compared to that of CN. The morphology of β-CE in re-assembled CN was characterized by atomic force microscopy. β-CE in re-assembled CN exhibited a more uniform and homogenous dispersion, with peak-to-valley differences of 6.8 nm rather than 91.3 nm for the pure β-CE. β-CE in re-assembled CN can effectively protect β-CE against degradation under conditions of heat and ultraviolet radiation. The antioxidant activity of β-CE when it was encapsulated in re-assembled CN was significantly higher than β-CE in control samples under both experimental conditions. The total release rate of β-CE was 76.7% and 63.4% by trypsin and pepsin hydrolysis, respectively. These results indicated a feasible application in the food industry.

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Introduction

Carotenoids make up the most widespread group of naturally occurring pigments which are extensively found in plants and microorganisms. Among these carotenoids, β-carotene (β-CE), with its antioxidant properties, is the most widespread.^1,2 β-CE as pro-vitamin A is also one of the most commonly used bioactive compounds to fortify functional foods.³ Studies have demonstrated that β-CE exhibits several biological functions, for example as an anticancer agent and antioxidant, and in the prevention of cardiovascular diseases.^4,5 However, the utilization of β-CE in the food industry is limited because of its poor water-solubility, low stability, high melting point, high susceptibility to chemical degradation and low bioaccessibility.⁶ To solve the abovementioned problems, various potential technologies, including encapsulation,⁷ nanoemulsification⁶ and self-assembly,³ have been introduced to improve the utilization, bioaccessibility and stability of β-CE in foods.

Milk proteins are ideal materials used to deliver bioactive compounds and drugs not only because they are inexpensive, readily available, easily digestible, non-toxic and highly stable, but also their ability to bind hydrophobic molecules, ions and biopolymers stabilizes emulsions and to some extent retards oxidation.^8,9 Milk proteins mainly consist of two proteins: casein and whey. Caseins, (CNs), a family of phosphoproteins, account for 80% of bovine milk protein and consist of the following four principle proteins: αs1-CN, αs2-CN, β-CN and κ-CN. The mass ratio of these four proteins is about 4 : 1 : 4 : 1.¹⁰ CNs form soluble aggregates because κ-CN molecules stabilize the micelle structure.

CN can be used to carry hydrophobic bioactive compounds because of its special supra-molecular structures.¹⁰ These structures not only allow the hydrophobic molecules to bind to CNs, but also effectively protect the β-CE from degradation under conditions of heat, light and oxygen.¹¹ Semo et al. suggested that CN can re-assemble in vitro and have a strong tendency to bind hydrophobic molecules, still exhibiting similar properties to those of the originally occurring CN micelles (CM).⁸ Extensive research efforts have been devoted to the utilization of re-assembled CM to deliver hydrophobic compounds. Re-assembled CM could encapsulate and deliver vitamin D2, and be used as a nano-delivery system to protect against photochemical degradation of the vitamin. CN as nano-vehicles was also found to protect against docosahexaenoic acid oxidation and exhibited good bioactive conservation throughout its shelf life.⁸ CN can bind β-CE to form β-CE nanomicelles by self-assembly. This nanostructure effectively protects β-CE against degradation during its treatment, such as sterilization, pasteurization, high hydrostatic pressure and baking.³ CN has also been used to deliver drugs in medical applications.¹² Several literature reports have indicated the roles of hydrophobic nutraceutical compounds in re-assembling CMs as well as the roles of calcium and phosphate nanoclusters in bridging between serine-phosphate groups of the CN and forming the micelles.^13–17

In order to expand the application of β-CE in functional foods, an improvement in the stability and bioaccessibility of β-CE is highly desirable. Thus, the main objective of this research was to study β-CE in re-assembled CN in order to improve its stability and bioaccessibility. β-CE in re-assembled CN was characterized by Atomic Force Microscopy (AFM) and particle electrophoresis. In order to investigate the stability against heat and ultraviolet (UV) radiation, the concentration changes of β-CE in re-assembled CN were determined by reversed phase-high performance liquid chromatography (RP-HPLC). Furthermore, the antioxidant activity of β-CE in re-assembled CN against heat and UV radiation was determined by hydroxyl radical scavenging. The release concentration of β-CE re-assembled in CN in the digestive system simulated by pepsin and trypsin proteolysis was determined by RP-HPLC. Thus, this study provides a novel perspective in the understanding of bioaccessibility of β-CE in re-assembled CN.

Materials and methods

Materials

β-CE, sodium caseinate from bovine milk, trypsin from porcine pancreas, pepsin from porcine gastric mucosa and HPLC-grade solvents (methanol, acetonitrile, and dichloromethane) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). All other chemicals used were of analytical grade.

Re-assembly of β-CE in CN

β-CE in CN was re-assembled following the procedure described by Semo et al.⁸ β-CE in acetone (0.3 mM, 18 mL), previously filtered using a 0.45 μm filter, and tri-sodium citrate (1 M, 4 mL) were added to sodium caseinate (200 mL, 5%). Subsequently, a total volume of 24 mL of K₂HPO₄ (0.2 M) and 20 mL of CaCl₂ (0.2 M) were added in eight consecutive additions every 15 min. The dispersions were stirred in a thermostatic bath at 37 °C. During the latter additions, the pH was adjusted to 6.7 using HCl (0.1 M) or NaOH (0.1 M). Finally, deionized water was added to a final volume of 400 mL. The pH was again adjusted to 6.7 and the final dispersion was gently stirred for 1 h.

Zeta-potential of β-CE in re-assembled CN

The zeta-potential of β-CE in re-assembled CN, CN and pure β-CE was determined using a Nano-ZS Malvern Instrument (Nano-ZS, Malvern Instruments, Worcestershire, UK).

Morphological characteristics of β-CE in re-assembled CN

The AFM was used to compare the morphological characteristics of β-CE in re-assembled CN, CN and pure β-CE. In practice, a sample solution (2 mL) was placed on a freshly cleaved mica slide (SPIChem TM Mica, Grade V-4, 9.9 mm discs of 0.15 mm thickness; West Chester, PA). Specimen slides were then stored in a desiccator prior to scanning. The air dried mica specimens were immersed in butanol for scanning, as described before in the literature.^18,19 Butanol repulses all the bound water from the sample surface, and protects hydrophilic structures from swelling due to atmospheric water vapour. Thus, butanol is widely used as an inert medium for improving image quality. The scans were performed using an AF microscope (Nanoscope IIIA Digital instruments Ltd, America) mounted with a silicon tip.

HPLC analysis of β-CE

HPLC quantification of β-CE was done based on the procedure reported by Yi et al. using an Agilent 1100 HPLC system with a DAD UV-Vis absorption detector (Agilent, Santa Clara, CA).²⁰ RP-HPLC was performed using a polymeric carotene C30 reverse-phase analytical column (250 × 4.6 mm ID, 5 mm, YMC, Inc., Wilmington, NC) to separate β-CE with linear gradients of buffer A in buffer B at a flow rate of 1 mL min⁻¹ at room temperature. The guard column was a reverse-phase C18 column (50 × 3.0 mm ID, 5 mm, YMC, Inc.). The injection volume was 20 μL and the detection wavelength was 450 nm. The chromatography conditions were as follows: solvent A: methanol : acetonitrile : H₂O (84 : 14 : 2, v/v/v) and solvent B: dichloromethane. The solvent gradient program was: 80% A and 20% B at 0 min and was changed linearly to 45% A and 55% B in 15 min, and maintained for 5 min. This was followed by a linear return to 80% A and 20% B in 25 min.

Hydroxyl radical scavenging of β-CE in re-assembled CN

The ability of the samples to scavenge hydroxyl radicals generated by the Fenton reaction was measured according to the modified method provided by Siriwardhana et al.²¹ The Fenton reaction mixture containing FeSO₄·7H₂O (10 mM, 20 μL), EDTA (10 mM, 20 μL) and 2-deoxyribose (10 mM, 20 μL) was mixed with sodium phosphate buffer (0.05 M, 120 μL, pH 7.0) containing 20 μL of sample. Thereafter, H₂O₂ (10 mM, 20 μL) was added prior to the incubation at 37 °C for 4 h. TCA (100 μL of 2.8%) and TBA (100 μL of 1%) were mixed together and placed in the boiling water bath for 10 min. After the resultant mixture was cooled to room temperature, the absorbance was recorded at 536 nm. The percentage of hydroxyl radical scavenged was calculated by using the following equation:

˙OH_scavenging = (1 − A_Sample/A_Control) × 100

where A_Control is the absorbance of the solution without sample and A_Sample is the absorbance in the presence of sample.

Heat stability of β-CE in re-assembled CN

β-CE in re-assembled CN and pure β-CE were heated at 85 °C for 25 min. Subsequently, β-CE in re-assembled CN (10 mL) was homogenized for 10 min at 5000 × g using a homogenizer (Scientz Co., Ltd, China), and hexane (4 mL) was added to extract the β-CE. The concentration of β-CE obtained from heated β-CE in re-assembled CN and pure β-CE was analyzed by HPLC. The degradation of β-CE is expressed in terms of the ratio of β-CE concentration obtained from heated β-CE in re-assembled CN and pure β-CE (C) to initial β-CE concentration (C₀). Hydroxyl radical scavenging of heated β-CE in re-assembled CN and pure β-CE was assayed.

UV resistance of β-CE in re-assembled CN

β-CE in re-assembled CN and pure β-CE were exposed to UV light for 90 min and UV light induced degradation of β-CE was evaluated. Following the UV exposure, the concentration of residual β-CE encapsulated in re-assembled CN and concentration of pure β-CE were analyzed by HPLC. The degradation of β-CE is expressed in terms of the ratio of residual β-CE concentration (C) in β-CE in re-assembled CN and pure β-CE to initial β-CE concentration (C₀).

Release of β-CE through pepsin or trypsin hydrolysis

β-CE in re-assembled CN was dissolved/dispersed in HCl (1 mL, 0.1 M). The solution was incubated at 37 °C and then pepsin solution (0.2 mg pepsin in HCl (1 mL 0.1 M), 50 μL) was added to initiate the hydrolysis.²² Aliquots of the hydrolysis solution were taken out at intervals of 30 min. The hydrolysis was quenched by adding NaOH solution (0.5 mL, 1 M). Then, n-hexane (2.0 mL) was added and the resulting solution was stirred gently for 1 h to extract the released β-CE. The release of β-CE through trypsin hydrolysis was performed by using Tris–HCl buffer (0.2 M, pH 8.0) and the hydrolysis was stopped by adding HCl (0.5 mL, 0.2 M); the other conditions were the same as in the pepsin hydrolysis.²³ The release of β-CE through pepsin or trypsin hydrolysis was analyzed by HPLC.

Statistical analysis

Data were analyzed by the ANOVA method using SPSS software, version 10.0 (SPSS, Inc., Chicago, IL, USA). The differences among the means of the analysis data were compared at a significance level of p < 0.05.

Results and discussion

Zeta-potential of β-CE in re-assembled CN

Fig. 1 shows the zeta-potential of β-CE in re-assembled CN, CN and pure β-CE at different pH values. Fig. 1 clearly exhibits that the zeta-potential of β-CE in re-assembled CN undergoes significant changes over pH 2.0–10.0 compared to CN and pure β-CE. In general, CN is highly liable to aggregation around the isoelectric point (PI = 4.6). Interestingly, at pH 4.6, the zeta-potential of β-CE in re-assembled CN significantly decreases to a negative value compared to CN. Furthermore, the zeta-potential of β-CE in re-assembled CN at pH 3.6 becomes almost zero. The differences of PI between CN and β-CE in re-assembled CN might be attributed to the large negative charges on β-CE, which neutralized some of the positive charges of CN when the bonding occurred between β-CE and CN. Thus, β-CE, encapsulated and stabilized in re-assembled CN, has a lowered zeta-potential around the isoelectric point of the protein compared to CN alone, leading to an improvement in its solubility at this point.

Fig. 1 Zeta-potential of β-CE in re-assembled CN, CN and pure β-CE at different pH values.

Morphological characteristics of β-CE in re-assembled CN

Structural characterization of β-CE in re-assembled CN was performed using intermittent AFM imaging in a butanol environment following the literature method.^24,25 AFM images of β-CE in re-assembled CN, pure β-CE and CN are shown in Fig. 2. The morphology of β-CE in re-assembled CN exhibits a fine and homogenous dispersion with peak-to-valley differences exceeding 6.8 nm. The result indicated that β-CE in re-assembled CN was liable to dissolve in water. This could be attributed to the fact that hydrophobic β-CE was entrapped within CMs by association with the hydrophobic domains of soluble CN. The AFM image of pure β-CE exhibits a more heterogeneous and rougher array with peak-to-valley differences exceeding 91.3 nm. Moreover, the AFM image of pure β-CE shows its poor water-solubility. The AFM image of CN displays small particles with a size of 7.5 nm. The particles of CN retained their original structure and morphology when β-CE was incorporated into the re-assembled CN. These results indicated that the incorporation of β-CE has an insignificant effect on the morphology of CN. This result was in good agreement with the results obtained by Semo et al., who reported that CN can re-assemble in vitro; however, it still exhibits properties similar to those of the originally occurring CMs.⁸ Thus, the properties of the micelles, including size and morphology, were preserved when β-CE was encapsulated in the re-assembled CN.

Fig. 2 AFM images of samples under butanol at intermittent contact mode. (A) β-CE in re-assembled CN; (B) CN and (C) pure β-CE.

Stability of β-CE in re-assembled CN

The β-CE concentration in heated samples and samples exposed to UV radiation is shown in Fig. 3A and B, respectively. During the heating process at 85 °C for 25 min, control samples show that the total β-CE concentration decreases by about 63.3%; however, the total β-CE concentration of β-CE in re-assembled CN decreases by only about 9% (Fig. 3A). Sáiz-Abajo et al. reported that β-CE concentration in the control samples decreased by about 80% and in re-assembled CN decreased by about 30% after 8 h of heating.³ β-CE concentration showed significant differences between the control samples and the re-assembled CN samples. The results revealed that β-CE encapsulated in re-assembled CN could be easily protected against heat degradation.

Fig. 3 (A) β-CE concentration in heated samples and (B) samples exposed to UV radiation.

Fig. 3B shows that β-CE concentration in the control samples decreases by 80% when the sample is exposed to UV radiation; however, the total β-CE concentration of β-CE in re-assembled CN decreases by only 24% during the period of 90 min exposure to UV radiation. The β-CE concentration exhibited significant differences between the control samples and the re-assembled CN samples. The results revealed that CMs protected β-CE from degradation during exposure to UV radiation.

Hydroxyl radical scavenging of β-CE in re-assembled CN

Fig. 4A and B, respectively, exhibit the hydroxyl radical scavenging of β-CE in heated samples and samples exposed to UV radiation. Fig. 4A shows that the hydroxyl radical scavenging of β-CE in the control samples decreases by about 50% during the heating process at 85 °C for 25 min; however, the hydroxyl radical scavenging of β-CE in the re-assembled samples decreases by only 10%. The decrease of hydroxyl radical scavenging in the two samples showed significant differences, which might be attributed to the degradation of β-CE. The concentration of β-CE in the control samples significantly decreased compared with that of the re-assembled samples. The hydroxyl radical scavenging in the control samples was significantly lower than in the re-assembled samples because the antioxidant activity of β-CE is positively related to the content of β-CE.²⁶

Fig. 4 Hydroxyl radical scavenging of β-CE in (A) heated samples and (B) samples exposed to UV radiation.

Fig. 4B shows that the hydroxyl radical scavenging of β-CE in the control samples decreases by about 80% when the sample is exposed to UV radiation for 90 min. The hydroxyl radical scavenging of β-CE in the re-assembled samples decreases by only 20%. The decrease of hydroxyl radical scavenging in the control samples was also in relation to the β-CE degradation; however, β-CE in the re-assembled samples was protected against UV radiation, which prevented the degradation of β-CE. Thus, the hydroxyl radical scavenging in the control samples was significantly lower than that in the re-assembled CN samples.

Release of β-CE in vitro

The release of β-CE in simulated gastric and intestinal juices has been commonly used to evaluate the bioaccessibility of β-CE in vitro.²⁷ The release of β-CE in re-assembled CN treated with proteases over 120 min is shown in Fig. 5. Without protease treatment, a negligible amount of β-CE is released in the simulated gastrointestinal juices. The release of β-CE increases rapidly during the initial 30 min in both trypsin and pepsin. The release rate of β-CE in re-assembled CN increases with an increase in hydrolysis time. The total release rate of β-CE was 76.7% and 63.4% by trypsin and pepsin hydrolysis, respectively. The result was similar to that reported for β-CE encapsulated in sodium caseinate by Yi et al.²⁰

Fig. 5 Release of β-CE when treated with pepsin (1.0 mg mL⁻¹) and trypsin (1.0 mg mL⁻¹) in simulated gastrointestinal juices.

The higher release rate of β-CE in re-assembled CN might be attributed to the capability of β-CE encapsulated in re-assembled CN to dissolve in water with a small particle size. Besides, CN has been proven to be easily digestible in the stomach, thus CN hydrolysis by proteases enabled hydrophobic amino acid exposure and β-CE was released from CN. These results indicated a feasible application in the food industry, ensuring the release of β-CE during the digestion stage.^28,29

Conclusions

In this study, the results indicated that β-CE in re-assembled CN could significantly alter the zeta-potential compared to that of CN. AFM images showed that β-CE in re-assembled CN could improve the water-solubility of β-CE compared to pure β-CE. β-CE encapsulated in re-assembled CN was protected against heat and UV radiation degradation. The antioxidant properties of β-CE were preserved when it was encapsulated in re-assembled CN. The release of β-CE in simulated gastric and intestinal juices showed that β-CE in re-assembled CN was provided with perfect bioaccessibility. Thus, this study provided a novel perspective to the understanding of the bioaccessibility of β-CE in re-assembled CN.

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