Article pubs.acs.org/JAFC
Effects of Environmental Stresses and in Vitro Digestion on the Release of Tocotrienols Encapsulated Within Chitosan-Alginate Microcapsules Phui Yee Tan,† Tai Boon Tan,† Hon Weng Chang,† Beng Ti Tey,‡ Eng Seng Chan,‡ Oi Ming Lai,§ Badlishah Sham Baharin,† Imededdine Arbi Nehdi,∥ and Chin Ping Tan*,† †
Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia ‡ Chemical Engineering Discipline, School of Engineering, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia § Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia ∥ King Saud University, College of Science, Chemistry Department, Riyadh, Saudi Arabia ABSTRACT: Considering the health benefits of tocotrienols, continuous works have been done on the encapsulation and delivery of these compounds. In this study, we encapsulated tocotrienols in chitosan-alginate microcapsules and evaluated their release profile. Generally, these tocotrienols microcapsules (TM) displayed high thermal stability. When subjected to pH adjustments (pH 1−9), we observed that the release of tocotrienols was the highest (33.78 ± 0.18%) under basic conditions. The TM were also unstable against the effect of ionic strength, with a high release (70.73 ± 0.04%) of tocotrienols even at a low sodium chloride concentration (50 mM). As for the individual isomers, δ-tocotrienol was the most sensitive to pH and ionic strength. In contrast, β-/γ-tocotrienols were the most ionic-stable isomers but more responsive toward thermal treatment. Simulated gastrointestinal model showed that the chitosan-alginate-based TM could be used to retain tocotrienols in the gastric and subsequently release them in the intestines for possible absorption. KEYWORDS: alginate, chitosan, gelation, microencapsulation, self-assembled, release profile
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submicron particles.6 The resulting tocotrienols Pickering emulsion (TPE) was then used as the template for the formation of tocotrienols microcapsules via hydrocolloids-based ionotropic gelation. In order to enhance the structure’s stability and rigidity, we coated the gel microspheres with an external layer of chitosan to fabricate a more rigid structure that could load more palm olein and, consequently, entrap a higher amount of palm tocotrienols. Given that the structural components (CaCO3, alginate and chitosan) of the tocotrienol microcapsules (TM) are mostly pH-sensitive, we believe that the environmental stresses, especially pH and ionic strength, would have important impacts on our TM’s stability. With the use of the biocompatible hydrocolloids (alginate and chitosan), it would be interesting to investigate the in vitro release of the chitosan-alginate microcapsule in order to explore the potential of the hydrocolloid-based system as a delivery system for tocotrienols. To date, no research has been done on the release of palm tocotrienols from polysaccharide-based microcapsules in response to environmental stresses and in vitro digestion. Therefore, the primary aim of our study was to investigate the
INTRODUCTION As members of the Vitamin E family, tocotrienols have been receiving increasing attention due to their neuroprotective, cholesterol-lowering and tumor growth suppression properties.1 In fact, tocotrienols have been proven to impose better health effects in comparison to other Vitamin E members, such as tocopherols.2 Hence, this micronutrient can be applied for possible use in functional food. Unfortunately, tocotrienols are only abundant in certain types of plant or food source, such as palm oil. The bioavailability of these compounds is therefore very low.3 In addition, tocotrienols are more easily oxidized and degraded as compared to tocopherols.4 Thus, in search of possible ways to protect and improve the bioavailability of tocotrienols, we have continuously worked on the development of a suitable delivery tool to encapsulate and deliver these lipophilic compounds. In the present study, the precursor emulsion of tocotrienols was formed through the Pickering emulsion approach. Pickering emulsion is a type of highly stable emulsion system that relies on the stabilizing effect of solid particles. The use of these solid particles as stabilizers eliminate the need for synthetic surfactants which are commonly used in normal emulsion systems.5 Therefore, the Pickering emulsion is more biocompatible than the common emulsion. With the help of a low-energy homogenization method, an oil-in-water Pickering emulsion consisting of palm tocotrienols-rich fraction (TRF) was fabricated and stabilized by calcium carbonate (CaCO3) © 2017 American Chemical Society
Received: Revised: Accepted: Published: 10651
July 29, 2017 November 9, 2017 November 10, 2017 November 10, 2017 DOI: 10.1021/acs.jafc.7b03521 J. Agric. Food Chem. 2017, 65, 10651−10657
Article
Journal of Agricultural and Food Chemistry
added into 10 mL of deionized water and heated in a water bath under the following prefixed batch pasteurization and rapid thermal treatment conditions: 63 °C for 30 min and 90 °C for 10 min.8 Then, the solutions were filtered, and the filtrates were subjected to an extraction procedure and HPLC analysis as outlined in the Tocotrienol Analysis section, respectively. Effect of Ionic Strength. The test on the stability of TM against ionic strength was carried out by adding 100 mg of TM into 10 mL of solution with varied concentrations of sodium chloride (0, 50, 100, 150, 200 mM) and leaving the respective solutions to stand for 1 h. After 1 h, the solutions were filtered, and the filtrates were subjected to an extraction procedure and HPLC analysis as outlined in the Tocotrienol Analysis section, respectively. In Vitro Release of TM. The in vitro release of TM was evaluated based on the procedure described by Karaca et al.,9 with slight modifications. The simulated gastric fluid was prepared by dissolving 2 g of sodium chloride and 7 mL of concentrated HCl in 900 mL of deionized water. The mixture was then added with 3.2 g of pepsin and adjusted to pH 1.2 using 0.1 M HCl. The volume of the solution was finally topped up to 1000 mL with deionized water. As for the preparation of simulated intestinal fluid, 6.8 g of K2HPO4 was dissolved in 800 mL of water, followed by the addition of 0.2 M NaOH (77 mL) and 100 g of pancreatin. The mixture was then stirred overnight at 4 °C. The in vitro test was performed by adding 100 mg of TM to 10 mL of the simulated gastric fluid. Then, the mixture was incubated in a water bath set at 37 °C for 2 h under shaking at 100 rpm. For sequential gastric and intestinal digestion simulation, the aforementioned steps were repeated. After the 2 h incubation, the pH of the mixture was adjusted to pH 6.8 using 1 M NaOH. Thereafter, the simulated intestinal fluid (10 mL) was added into the mixture, followed by further incubation under the same conditions for 3 h. The simulated gastric and intestinal phases were each prepared in five sets. Each set was taken out from the water bath at a fixed time interval of 30 min. Then, the solutions were filtered, and the filtrates were subjected to an extraction procedure and HPLC analysis to determine the amount of tocotrienols released from the TM. Tocotrienol Analysis. Extraction. The tocotrienols content in the filtered solutions (pH solutions, water, sodium chloride solutions, simulated gastric and intestinal solutions) was determined using the method described by Xu10 and Xu et al.,11 with slight modifications. Briefly, the sample solution was extracted with 10 mL of hexane and vortexed. The hexane layer was then purged to dryness using nitrogen gas and reconstituted in 2 mL of acetonitrile, which was subsequently filtered (0.45 μm nylon syringe filter) for HPLC analysis. HPLC Analysis. The presence of tocotrienols was determined by using a Shimadzu HPLC system equipped with a fluorescence detector (Shimadzu, Prominence LC-20AD). The mobile phase consisting of methanol/acetonitrile/dichloromethane (25:23:2, v/v/v) were run using an isocratic flow of 1 mL/min. An injection volume of 20 μL was applied, and the peaks were separated on a reverse phase C18 column (250 mm × 4.6 mm, 5 μm, Phenomenex), with the temperature set at 30 °C. The excitation and emission wavelengths of the fluorescence detector were fixed at 290 and 330 nm. An external calibration curve (R2 ≥ 0.99) was used to quantify the tocotrienols isomers (α-, β-/γ-, δ). The total tocotrienols content (μg/g TM) was then determined by adding up all the isomers concentration. Because of the loss encountered during the microencapsulation process, the encapsulation efficiency of the tocotrienols was found to be in the range of 56.68− 76.30%. By using the initial tocotrienols’ concentrations in TM (522.78 α-tocotrienol, 548.86 μg/g β-/γ-tocotrienols, 312.90 μg/g δtocotrieol, and 1384.54 μg/g total tocotrienols), the release percentage of the tocotrienol isomers and total tocotrienols were calculated based on eq 1.
effects of environmental stresses (pH and ionic strength) and in vitro digestion on the release profile of chitosan-alginate TM. In addition, we also studied their stability as affected by thermal treatment (in the form of batch pasteurization and rapid thermal treatment conditions) in order to assess the suitability of future incorporation of our TM into food products.
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MATERIALS AND METHODS
Materials. Palm TRF and refined, bleached, and deodorized (RBD) palm olein (Elaeis guineenis var. tenera) were supplied by Supervitamins Company (Johor, Malaysia) and Moi Foods Malaysia Sdn. Bhd. (Selangor, Malaysia), respectively. Precipitated CaCO3 nanoparticles were obtained from NanoMaterials Technology Company (Singapore). Sodium alginate (Manugel GHB) and chitosan (molecular weight 100 000−300 000) were purchased from FMC Bioplymers (U.K.) and Fisher Scientific (USA), respectively. Tocotrienols and tocopherols (α-, β-, δ-, γ-) mixed standard was obtained from LGC Standard (Teddington, U.K.). Sodium hydroxide (NaOH) was supplied by Friendemann Schmidt (Australia). Dipotassium hydrogen phosphate (K2HPO4) of analytical grade was obtained from Merck (Darmstadt, Germany). Analytical grade glacial acetic acid, concentrated hydrochloric acid (HCl, 36%), sodium carbonate, sodium chloride, sodium hydrogen carbonate, hexane; and HPLC grade methanol, acetonitrile and dichloromethane were all obtained from Fisher Chemical (U.K.). Preparation of CaCO3 Dispersion. The preparation and fabrication of tocotrienols microcapsules were carried out based on previous studies.7 A coarse dispersion of 5% (w/v) CaCO3 was first prepared by dispersing precipitated CaCO3 nanoparticles in deionized water at 5000 rpm for 30 min using Ultra-Turrax rotor-stator homogenizer (IKA, Staufen, Germany). The dispersion was then passed through a high pressure homogenizer (Microfluidizer M-110L, Microfluidics, MA) at 22 000 psi for 4 passes. The resulting dispersion was kept diluted (0.75%) for use in the subsequent emulsification process. Formation of TPE Template. RBD palm olein (10 mL) containing 2% (w/w) TRF was added into 0.75% CaCO3 dispersion and homogenized using a high shear homogenizer (Silverson, MA) at 5000 rpm for 15 min. The resulting emulsion was allowed to stand for 30 min to settle and form the tocotrienol-rich template on the top layer which was then used for the gelation process. Formation of TM. Alginate Gelation. TPE (2 mL) was added into a 2% (w/v) alginate solution which was being gently agitated. Acetic acid (1.0 M) was slowly added to reduce the pH of the mixture. The mixing was immediately stopped as soon as pH 4 was achieved. Then, the mixture was left to stand for 16 h for the curing of the microcapsules. After 16 h, the microcapsules were collected from the mixture using a sieve (80 μm) and washed with deionized water to remove excess alginate. Chitosan Coating. The collected TM were dispersed in 0.1% calcium chloride prior to layer coating using chitosan. The chitosan solution (0.5%; w/v) was prepared by dissolving the chitosan in deionized water, followed by the addition of acetic acid, all while under magnetic stirring. The solution was then added with 0.1 M NaOH to adjust the pH to 5.5−6.0 and filtered thereafter. The TM was then coated by dispersing the TM in the chitosan solution at a 1:2 ratio (v/ v) for 15 min. The coated TM were then sieved, washed with deionized water, and air-dried. The final TM were kept in an amber bottle and stored at 4 °C for further analyses. Experimental Design. Effect of Environmental Stresses on the Release of Tocotrienols. Effect of pH. In order to assess the release of tocotrienols in different pH conditions, TM (100 mg) were immersed in 10 mL of buffer medium (pH 1−9) for 1 h. Then, the solutions were filtered to remove intact TM. The filtrates were subsequently subjected to an extraction procedure and HPLC analysis as outlined in the Tocotrienol Analysis section, respectively. Effect of Heating. Since the TM were developed for possible food applications, the effect of common food preparation conditions on the tocotrienols’ release profile was also investigated. TM (100 mg) was
released content/initial content × 100%
(1)
Statistical Analyses. The data were expressed as mean ± standard deviation, calculated from triplicate analyses of duplicate samples. One-way analysis of variance (ANOVA) was applied to determine the 10652
DOI: 10.1021/acs.jafc.7b03521 J. Agric. Food Chem. 2017, 65, 10651−10657
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Journal of Agricultural and Food Chemistry significant difference (p < 0.05) between the data using Minitab (version 16.2.2; Minitab Inc., State College, PA).
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RESULTS AND DISCUSSION Effect of pH on Tocotrienols Release. Because of its hydrophilic nature, alginate shrinks at lower pH and absorbs water at higher pH. In the context of this study, water would, under basic conditions, penetrate slowly toward the center core, causing the microcapsules to swell. At this point, a concentration gradient exists between the oil phase and the aqueous phase, thereby inducing the oil containing tocotrienols to diffuse across the hydrocolloids-based shell wall and be released into the external medium.12 Given that the diffusion of tocotrienols depends on the wall thickness and structure (porosity) of the microcapsules, the release of tocotrienols is very much affected by the dissolution of the microcapsules. In this context, the alginate gelation and chitosan coating formed thick, double layers on the surface of the microcapsules, thus accounting for the enhanced protection of the tocotrienols. In addition, the cross-linking between Ca2+, alginate and chitosan reduced the porosity of the microcapsules’ wall and therefore delayed the release of the tocotrienols entrapped within the core. Our findings, as shown in Figure 1a, revealed a decrease in the release of tocotrienols as the pH decreased to below 7, with the minimum release observed at pH 1. When the pH was reduced, the amine groups of chitosan were protonated (−NH3+), and this led to charge repulsion between the ionized groups.13 The ionic repulsion further stabilized the chitosanalginate hydrocolloids network on the microcapsules’ surface, thus preventing the shrinking of alginate at low pH. Interestingly, the amount of tocotrienols released at pH 3 was slightly higher than that at pH 5. This anomaly was most likely due to the increased liberation of Ca2+ from the excess CaCO3 and CaCl2 in the microcapsules which then diffused through the ion-permeable chitosan layer,14 causing partial destabilization of the hydrocolloids network. However, the chitosan-alginate complex was found to be stable enough to even resist the extremely acidic condition at pH 1. As such, this finding suggests the possibility of the TM to retain higher amount of tocotrienols in the gastrointestinal environment. The release pattern of TM showed a relatively higher amount of tocotrienols being released at neutral pH (pH 7) as compared to that at lower pH. This was most likely due to the chelating effect of Na+ from the sodium phosphate buffer in the medium. As the chitosan layer is permeable to ions, the monovalent Na+ would diffuse through the chitosan shell and replace the Ca2+ within the hydrocolloids network to bind with the carboxylate groups of alginate.15 In addition to the Na+ ionchelating effect, the amino group of chitosan would start to deprotonate as the pH was increased to its pKa value of pH 6.5.13 These two events would eventually lead to the destruction of the microcapsules’ wall complex and allow more water to be absorbed into the alginate gel. Consequently, the alginate layer and the overall microcapsule structure would swell, burst and release more tocotrienols. In comparison to that of low pH (pH 1), the more alkaline pH 9 triggered an 18% increment in the total tocotrienols release. The increment in pH led to an increase in the OH− present in the medium, which then progressively deprotonated the amino group of the chitosan, thus causing the dissociation of the amino-carboxylate bond between chitosan and alginate polymers. As a result, this phenomenon would again lead to the
Figure 1. Total tocotrienols released in response to (a) pH, (b) heating, and (c) ionic strength. Different letters indicate a significant difference (p < 0.05) between data.
disintegration of the chitosan-alginate wall structure and facilitate water penetration, followed by the swelling of alginate.13 The resulting swollen microcapsules would eventually burst and release more tocotrienols. Therefore, at higher pH, the shell wall disintegration rate would be boosted as more amino groups of chitosan deprotonate. This explains the highest release of tocotrienols observed at pH 9. In view of the different tocotrienols isomers, the isomers exhibited different release profiles in response to the changes in pH. Within the fixed duration of incubation (1 h), δ-tocotrienol displayed the highest release across the pH range studied (Table 1), indicating a higher sensitivity toward pH. To be more specific, the increase from pH 1 to 9 triggered a 42% increment in the release of δ-tocotrienol from the microcapsules. On the other hand, β-/γ-tocotrienols showed the lowest released amount as compared to that of other isomers. It is therefore evident that β-/γ-tocotrienols are of higher encapsulation stability, with δ-tocotrienol being the least stable isomer when subjected to pH adjustments. As for α-tocotrienol, the isomer exhibited moderate sensitivity to pH change, thus a moderately high release of the isomer was observed after the 1 10653
DOI: 10.1021/acs.jafc.7b03521 J. Agric. Food Chem. 2017, 65, 10651−10657
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Journal of Agricultural and Food Chemistry Table 1. Release of Tocotrienol Isomers from Tocotrienol Microcapsules As Affected by Environmental Stressesa release (%) stress factors
conditions
α-tocotrienol
δ-tocotrienol
4.05 ± 0.03 eC 6.72 ± 0.03 cC 5.84 ± 0.04 dC 12.82 ± 0.04 bC 13.08 ± 0.10 aC
34.06 41.92 40.77 52.33 76.06
N/A N/A
22.46 ± 0.02 bA 25.51 ± 0.04 aA
5.68 ± 0.04 bB 6.19 ± 0.04 aB
0.87 ± 0.09 eC 35.72 ± 0.06 dB 37.45 ± 0.30 cB 42.03 ± 0.06 bB 44.22 ± 0.37 aB
1.07 ± 0.02 dB 14.56 ± 0.03 cC 14.61 ± 0.16 cC 16.62 ± 0.04 bC 17.47 ± 0.04 aC
5.60 ± 0.07 eA 75.92 ± 0.06 dA 80.71 ± 0.04 cA 74.10 ± 0.08 bA 96.74 ± 0.05 aA
pH
1 3 5 7 9
16.54 15.91 16.12 33.98 30.20
heating
60 °C, 30 min 90 °C, 10 min
NaCl (mM)
0 50 100 150 200
± ± ± ± ±
β-/γ-tocotrienols
0.20 0.21 0.16 0.18 0.33
dB dB cB aB bB
± ± ± ± ±
0.09 0.08 0.04 0.10 0.19
eA cA dA bA aA
The reported values represent the mean ± standard deviation of six measurements from two replications. Lowercase letters represent a significant difference (p < 0.05) within the column. Uppercase letters represent a significant difference (p < 0.05) between columns. N/A is not available.
a
would eventually lead to a greater extent of structure disruption and more substantial release of the encapsulated tocotrienols. Similar to the release pattern observed when the TM were subjected to the effect of pH, the change in ionic strength triggered the highest release of δ-tocotrienol, which was twice the release percentage of α-tocotrienol. Again, α-tocotrienol exhibited moderate sensitivity to the ionic strength. On the other hand, β-/γ-tocotrienols showed greater stability, as evident by their lowest release (Table 1). Hence, it is believed that δ-tocotrienol is more responsive toward ionic strength as compared to the other isomers. On the basis of all these findings, α-tocotrienol appeared to be strongly bound within the microcapsule network, thus making it the most stable isomer against heat while being moderately resistant against the changes in pH and ionic strength. Conversely, β-/γ-tocotrienols in the microcapsules were easily liberated when subjected to high temperature treatment but displayed the highest resistance against changes in pH and ionic strength. Lastly, unlike the aforementioned isomers, the release of δ-tocotrienol was easily triggered through pH and ionic strength adjustments. In Vitro Digestion. Simulated Gastric Phase. As denoted in Figure 2, the release of the total tocotrienols from TM
h incubation. Generally, the stability of the isomers could be affected by the system in which they exist.16 The higher stability of β-/γ-tocotrienols was probably due to the higher affinity of the β-/γ-tocotrienols toward the chitosan-alginate network.17 Effect of Heating on Tocotrienols Release. As implied by the results shown in Figure 1b, the chitosan-coated TM exhibited acceptable thermal stability with only 10−11% of tocotrienols released after exposure to the heating conditions. Generally, the exposure to a higher temperature may accelerate the penetration of water into alginate microcapsules and therefore promote the swelling of the hydrocolloid. However, by coating an extra chitosan layer on the alginate surface, the resulting entanglement formed a hydrocolloids network which is strong enough to withstand even the high temperature of 90 °C. As pointed out in the study done by Abbaszadeh et al.,18 the usage of chitosan-alginate as the encapsulation matrix provided better protection that contributed to the survival of probiotic bacteria against heat. Hence, it is believed that this chitosan-coated TM could be of great use in protecting tocotrienols from heat degradation. Again, the different isomers of tocotrienol exhibited different degrees of stability against different heating conditions. β-/γTocotrienols displayed rapid release with 4-times higher release as compared to δ-tocotrienol, while no apparent release was observed for α-tocotrienol. Apparently, α-tocotrienol possesses the highest thermal stability among all the isomers within the microcapsule, with β-/γ-tocotrienols being the least heatresistant form. Effect of Ionic Strength on Tocotrienols Release. In contrast to their high resistance against low pH and temperature, the TM seemed to be rather unstable in the presence of NaCl. As expected, minimal release of tocotrienols was observed at 0 mM, after which the amount drastically increased, with a 66% rise in tocotrienols release when 50 mM NaCl was added. The highest release (89.21 ± 0.16%) was observed when the NaCl concentration was increased to 200 mM (Figure 1c). The increment in ionic strength led to an elevated osmotic effect, which caused the Na+ to diffuse through the surface of the microcapsules. Consequently, the Na+ would displace the Ca2+, leading to the destabilization of the “egg-box” structure and swelling of the microcapsules.9,15 With a higher ionic strength, the increased Ca2+ displacement
Figure 2. Cumulative release of tocotrienols in simulated gastric and intestinal phases. Dotted line marks the 120 min time interval. Different letters indicate significant difference (p < 0.05) between data. 10654
DOI: 10.1021/acs.jafc.7b03521 J. Agric. Food Chem. 2017, 65, 10651−10657
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Journal of Agricultural and Food Chemistry Table 2. Cumulative Release of Tocotrienols Isomers in Simulated Gastric and Intestinal Phasesa cumulative release (%) incubation time (min)
α-tocotrienol
gastric
30 60 90 120
17.49 ± 0.16 hC N/A 19.43 ± 0.31 fC 13.24 ± 0.05 iC
intestinal
150 180 210 240 270 300
17.79 17.79 17.79 17.79 68.78 75.23
phase
± ± ± ± ± ±
0.04 0.04 0.04 0.04 0.69 0.63
gC eC dC bC cB aB
β-/γ-tocotrienols
δ-tocotrienol
18.72 27.05 20.53 18.73
± ± ± ±
0.02 0.10 0.03 0.04
iB dA hB iB
26.36 14.38 29.58 21.76
± ± ± ±
0.12 0.15 0.10 0.04
dA eB cA eA
23.16 23.85 27.99 32.16 25.77 30.79
± ± ± ± ± ±
0.06 0.07 0.07 0.08 0.15 0.19
gB fB cB aB eC bC
28.46 36.18 66.22 92.96 81.56 99.89
± ± ± ± ± ±
0.05 0.05 0.12 0.14 0.41 0.89
dA dA dA dA bA aA
The reported values represent the mean ± standard deviation of six measurements from two replications. Lowercase letters respresent a significant difference (p < 0.05) within column. Uppercase letters represent a significant difference (p < 0.05) between columns. N/A is not available.
a
among all isomers, it possesses the highest sensitivity to acidic pH and ionic strength. Despite its higher release, δ-tocotrienol exhibited fluctuating trend during the incubation in gastric phase (30−120 min). As mentioned earlier, the degradation of the released δ-tocotrienol in the acidic medium created a gradient that allowed the isomer to diffuse out to the medium.4 Unfortunately, the diffusion of the isomer was hindered by the microcapsule wall.19 This phenomenon thereby caused the fluctuating trend observed in the δ-tocotrienol release. Simulated Intestinal Phase. After being treated with simulated gastric fluid, the tocotrienols within the coated TM continued to be released in the simulated intestinal fluid (see Table 2). Generally, the cumulative release of tocotrienols began with a relatively low percentage in the initial period, followed by a progressive inclination as the incubation period proceeded to 300 min. Specifically, the extended incubation time from 120 to 300 min sped up the release of tocotrienols, with a consistent increment of 2−14% in the cumulative release. As mentioned earlier in the section on the effect of pH on tocotrienols release, higher pH value would trigger a higher release of tocotrienols from the chitosan-coated TM. Hence, the incubation of TM in pH 6.8 triggered the consistent dissolution and swelling of the microcapsules, which in turn led to the steady release of tocotrienols over time. Besides, the addition of pancreatin in the medium aided the disintegration of the microcapsules’ wall, and this accelerated the release of the tocotrienols as well. Unlike its release pattern in the simulated gastric phase, αtocotrienol displayed high stability in the simulated intestinal phase, with no observable release during the first 90 min of incubation (150−240 min). As aforementioned, the surface tocotrienols including α-tocotrienol were actively released in the first 90 min incubation of gastric phase. Thus, as the digestion proceeded to the intestinal phase, the remaining αtocotrienol within the microcapsule would require sufficient time to diffuse through the microcapsule wall out to the medium. Therefore, as the incubation period was prolonged to 270 min, the release of α-tocotrienol increased to 68.78 ± 0.69%. On the other hand, the release of β-/γ-tocotrienols only experienced a small increment of 4−5% throughout the whole incubation period, thus indicating the highest stability of these isomers in the simulated intestinal environment. Again, δtocotrienol showed the highest release from the TM, with a complete release achieved within the 3 h incubation period.
showed an initial release of about 20%, which was probably due to the rapid diffusion of the compounds on the TM surface.19 However, prolonged incubation of TM in the simulated gastric fluid did not cause an increase in the tocotrienols release. Instead, the release of tocotrienols remained low, ranging from approximately 14−22% in the acidic gastric medium, with some fluctuations observed during the first 120 min of incubation time, whereby the release of tocotrienols dropped about 5−6% at the 60th and 120th min intervals. The observed fluctuations could be explained by the degradation of the released tocotrienols in the acidic medium,4 which consequently caused a higher concentration gradient to exist and induced more tocotrienols to be released from the TM core. However, because of the double-layer surface of the TM, the swelling of the microcapsules was rather inhibited, thus hindering the diffusion of the tocotrienols.19 As a result, lower amount of tocotrienols were actually released from the TM core. Additionally, the high surface rigidity also played a part in slowing down the enzyme’s penetration into the TM, thereby decelerating the microcapsules’ solubility.15 Our observations were in agreement with previous findings,20 which emphasized the excellent ability of chitosan in protecting the encapsulated active component against gastric fluid. The overall release of the tocotrienol isomers was considerably low, possibly due to the insufficient incubation time. Even though the 2 h period is the commonly applied incubation time in studies involving simulated gastric phase, it should be noted that the normal gastric emptying time of human body is 4 h.21 In this context, the release of tocotrienols could possibly be increased by prolonging the incubation time. Given that the chitosan-coated TM possessed higher stability against thermal treatment (as discussed earlier in the section on effect of heating on tocotrienols release), the temperature of 37 °C used to mimic the human body’s temperature did not really exert any effect on the tocotrienols release. Hence, the release of tocotrienols in the simulated gastric environment was dependent on the surrounding pH and the effect of ionic strength (NaCl). α-Tocotrienol, which exhibited moderately high stability in acidic and ionic mediums, as was discussed earlier, contributed to the lowest release and can therefore be said to possess the highest stability in the simulated gastric fluid which is of pH 1.2. Meanwhile, β-/γ-tocotrienols showed the second highest release in the simulated gastric phase, while δtocotrienol again exhibited the most rapid release in the simulated gastric condition, further reinforcing the fact that 10655
DOI: 10.1021/acs.jafc.7b03521 J. Agric. Food Chem. 2017, 65, 10651−10657
Article
Journal of Agricultural and Food Chemistry
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In the current study, we have demonstrated that the doublelayered chitosan-alginate TM were able to provide excellent protection against external stimuli, especially heat treatment and low-pH gastric fluid. However, changes in ionic strength were able to trigger immediate release of tocotrienols from the TM, even at low NaCl concentration (50 mM). Despite this low stability in the presence of NaCl, we have shown that a more consistent release of tocotrienols could be achieved at neutral and basic pH conditions. These findings therefore suggest the possibility of our chitosan-alginate microcapsules to be used as a potential delivery tool for palm tocotrienols. Our chitosan-coated TM were able to protect the tocotrienols by bypassing the simulated gastric phase (at pH 1.2) without losing substantial amount of the tocotrienols and ideally liberate a consistent amount of tocotrienols in the subsequent simulated intestinal phase (at pH 6.8). Therefore, by using the present microencapsulation method to encapsulate the palm tocotrienols within chitosan-alginate microcapsules, the tocotrienols would have a higher possibility of being absorbed into the blood system and confer their benefits to human health in a more effective manner.
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AUTHOR INFORMATION
Corresponding Author
*Phone: 603-89468418. Fax: 603-89423552. E-mail: tancp@ upm.edu.my. ORCID
Chin Ping Tan: 0000-0003-4177-4072 Funding
The research grant provided by the Science Fund program (Grant 06-02-10-SF0157) of Ministry of Science, Technology and Innovation, Malaysia, is deeply appreciated. The authors extend their appreciation to the International Scientific Partnership Program ISPP at King Saud University for funding this research work through ISPP Grant No. 0055. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The kind support of the specialist team from Shimadzu (Malaysia) Sdn. Bhd. on tocotrienols HPLC analysis is sincerely acknowledged.
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ABBREVIATIONS USED RBD, refined, bleached, and deodorized; TM, tocotrienol microcapsules; TPE, tocotrienol Pickering emulsion; TRF, tocotrienol rich fraction
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REFERENCES
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DOI: 10.1021/acs.jafc.7b03521 J. Agric. Food Chem. 2017, 65, 10651−10657
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DOI: 10.1021/acs.jafc.7b03521 J. Agric. Food Chem. 2017, 65, 10651−10657
