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Complexation of curcumin with Lepidium sativum protein hydrolysate as a novel curcumin delivery system
Food Chemistry 298 (2019) 125091
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Complexation of curcumin with Lepidium sativum protein hydrolysate as a novel curcumin delivery system Deepak Kadam, Shanooba Palamthodi, S.S. Lele
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Department of Food Engineering and Technology, Institute of Chemical Technology, Matunga, Mumbai 400019, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Protein hydrolysate Curcumin Complexation Bioaccessibility/stability
The complexation of Lepidium sativum protein hydrolysate (LSPH) with a lipophilic molecule, curcumin (CUR), and its effect on curcumin in vitro bioaccessibility/stability, functional and antioxidant activity were investigated. Fluorescence spectroscopy of the LSPH/CUR complex confirmed the presence of hydrophobic interactions that led to the complex formation. The LSPH (10–30 kDa) fraction showed a compact complexation with curcumin at pH 3.0 with excellent aqueous solubility, stability, and bioaccessibility. Further, complexation enhanced the aqueous solubility of curcumin more than 856-fold. In vitro sequential simulated gastric and intestinal digestion indicated that the bioaccessibility of curcumin was increased from 67% to 95% post complexation. The functional attributes suggest that the LSPH/CUR complex has good foam-forming capacity and emulsion stability, which are crucial for food product formulations. The results indicate that, since LSPH is a dietary protein, it might possibly be formulated as a functional food and as an excellent lipophilic bioactive molecule delivery vehicle in food formulations.
1. Introduction In the past decade, complexations between proteins and lipophilic molecules, especially polyphenolic compounds, have attracted attention of many researchers and nutraceutical industries. The increased interest is mainly due to the ability of formed complexes to enhance water solubility, stability and bioavailability of the bioactive compounds with a targetted release in the GI tract (Ahmed, Li, McClements, & Xiao, 2012). Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)1,6heptadiene-3,5-dione], an important lipophilic polyphenol, is found in the rhizome of turmeric (Curcuma longa) and the crude extract is used indigenously in numerous herbal medicines; it is commonly comprised of three key compounds: curcumin, demethoxy-curcumin, and bisdemethoxycurcumin (Chen, Li, & Tang, 2015). Among the three, curcumin is the major active constituent that contributes to several biological and pharmacological activities, such as anti-inflammatory, antioxidant, anti-cancer, antiproliferative, antimicrobial and antiangiogenic properties (Bhatia et al., 2016). However, the applications of curcumin in functional food and nutraceutical formulations are limited due to its limited water solubility and poor bioavailability. Many studies have shown the potential of animal-derived protein molecules to act as a carrier to improve the solubility and bioavailability of curcumin. Previous reports concluded that casein (Rahimi
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Yazdi & Corredig, 2012; Sahu, Kasoju, & Bora, 2008), β-lactoglobulin nanoparticle (Sneharani, Karakkat, Singh, & Rao, 2010), β-casein micelle (Esmaili et al., 2011; Pan, Zhong, & Baek, 2013) and bovine serum albumin (Yang, Wu, Li, Zhou, & Wang, 2013) increased the solubility of various molecules by balancing the ratio of hydrophilic-to-hydrophobic amino acids with a peculiar micellar structure that aids complexation. Beside the animal-derived proteins, plant-based proteins are also regarded as safe and economical with significant health benefits (Satija & Hu, 2018). The complexation with soy protein isolate (Chen et al., 2015; Tang & Li, 2013; Tapal & Tiku, 2012), pea protein (Donsì, Senatore, Huang, & Ferrari, 2010), flaxseed hydrolysate (Akbarbaglu et al., 2019) and zein colloidal particles (Patel, Hu, Tiwari, & Velikov, 2010) has demonstrated the improvement in solubility and stability of curcumin. However, many of these proteins in native form failed to show proper functional characteristics for food applications. Therefore, the natural proteins are modified by various methods, such as enzymatic hydrolysis, to improve their solubility, emulsification capability and foaming capacity (Akbarbaglu et al., 2019). The plant-based proteins are environmentally sustainable dietary sources of proteins and their demand from vegans is increasing, with only limited experimental results available to address the possible application of the proteins in this respect (Karaca, Low, & Nickerson, 2011). In our study, Lepidium sativum seed protein isolates were used as
Corresponding author. E-mail address: [email protected] (S.S. Lele).
https://doi.org/10.1016/j.foodchem.2019.125091 Received 13 January 2019; Received in revised form 21 June 2019; Accepted 26 June 2019 Available online 27 June 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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2.4. Characterization of curcumin complex
the carrier for curcumin complexation. Lepidium sativum, a shrub native to the Indian subcontinent, belongs to the Cruciferae family and is a cheap and abundant renewable source of proteins. The object of our research was to evaluate the feasibility of molecular weight-based Lepidium sativum protein hydrolysate fractions as potential complexation agents for curcumin. This study systematically investigated the preparation, characterization and functional evaluation of protein-based curcumin complex as an effective nutraceutical and functional food ingredient.
2.4.1. Dynamic light scattering (DLS), ζ-potential and polydispersity index (PDI) of LSPH and LSPH/CUR The ζ-potential, Z-average diameter (Dz) and polydispersity index (PDI) of LSPH/CUR solution at 3.0, 3.4 and 4.0 were measured, using a dynamic light scattering (DLS) instrument (Zetasizer Nano ZS90, Malvern Instrument Ltd., Malvern, Worcestershire, UK). Briefly, LSPH/ CUR solution (0.1% w/v) of the same pH was filtered through a 0.22 µM Millipore membrane. The particle size analysis was done at a scattering angle of 90° at 25 °C and with an assumption that all particles are spherical.
2. Materials and methods 2.1. Material
2.5. Fluorescence measurement
Curcumin (CUR, 95%) was purchased from ICN Biomedicals, Inc. (Aurora, Ohio). Lepidium sativum seeds were purchased from APMC market, Mumbai. 2,2-Diphenyl-1-picrylhydrazyl, 2,4,6-Tris(2-pyridyl)s-triazine (TPTZ), was from Sigma Aldrich and the rest of the chemicals were of analytical grade from the Merck Company.
Quantification of LSPH complexation with curcumin was done by fluorescence spectrophotometry (Shimadzu RF-5000 spectrofluorimeter, Kyoto, Japan). Fluorescence emission spectra of 5 µM curcumin in deionized water (pH 3.0) at varying LSPH concentrations (0.1–0.5 mg/ml) were recorded from 450 to 650 nm with 420 nm as the excitation wavelength. The slit widths were 2.5 and 5 nm for excitation and emission, respectively. LSPH dispersed solutions (pH 3.0) without curcumin served as controls. Intrinsic fluorescence of protein was estimated at 0–5 µM curcumin with 0.5 mg/ml of LSPH individually from 310 to 450 nm at an excitation wavelength of 280 nm.
2.2. Production of enzymatic hydrolysate To prepare LSPH/CUR complex, a freeze-dried Lepidium sativum protein hydrolyzed molecular fraction was used. Prior to enzymatic hydrolysis, defatted Lepidium sativum seedcake was extracted to obtain protein isolate and this was further hydrolyzed with pepsin according to the method described by Siow and Gan (2013). Briefly, 1 g of seedcake flour was dispersed in 45 ml of 0.17 M NaOH aqueous solution and stirred at 500 rpm for 45 min at 25 °C. The resultant slurry was then centrifuged at 2459×g at 25 °C for 20 min and the pH of the supernatant collected was adjusted to the isoelectric point (pI: 4.5) and centrifuged again. The precipitated protein was dialyzed against deionized water and adjusted to pH 7.0. The dialyzed protein isolate was then enzymatically hydrolyzed with pepsin at an enzyme to substrate ratio (E/S) of 2% in deionized water (adjusted to pH 3) for 2 h at 37 °C to attain maximum hydrolysis. The mixture was further heated at 100 °C for 10 min to inactivate the enzyme, followed by rapid cooling to 20–25 °C. The mixture was sequentially passed through ultrafiltration membranes with molecular weight cut-offs (MWCO) of 30 kDa, 10 kDa and 5 kDa (Millipore Co., Billerica, MA) at 25 °C; the pressure was 1.0–1.5 bar and recirculation rate 0.03 l/min. The molecular fractions of > 30 kDa, 10–30 kDa, 5–10 kDa and < 5 kDa were freeze-dried and stored at −20 °C until used.
2.6. In vitro bioaccessibility and stability 2.6.1. Sequential in vitro gastric and intestinal digestion The potential gastrointestinal fate of the LSPH/CUR complex was evaluated by the in vitro model of sequential simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) digestion, as described by Chen et al. (2015) with a few modifications. 2.6.1.1. Stomach phase. SGF was prepared by adding 0.2% NaCl, 0.32% of pepsin in distilled water adjusted to pH 1.5 by 0.1 M HCl and then incubating to 37 °C. Then 10 ml of LSPH/CUR complex solution (1.0% w/v) was added to 10 ml of SGF, adjusted to pH 2.5 and kept on a platform shaker at 100 rpm for 1 h. 2.6.1.2. Small intestine phase. Samples from the simulated gastric phase were immediately adjusted to pH 7.0 and mixed with SIF solution containing bile extract (50 mg/ml, 3.5 ml), pancreatin (24 mg/ml, 2.5 ml), and saline solution (0.5 M CaCl2 and 7.5 M NaCl, 1.5 ml). The resultant mixture was further maintained at constant pH 7.0 using 50 mM NaOH for 2 h.
2.3. Preparation of complex of curcumin with LSPH The LSPH molecular fractions of > 30 kDa, 10–30 kDa, 5–10 kDa and < 5 kDa (1% w/v) were dispersed in deionized water and stirred for 2 h; subsequently, the pH values of the solutions were adjusted to 3.0, 3.5 and 4.0 and they were stirred for 2 h. An excessive amount of curcumin was incorporated in all molecular fraction solutions and homogenized at 4382×g for 5 min with a high-speed homogenizer. After overnight incubation on a magnetic stirrer at 25 °C, free curcumin was separated by centrifugation at 2459×g for 20 min. The supernatant obtained was analyzed for particle properties and lyophilized to yield LSPH/CUR complex powder. The encapsulated amount of curcumin (µg/ml) in the complex with LSPH was measured by the following method. Briefly, 10 mg of LSPHcurcumin powder was extracted twice with 2 ml of ethyl acetate under vortex for 5 min and subsequent centrifuged at 5031×g for 5 min. The supernatants were pooled and absorbance was measured at 420 nm (Chen, Zhang, & Tang, 2016).
2.6.2. Determination of curcumin concentration Approximately, 500 µl of an ethanolic solution of curcumin (4.5 g/l) were dispersed in 25 ml of deionized water and exposed to digestion as described earlier. After completion of the digestion, 1 ml of digest dispersion collected was centrifuged and the amount of curcumin in the resultant supernatants was calculated (see Section 2.3). 2.6.3. In vitro digestibility of LSPH The hydrolyzed LSPH and complexation with curcumin were characterized according to the method reported by using the release of trichloroacetic acid (TCA)-soluble nitrogen. During digestion, specific volumes of digests were withdrawn at particular time intervals, combined with 20% TCA solution and kept undisturbed for 10 min. Later, the mixtures were centrifuged at 500 g for 5 min and supernatants obtained were analyzed by the micro Kjeldahl method to measure the nitrogen content (Chen et al., 2015). 2
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employed to plot the calibration curve. It was estimated that the 10–30 kDa fraction of LSPH would exhibit a higher encapsulation capacity for curcumin at pH 3.0. (Fig. 1). The absolute concentration of curcumin was 9.42 µg/ml, compared with an estimated water solubility of 11 ng/ml (Tapal & Tiku, 2012). This shows that LSPH at pH 3.0 increased the solubility of curcumin 856-fold. This is due to the complexation of curcumin with protein via hydrophobic interactions (Chen et al., 2015). The freeze-dried LSPH/CUR powder reconstituted in water very quickly and easily dissolved to give a clear solution. Similar findings were also observerd for the complexation of curcumin with casein (Esmaili et al., 2011; Rahimi Yazdi & Corredig, 2012), soy globulin and vitamin B12 (Zhang, Tian, Liang, Subirade, & Chen, 2013), saponin (Peng et al., 2018), casein and soy polysaccharide complex (Xu, Wang, & Yao, 2017). Tapal and Tiku (2012) and Yu and Huang (2010) reported enhanced (812- and 1670-fold) water solubility of hydrophobically modified starch and soy protein isolate-encapsulated curcumin.
2.7. Antioxidant activity (ABTS, DPPH, FRAP) Antioxidant activity of the LSPH/CUR complex was measured using DPPH (Kadam, Shah, Palamthodi, & Lele, 2018), ABTS radicalscavenging and ferric equivalent antioxidant power (FRAP) methods. 2.8. Foaming and emulsion capacity Foaming capacity (FC) was evaluated as described by Timilsena, Adhikari, Barrow, and Adhikari (2016). Samples (0.2%, w/v) were dispersed in 50 ml of Milli-Q water and whipped in a mechanical homogenizer at 10000 rpm for 3 min at (25 ± 2 °C). The emulsion activity index (EAI) of samples was determined as by Hou et al. (2017) with a few revisions. Concisely, 50 ml of 0.20% (w/v) of LSPH solution were mixed with sunflower oil (10 ml), pH was brought to 3 and homogenized in a mechanical homogenizer at 4000 rpm. About 50 µl sample fractions were drawn from the bottom of the beaker and mixed with 5 ml of 0.1% (w/v) SDS. Immediate (A0) and after 10 min (A10) emulsion formation absorbances were measured.
3.2. Curcumin-LSPH interaction
3. Results and discussion
The interaction between LSPH (10–30 kDa fraction) and curcumin was investigated by measuring fluorescence spectra. Curcumin itself is a fluorescent compound, which exhibited very low fluorescence in an aqueous solution. Experimental data showed that free curcumin has a low-intensity broad fluorescence peak at around 550 nm in aqueous solution, while the peak shifted to 530 nm for curcumin encapsulated in LSPH solution when it was excited at 420 nm (Fig. 2a). This phenomenon suggested an upturn in the solubility of curcumin owing to its hydrophobic interactions with LSPH. As shown in (Fig. 2b), the increment of LSPH concentration reveals a sharper emission peak with higher intensity. Additionally, a shift in the emission maximum of curcumin indicates the movement of curcumin from a polar to a less polar environment. The fluorescence emission of curcumin is usually affected by the polarity of the surrounding environment. Parallel results were observed in the complexation of curcumin with soy protein isolate (Tapal & Tiku, 2012), hydrophobically modified starch (Yu & Huang, 2010), human serum albumin (Leung & Kee, 2009) and casein micelles (Sneharani, Singh, & Appu Rao, 2009). Studies suggested that the curcumin binds to hydrophobic regions of protein molecules and LSPH possesses a high degree of hydrophobicity. Binding of a drug molecule to a protein molecule in solution is often investigated by intrinsic fluorescence of tryptophan and tyrosine amino acids at the excitation wavelength of 280 nm. As shown in Fig. 2b, the
3.1. Complexation of curcumin with LSPI Curcumin (diferuloylmethane), a lipophilic polyphenol found in turmeric (Curcuma longa), exhibits potential biological and pharmacological activities. However, its applicability as a health-promoting agent in various formulation developments is often limited due to its poor water-solubility and chemical instability, as well as low and variable bioavailability. In this study, we used LSPH (MWCO > 30 kDa, 10–30 kDa, 5–10 kDa and < 5 kDa) as an emulsifier and stabilizer at pH 3.0, 3.5 and 4.0 to produce a water emulsion. The solubility of curcumin significantly (p > 0.05) differed as a function of MWCO fraction of LSPH and pH, indicating the importance of surface charge on solubility (Fig. 1). The solubility of LSPH reached 86–90% at pH 3.0 indicating that, under acidic conditions, LSPH can be added to food formulations. LSPH solutions of > 30 kDa, 10–30 kDa, 5–10 kDa and < 5 kDa MWCO fractions (1%, w/v; at pH 3.0, 3.5 and 4.0) were prepared in water to develop a complex with curcumin under high-speed homogenization, followed by stirring on a magnetic stirrer overnight. Free curcumin was removed to obtain a clear yellowish solution of LSPH/ CUR. To determine the amount of curcumin in LSPH micelles, curcumin was extracted with methanol. A curcumin-methanol solution was
Fig. 1. Encapsulating activity of LSPH. 3
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LSPH-curcumin complex exhibited a robust fluorescence emission with a peak at around 340 nm. However, the intensity of the fluorescence emission of LSPH steadily increased with a decrease in the curcumin concentration. This indicated the binding of curcumin to some or all of the Trp or Tyr residues, though the exact position of binding is unclear. Fig. 2c depicts the fluorescence emission spectra of the LSPH/CUR complex and curcumin in water. From the graph, it is clear that solubility of curcumin has increased when complexed with LSPH, which could be a result of the hydrophobic interactions of curcumin and LSPH. These results suggest that curcumin can be formulated with LSPH to increase its solubility. 3.3. Dynamic light scattering (DLS), ζ-potential and polydispersity index (PDI) of LSPH and LSPH/CUR The particle sizes of LSPH and LSPH/CUR complex at pH 3.0, 3.5 and 4.0 were evaluated by the DLS technique. Our study demonstrates a monomodal particle size distribution profile for all the test samples, for a particle size range of 130–220 nm, with a relatively narrow polydispersity index (Fig. 3). The z-average diameter (Dz) and polydispersity (PDI) values of LSPH particles in dispersion significantly increased when complexed with curcumin. This might be due to the incorporation of a considerable amount of curcumin molecules into the hydrophobic interiors of LSPH micelles. Further, the Dz and PDI values of both LSPH and LSPH/CUR complex decreased with increasing pH. The results confirmed the pH dependence of protein–protein interactions or the aggregation in dispersions. The result is in agreement with the fluorescence emission observations; the Dz value was much higher at pH 3.0 due to more aggregate in the solution with a compact structure. A similar phenomenon was also observed by Chen et al. (2016) who reported that higher Dz value indicates structural rearrangement to a more compacted structure through interactions of various unfolded molecules, mainly inter-particle hydrophobic interactions. Surface characteristics (ζ-potential) exhibit the electrokinetic potential, which is important for the stability of particle dispersion. LSPH particles at pH 3.0, 3.5 and 4.0 (pI around 4.5) were positively charged and were consistently insoluble, indicating that electrostatic dispersion dominates particle stability at pH 3.0. The complexation of LSPH with curcumin exhibited a minor, yet substantial increase in the absolute magnitude of ζ-potential compared to the LSPH. As discussed earlier, the LSPH and LSPH/curcumin complex had a relatively high positive surface potential in the dispersion at pH 3.0 (+28.4 and +31.3 mV, respectively) and this decreased slightly with increase in pH value. The variations could be attributed to the changes in the degree of curcumin binding-induced particle aggregation. These results are in agreement with Chen et al. (2016), who reported that, at pH 3.0, proteins were more compacted due to the association of different unfolded protein (i.e. more hydrophobic groups buried inside the molecule). 3.4. Sequential stimulated gastric and intestinal digestion 3.4.1. Bioaccessibility and stability of curcumin The encapsulated curcumin-LSPH complex might have undergone severe changes during the process due to the pH and ionic condition, the effect of proteases on the proteins and the presence of various active surfactants. Fig. 4A represents the measure of bioaccessible curcumin transferred to the aqueous phase or its encapsulated curcumin with LSPH, after sequential digestion in simulated digestive juices, in the absence or presence of proteases. The bioaccessibility of curcumin is primarily dependent on its stability all through the digestion process. In our study, we encapsulated the curcumin with LSPH, which might be susceptible to the digestion process and which could affect its bioaccessibility. Therefore, we studied the stability of curcumin upon digestion (Fig. 4B). The result indicated that the protease activity in digestion
Fig. 2. (a) Fluorescence emission spectra of 5 µM curcumin in deionized water (3.0 pH) in the presence of LSPH (5 mg/ml) concentration. (b) Quenching of LSPH intrinsic fluorescence by curcumin. Fluorescence emission spectra of the LSPH suspension at an excitation wavelength of 280 nm in the presence of curcumin at different concentrations. Curcumin stock (0.5 mg/ml) was prepared in methanol. (c) Comparison of the fluorescence emission spectra of curcumin and the LSPH/CUR complex dissolved in water (3.0). The excitation wavelength was 420 nm, excitation and emission slit widths were 2.5 and 5 nm, respectively.
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Fig. 3. (a) Dynamic light scattering (DLS), (b) ζ-Potential and (c) Polydispersity index (PDI) of LSPH and LSPH/CUR complex. All data are expressed as a means ± standard deviation.
(SGF and SIF). The protease-facilitated hydrolysis of proteins in the LSPH/CUR curcumin complex was analyzed in terms of TCA-soluble nitrogen fractions, as shown in (Fig. 4C). It can be observed that digestion caused progressive hydrolysis of the LSPH and LSPH/CUR complex in the SGF digestion, whereas this was relatively less in SIF hydrolysis, as evidenced by the TCA method. The observation was similar to those reported for SPI-encapsulated curcumin; the protein components in SPI are more vulnerable to gastric protein hydrolysis (SGI) than to SIF (Chen et al., 2015). The same is true for green pea protein (Deshpande & Ddamodaran, 1989). In addition, the release of TCA-soluble nitrogen during the SGF digestion was much higher in the LSPH/CUR complex than in the LSPH. A similar phenomenon (pepsin digestibility) in simulated SGI has also been described for a few key food allergens when they interacted with green tea catechins (Tagliazucchi, Verzelloni, & Conte, 2005; Tantoush et al., 2012). But, some contrasting observations have also been made, showing adverse and insignificant influences for the digestibility of many proteins, such as β-lactoglobulin, and legume protein (Carbonaro, Grant, Cappelloni, & Pusztai, 2000; Stojadinovic et al., 2013; Venkatachalam & Sathe, 2003). Consequently, the effect of phenolic-protein complexes on the digestibility of proteins is multifaceted and associated with the nature of both the phenolic compounds and proteins.
significantly enhanced the stability of curcumin. Interestingly, it can be observed that, after 120 min of digestion without the addition of proteases, only about 15% of free curcumin was transferred to the aqueous phase, rendering it bioaccessible whereas, in the LSPH/CUR complex, it was about 72%, suggesting that state of curcumin (free or bound) affects the digestive stability of curcumin (Fig. 4B). The stability enhancement of curcumin by the protease activity during digestion might be largely due to the curcumin-protein interaction or complexation. In the presence of protease, curcumin in the LSPH/CUR complex did not decrease significantly during the digestion, whereas a significant amount of free curcumin survived the whole digestion from 15% to 83%. In the previous study reported by Chen et al. (2015) on the digestive stability of SPI-encapsulated curcumin, it was reported that presence of enzyme during the digestion enhanced curcumin stability due to the interaction between curcumin and protein or complexation. It is seen that the bioaccessibility of curcumin is greatly governed by the state of curcumin (free and bound) and the presence of enzymes (Fig. 4A). In the absence of enzymes, < 1% of curcumin was shifted to the aqueous phase as free curcumin exhibiting exceptionally low bioaccessibility, compared to approximately 67% of the curcumin encapsulated in LSPH (Fig. 4A). With digestion by enzymes, bioaccessibility of free curcumin increased up to 22% and, if the degradation of curcumin is considered into account, it may reach about 95%. Hence, in the presence of protease, the optimum bioaccessibility for free curcumin would be around 25%, whereas for curcumin encapsulated in LSPH it was significantly improved from 67% to 95% (Fig. 4A).
3.5. Antioxidant activity and metal chelating property The antioxidant potentials of LSPH and LSPH/CUR complex were studied by ABTS, DPPH, and FRAP assays. Metal binding activity was measured to study the inhibitory effect on radical generation. Ascorbic acid was used as a control. Fig. 5a and 5b show the DPPH, ABTS+ radical-scavenging, and FRAP reducing power activities of LSPH and LSPH/CUR complex. Results indicated that the antioxidant activities in terms of free radical-scavenging, hydrogen donation, as well as
3.4.2. Protein in vitro digestibility The bioaccessibility of curcumin in the encapsulated LSPH could be related to the enzymatic hydrolysis of the proteins during digestion. In our study, we saw that LSPH complexation with curcumin affected the digestibility of the proteins during the sequential simulated digestion 5
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Fig. 4. A) Percentage of curcumin remaining in the aqueous phase for free curcumin and LSPH/CUR complex after the whole simulated digestion of 120 min in the absence or presence of the protease. B) Percentage of curcumin in the whole digest of free curcumin and LSPH/CUR complex after the digestion of 120 min in the absence or presence of the protease. Data are presented as means of triplicate measurement on a separate sample. C) Release kinetics of TCA-soluble nitrogen during the sequential SGF and SIF digestion of LPSH and LPSH-curcumin complex.
influenced by its concentration, molecular size, ionic strength, solubility, temperature, and pH. Foaming contributes to the aeration and whipping property of an amphoteric molecule associated with the two-phase interface. Many studies report that protein molecules significantly help in the uniform distribution of air cells which provide a continuous inter-molecular cohesiveness and elasticity to the air bubble in the structure of foods (Hou et al., 2017). We determined the emulsifying and foaming activity of LSPH and LSPH/CUR complex to study the effect of complexation on the performance of the Lepidium sativum protein hydrolysate (10–30 kDa). Table 1 represents the emulsifying and foaming activity of both LSPH and LSPH/CUR complex, indicating no significant variation in the functional attributes of LSPH after complexation with curcumin. This study reveals that the LSPH/CUR complex was able to form emulsion and foam, and therefore it is possible to use the complex as a food and pharmaceutical ingredient and in novel formulation development in order to enhance acceptability. Our results are in agreement with the previous work reported by Tapal and Tiku (2012), showing no significant change in the emulsifying and foaming capacity of SPI-
reduction, are concentration-dependent. Antioxidant activity of proteins is contributed by Trp, Tyr, Phe, and His amino acids which have hydrogen donor groups, e.g. indole, phenyl and imidazole, or by Met, Cys, and Pro, amino acids, which are the endogenous protein antioxidants (Levine, Mosoni, Berlett, & Stadtman, 1996; Zhang, Wang, & Xu, 2008). Curcumin has a phenolic hydroxyl group that has the ability to scavenge radicals through H-atom donating (Tapal & Tiku, 2012). The antioxidant activity of LSPH/CUR complex is higher than that of LSPH due to the presence of curcumin in the LSPH/CUR complex. 3.6. Foaming and emulsion capacity Emulsion capacity (m2/g) measures the ability of protein and another amphoteric molecule to form a stable emulsion within a prescribed period of time. Hou et al. (2017) reported that hydrophilic-lipophilic properties of protein molecules significantly affect the emulsion property by lowering the tension at the oil-water interfaces and they control diffusion and aggregation of oil droplets by forming an adsorption layer. The diffusion of a protein molecule is prominently 6
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Fig. 5. (a) ABTS and DPPH activities; (b) FRAP antioxidant activity.
Declaration of Competing Interest
curcumin complexation at neural pH compared to the SPI.
None declared.
4. Conclusion This study has shown that the LSPH (10–30 kDa) fraction can undergo a compact complexation with curcumin at pH 3.0 that significantly improves its solubility (in the aqueous phase), stability and even bioaccessibility. The functional attributes of the LSPH/CUR complex, especially the foam and emulsion forming capacities, were promising for its applications in food product formulations. Furthermore, the antioxidant activity of LSPH increased after complexation with curcumin. The present work reveals that the LSPH complex can be formulated as a functional food and it can act as a useful vehicle for a lipophilic bioactive molecule. Nevertheless, the denatured proteins in LSPH might significantly influence the bioavailability of encapsulated bioactive ingredients. Therefore, further studies should be carried out to evaluate the efficacy of the curcumin-LSPH complex for in-vivo digestion models.
Acknowledgment The authors are thankful for the Basic Scientific Research Fellowship in Sciences by the University Grants Commission, Government of India, for providing financial assistance (Grant number: 2812/UGC-SAP) during this investigation.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125091.
Table 1 Foaming and emulsion capacities of LSPH and LSPH/CUR complex. Concentration of protein (mg/ml)
5 10 15
Foaming capacity (%)
Emulsion capacity
10–30 kDa fraction of LSPH
10–30 kDa fraction of LSPH-curcumin complex
10–30 kDa fraction of LSPH
10–30 kDa fraction of LSPH-curcumin complex
27.43 ± 2a 31.20 ± 1b 32.30 ± 1bc
23.30 ± 2a 26.40 ± 2ab 26.40 ± 2b
0.88 ± 0.02a 0.93 ± 0.01b 0.98 ± 0.03c
0.75 ± 0.1a 0.87 ± 0.2b 0.94 ± 0.2c
All data are expressed as means ± standard deviation. Means with different superscript letters in a column differ significantly (P < 0.05). 7
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References
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Complexation of curcumin with Lepidium sativum protein hydrolysate as a novel curcumin delivery system Deepak Kadam, Shanooba Palamthodi, S.S. Lele
T
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Department of Food Engineering and Technology, Institute of Chemical Technology, Matunga, Mumbai 400019, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Protein hydrolysate Curcumin Complexation Bioaccessibility/stability
The complexation of Lepidium sativum protein hydrolysate (LSPH) with a lipophilic molecule, curcumin (CUR), and its effect on curcumin in vitro bioaccessibility/stability, functional and antioxidant activity were investigated. Fluorescence spectroscopy of the LSPH/CUR complex confirmed the presence of hydrophobic interactions that led to the complex formation. The LSPH (10–30 kDa) fraction showed a compact complexation with curcumin at pH 3.0 with excellent aqueous solubility, stability, and bioaccessibility. Further, complexation enhanced the aqueous solubility of curcumin more than 856-fold. In vitro sequential simulated gastric and intestinal digestion indicated that the bioaccessibility of curcumin was increased from 67% to 95% post complexation. The functional attributes suggest that the LSPH/CUR complex has good foam-forming capacity and emulsion stability, which are crucial for food product formulations. The results indicate that, since LSPH is a dietary protein, it might possibly be formulated as a functional food and as an excellent lipophilic bioactive molecule delivery vehicle in food formulations.
1. Introduction In the past decade, complexations between proteins and lipophilic molecules, especially polyphenolic compounds, have attracted attention of many researchers and nutraceutical industries. The increased interest is mainly due to the ability of formed complexes to enhance water solubility, stability and bioavailability of the bioactive compounds with a targetted release in the GI tract (Ahmed, Li, McClements, & Xiao, 2012). Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)1,6heptadiene-3,5-dione], an important lipophilic polyphenol, is found in the rhizome of turmeric (Curcuma longa) and the crude extract is used indigenously in numerous herbal medicines; it is commonly comprised of three key compounds: curcumin, demethoxy-curcumin, and bisdemethoxycurcumin (Chen, Li, & Tang, 2015). Among the three, curcumin is the major active constituent that contributes to several biological and pharmacological activities, such as anti-inflammatory, antioxidant, anti-cancer, antiproliferative, antimicrobial and antiangiogenic properties (Bhatia et al., 2016). However, the applications of curcumin in functional food and nutraceutical formulations are limited due to its limited water solubility and poor bioavailability. Many studies have shown the potential of animal-derived protein molecules to act as a carrier to improve the solubility and bioavailability of curcumin. Previous reports concluded that casein (Rahimi
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Yazdi & Corredig, 2012; Sahu, Kasoju, & Bora, 2008), β-lactoglobulin nanoparticle (Sneharani, Karakkat, Singh, & Rao, 2010), β-casein micelle (Esmaili et al., 2011; Pan, Zhong, & Baek, 2013) and bovine serum albumin (Yang, Wu, Li, Zhou, & Wang, 2013) increased the solubility of various molecules by balancing the ratio of hydrophilic-to-hydrophobic amino acids with a peculiar micellar structure that aids complexation. Beside the animal-derived proteins, plant-based proteins are also regarded as safe and economical with significant health benefits (Satija & Hu, 2018). The complexation with soy protein isolate (Chen et al., 2015; Tang & Li, 2013; Tapal & Tiku, 2012), pea protein (Donsì, Senatore, Huang, & Ferrari, 2010), flaxseed hydrolysate (Akbarbaglu et al., 2019) and zein colloidal particles (Patel, Hu, Tiwari, & Velikov, 2010) has demonstrated the improvement in solubility and stability of curcumin. However, many of these proteins in native form failed to show proper functional characteristics for food applications. Therefore, the natural proteins are modified by various methods, such as enzymatic hydrolysis, to improve their solubility, emulsification capability and foaming capacity (Akbarbaglu et al., 2019). The plant-based proteins are environmentally sustainable dietary sources of proteins and their demand from vegans is increasing, with only limited experimental results available to address the possible application of the proteins in this respect (Karaca, Low, & Nickerson, 2011). In our study, Lepidium sativum seed protein isolates were used as
Corresponding author. E-mail address: [email protected] (S.S. Lele).
https://doi.org/10.1016/j.foodchem.2019.125091 Received 13 January 2019; Received in revised form 21 June 2019; Accepted 26 June 2019 Available online 27 June 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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2.4. Characterization of curcumin complex
the carrier for curcumin complexation. Lepidium sativum, a shrub native to the Indian subcontinent, belongs to the Cruciferae family and is a cheap and abundant renewable source of proteins. The object of our research was to evaluate the feasibility of molecular weight-based Lepidium sativum protein hydrolysate fractions as potential complexation agents for curcumin. This study systematically investigated the preparation, characterization and functional evaluation of protein-based curcumin complex as an effective nutraceutical and functional food ingredient.
2.4.1. Dynamic light scattering (DLS), ζ-potential and polydispersity index (PDI) of LSPH and LSPH/CUR The ζ-potential, Z-average diameter (Dz) and polydispersity index (PDI) of LSPH/CUR solution at 3.0, 3.4 and 4.0 were measured, using a dynamic light scattering (DLS) instrument (Zetasizer Nano ZS90, Malvern Instrument Ltd., Malvern, Worcestershire, UK). Briefly, LSPH/ CUR solution (0.1% w/v) of the same pH was filtered through a 0.22 µM Millipore membrane. The particle size analysis was done at a scattering angle of 90° at 25 °C and with an assumption that all particles are spherical.
2. Materials and methods 2.1. Material
2.5. Fluorescence measurement
Curcumin (CUR, 95%) was purchased from ICN Biomedicals, Inc. (Aurora, Ohio). Lepidium sativum seeds were purchased from APMC market, Mumbai. 2,2-Diphenyl-1-picrylhydrazyl, 2,4,6-Tris(2-pyridyl)s-triazine (TPTZ), was from Sigma Aldrich and the rest of the chemicals were of analytical grade from the Merck Company.
Quantification of LSPH complexation with curcumin was done by fluorescence spectrophotometry (Shimadzu RF-5000 spectrofluorimeter, Kyoto, Japan). Fluorescence emission spectra of 5 µM curcumin in deionized water (pH 3.0) at varying LSPH concentrations (0.1–0.5 mg/ml) were recorded from 450 to 650 nm with 420 nm as the excitation wavelength. The slit widths were 2.5 and 5 nm for excitation and emission, respectively. LSPH dispersed solutions (pH 3.0) without curcumin served as controls. Intrinsic fluorescence of protein was estimated at 0–5 µM curcumin with 0.5 mg/ml of LSPH individually from 310 to 450 nm at an excitation wavelength of 280 nm.
2.2. Production of enzymatic hydrolysate To prepare LSPH/CUR complex, a freeze-dried Lepidium sativum protein hydrolyzed molecular fraction was used. Prior to enzymatic hydrolysis, defatted Lepidium sativum seedcake was extracted to obtain protein isolate and this was further hydrolyzed with pepsin according to the method described by Siow and Gan (2013). Briefly, 1 g of seedcake flour was dispersed in 45 ml of 0.17 M NaOH aqueous solution and stirred at 500 rpm for 45 min at 25 °C. The resultant slurry was then centrifuged at 2459×g at 25 °C for 20 min and the pH of the supernatant collected was adjusted to the isoelectric point (pI: 4.5) and centrifuged again. The precipitated protein was dialyzed against deionized water and adjusted to pH 7.0. The dialyzed protein isolate was then enzymatically hydrolyzed with pepsin at an enzyme to substrate ratio (E/S) of 2% in deionized water (adjusted to pH 3) for 2 h at 37 °C to attain maximum hydrolysis. The mixture was further heated at 100 °C for 10 min to inactivate the enzyme, followed by rapid cooling to 20–25 °C. The mixture was sequentially passed through ultrafiltration membranes with molecular weight cut-offs (MWCO) of 30 kDa, 10 kDa and 5 kDa (Millipore Co., Billerica, MA) at 25 °C; the pressure was 1.0–1.5 bar and recirculation rate 0.03 l/min. The molecular fractions of > 30 kDa, 10–30 kDa, 5–10 kDa and < 5 kDa were freeze-dried and stored at −20 °C until used.
2.6. In vitro bioaccessibility and stability 2.6.1. Sequential in vitro gastric and intestinal digestion The potential gastrointestinal fate of the LSPH/CUR complex was evaluated by the in vitro model of sequential simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) digestion, as described by Chen et al. (2015) with a few modifications. 2.6.1.1. Stomach phase. SGF was prepared by adding 0.2% NaCl, 0.32% of pepsin in distilled water adjusted to pH 1.5 by 0.1 M HCl and then incubating to 37 °C. Then 10 ml of LSPH/CUR complex solution (1.0% w/v) was added to 10 ml of SGF, adjusted to pH 2.5 and kept on a platform shaker at 100 rpm for 1 h. 2.6.1.2. Small intestine phase. Samples from the simulated gastric phase were immediately adjusted to pH 7.0 and mixed with SIF solution containing bile extract (50 mg/ml, 3.5 ml), pancreatin (24 mg/ml, 2.5 ml), and saline solution (0.5 M CaCl2 and 7.5 M NaCl, 1.5 ml). The resultant mixture was further maintained at constant pH 7.0 using 50 mM NaOH for 2 h.
2.3. Preparation of complex of curcumin with LSPH The LSPH molecular fractions of > 30 kDa, 10–30 kDa, 5–10 kDa and < 5 kDa (1% w/v) were dispersed in deionized water and stirred for 2 h; subsequently, the pH values of the solutions were adjusted to 3.0, 3.5 and 4.0 and they were stirred for 2 h. An excessive amount of curcumin was incorporated in all molecular fraction solutions and homogenized at 4382×g for 5 min with a high-speed homogenizer. After overnight incubation on a magnetic stirrer at 25 °C, free curcumin was separated by centrifugation at 2459×g for 20 min. The supernatant obtained was analyzed for particle properties and lyophilized to yield LSPH/CUR complex powder. The encapsulated amount of curcumin (µg/ml) in the complex with LSPH was measured by the following method. Briefly, 10 mg of LSPHcurcumin powder was extracted twice with 2 ml of ethyl acetate under vortex for 5 min and subsequent centrifuged at 5031×g for 5 min. The supernatants were pooled and absorbance was measured at 420 nm (Chen, Zhang, & Tang, 2016).
2.6.2. Determination of curcumin concentration Approximately, 500 µl of an ethanolic solution of curcumin (4.5 g/l) were dispersed in 25 ml of deionized water and exposed to digestion as described earlier. After completion of the digestion, 1 ml of digest dispersion collected was centrifuged and the amount of curcumin in the resultant supernatants was calculated (see Section 2.3). 2.6.3. In vitro digestibility of LSPH The hydrolyzed LSPH and complexation with curcumin were characterized according to the method reported by using the release of trichloroacetic acid (TCA)-soluble nitrogen. During digestion, specific volumes of digests were withdrawn at particular time intervals, combined with 20% TCA solution and kept undisturbed for 10 min. Later, the mixtures were centrifuged at 500 g for 5 min and supernatants obtained were analyzed by the micro Kjeldahl method to measure the nitrogen content (Chen et al., 2015). 2
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employed to plot the calibration curve. It was estimated that the 10–30 kDa fraction of LSPH would exhibit a higher encapsulation capacity for curcumin at pH 3.0. (Fig. 1). The absolute concentration of curcumin was 9.42 µg/ml, compared with an estimated water solubility of 11 ng/ml (Tapal & Tiku, 2012). This shows that LSPH at pH 3.0 increased the solubility of curcumin 856-fold. This is due to the complexation of curcumin with protein via hydrophobic interactions (Chen et al., 2015). The freeze-dried LSPH/CUR powder reconstituted in water very quickly and easily dissolved to give a clear solution. Similar findings were also observerd for the complexation of curcumin with casein (Esmaili et al., 2011; Rahimi Yazdi & Corredig, 2012), soy globulin and vitamin B12 (Zhang, Tian, Liang, Subirade, & Chen, 2013), saponin (Peng et al., 2018), casein and soy polysaccharide complex (Xu, Wang, & Yao, 2017). Tapal and Tiku (2012) and Yu and Huang (2010) reported enhanced (812- and 1670-fold) water solubility of hydrophobically modified starch and soy protein isolate-encapsulated curcumin.
2.7. Antioxidant activity (ABTS, DPPH, FRAP) Antioxidant activity of the LSPH/CUR complex was measured using DPPH (Kadam, Shah, Palamthodi, & Lele, 2018), ABTS radicalscavenging and ferric equivalent antioxidant power (FRAP) methods. 2.8. Foaming and emulsion capacity Foaming capacity (FC) was evaluated as described by Timilsena, Adhikari, Barrow, and Adhikari (2016). Samples (0.2%, w/v) were dispersed in 50 ml of Milli-Q water and whipped in a mechanical homogenizer at 10000 rpm for 3 min at (25 ± 2 °C). The emulsion activity index (EAI) of samples was determined as by Hou et al. (2017) with a few revisions. Concisely, 50 ml of 0.20% (w/v) of LSPH solution were mixed with sunflower oil (10 ml), pH was brought to 3 and homogenized in a mechanical homogenizer at 4000 rpm. About 50 µl sample fractions were drawn from the bottom of the beaker and mixed with 5 ml of 0.1% (w/v) SDS. Immediate (A0) and after 10 min (A10) emulsion formation absorbances were measured.
3.2. Curcumin-LSPH interaction
3. Results and discussion
The interaction between LSPH (10–30 kDa fraction) and curcumin was investigated by measuring fluorescence spectra. Curcumin itself is a fluorescent compound, which exhibited very low fluorescence in an aqueous solution. Experimental data showed that free curcumin has a low-intensity broad fluorescence peak at around 550 nm in aqueous solution, while the peak shifted to 530 nm for curcumin encapsulated in LSPH solution when it was excited at 420 nm (Fig. 2a). This phenomenon suggested an upturn in the solubility of curcumin owing to its hydrophobic interactions with LSPH. As shown in (Fig. 2b), the increment of LSPH concentration reveals a sharper emission peak with higher intensity. Additionally, a shift in the emission maximum of curcumin indicates the movement of curcumin from a polar to a less polar environment. The fluorescence emission of curcumin is usually affected by the polarity of the surrounding environment. Parallel results were observed in the complexation of curcumin with soy protein isolate (Tapal & Tiku, 2012), hydrophobically modified starch (Yu & Huang, 2010), human serum albumin (Leung & Kee, 2009) and casein micelles (Sneharani, Singh, & Appu Rao, 2009). Studies suggested that the curcumin binds to hydrophobic regions of protein molecules and LSPH possesses a high degree of hydrophobicity. Binding of a drug molecule to a protein molecule in solution is often investigated by intrinsic fluorescence of tryptophan and tyrosine amino acids at the excitation wavelength of 280 nm. As shown in Fig. 2b, the
3.1. Complexation of curcumin with LSPI Curcumin (diferuloylmethane), a lipophilic polyphenol found in turmeric (Curcuma longa), exhibits potential biological and pharmacological activities. However, its applicability as a health-promoting agent in various formulation developments is often limited due to its poor water-solubility and chemical instability, as well as low and variable bioavailability. In this study, we used LSPH (MWCO > 30 kDa, 10–30 kDa, 5–10 kDa and < 5 kDa) as an emulsifier and stabilizer at pH 3.0, 3.5 and 4.0 to produce a water emulsion. The solubility of curcumin significantly (p > 0.05) differed as a function of MWCO fraction of LSPH and pH, indicating the importance of surface charge on solubility (Fig. 1). The solubility of LSPH reached 86–90% at pH 3.0 indicating that, under acidic conditions, LSPH can be added to food formulations. LSPH solutions of > 30 kDa, 10–30 kDa, 5–10 kDa and < 5 kDa MWCO fractions (1%, w/v; at pH 3.0, 3.5 and 4.0) were prepared in water to develop a complex with curcumin under high-speed homogenization, followed by stirring on a magnetic stirrer overnight. Free curcumin was removed to obtain a clear yellowish solution of LSPH/ CUR. To determine the amount of curcumin in LSPH micelles, curcumin was extracted with methanol. A curcumin-methanol solution was
Fig. 1. Encapsulating activity of LSPH. 3
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LSPH-curcumin complex exhibited a robust fluorescence emission with a peak at around 340 nm. However, the intensity of the fluorescence emission of LSPH steadily increased with a decrease in the curcumin concentration. This indicated the binding of curcumin to some or all of the Trp or Tyr residues, though the exact position of binding is unclear. Fig. 2c depicts the fluorescence emission spectra of the LSPH/CUR complex and curcumin in water. From the graph, it is clear that solubility of curcumin has increased when complexed with LSPH, which could be a result of the hydrophobic interactions of curcumin and LSPH. These results suggest that curcumin can be formulated with LSPH to increase its solubility. 3.3. Dynamic light scattering (DLS), ζ-potential and polydispersity index (PDI) of LSPH and LSPH/CUR The particle sizes of LSPH and LSPH/CUR complex at pH 3.0, 3.5 and 4.0 were evaluated by the DLS technique. Our study demonstrates a monomodal particle size distribution profile for all the test samples, for a particle size range of 130–220 nm, with a relatively narrow polydispersity index (Fig. 3). The z-average diameter (Dz) and polydispersity (PDI) values of LSPH particles in dispersion significantly increased when complexed with curcumin. This might be due to the incorporation of a considerable amount of curcumin molecules into the hydrophobic interiors of LSPH micelles. Further, the Dz and PDI values of both LSPH and LSPH/CUR complex decreased with increasing pH. The results confirmed the pH dependence of protein–protein interactions or the aggregation in dispersions. The result is in agreement with the fluorescence emission observations; the Dz value was much higher at pH 3.0 due to more aggregate in the solution with a compact structure. A similar phenomenon was also observed by Chen et al. (2016) who reported that higher Dz value indicates structural rearrangement to a more compacted structure through interactions of various unfolded molecules, mainly inter-particle hydrophobic interactions. Surface characteristics (ζ-potential) exhibit the electrokinetic potential, which is important for the stability of particle dispersion. LSPH particles at pH 3.0, 3.5 and 4.0 (pI around 4.5) were positively charged and were consistently insoluble, indicating that electrostatic dispersion dominates particle stability at pH 3.0. The complexation of LSPH with curcumin exhibited a minor, yet substantial increase in the absolute magnitude of ζ-potential compared to the LSPH. As discussed earlier, the LSPH and LSPH/curcumin complex had a relatively high positive surface potential in the dispersion at pH 3.0 (+28.4 and +31.3 mV, respectively) and this decreased slightly with increase in pH value. The variations could be attributed to the changes in the degree of curcumin binding-induced particle aggregation. These results are in agreement with Chen et al. (2016), who reported that, at pH 3.0, proteins were more compacted due to the association of different unfolded protein (i.e. more hydrophobic groups buried inside the molecule). 3.4. Sequential stimulated gastric and intestinal digestion 3.4.1. Bioaccessibility and stability of curcumin The encapsulated curcumin-LSPH complex might have undergone severe changes during the process due to the pH and ionic condition, the effect of proteases on the proteins and the presence of various active surfactants. Fig. 4A represents the measure of bioaccessible curcumin transferred to the aqueous phase or its encapsulated curcumin with LSPH, after sequential digestion in simulated digestive juices, in the absence or presence of proteases. The bioaccessibility of curcumin is primarily dependent on its stability all through the digestion process. In our study, we encapsulated the curcumin with LSPH, which might be susceptible to the digestion process and which could affect its bioaccessibility. Therefore, we studied the stability of curcumin upon digestion (Fig. 4B). The result indicated that the protease activity in digestion
Fig. 2. (a) Fluorescence emission spectra of 5 µM curcumin in deionized water (3.0 pH) in the presence of LSPH (5 mg/ml) concentration. (b) Quenching of LSPH intrinsic fluorescence by curcumin. Fluorescence emission spectra of the LSPH suspension at an excitation wavelength of 280 nm in the presence of curcumin at different concentrations. Curcumin stock (0.5 mg/ml) was prepared in methanol. (c) Comparison of the fluorescence emission spectra of curcumin and the LSPH/CUR complex dissolved in water (3.0). The excitation wavelength was 420 nm, excitation and emission slit widths were 2.5 and 5 nm, respectively.
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Fig. 3. (a) Dynamic light scattering (DLS), (b) ζ-Potential and (c) Polydispersity index (PDI) of LSPH and LSPH/CUR complex. All data are expressed as a means ± standard deviation.
(SGF and SIF). The protease-facilitated hydrolysis of proteins in the LSPH/CUR curcumin complex was analyzed in terms of TCA-soluble nitrogen fractions, as shown in (Fig. 4C). It can be observed that digestion caused progressive hydrolysis of the LSPH and LSPH/CUR complex in the SGF digestion, whereas this was relatively less in SIF hydrolysis, as evidenced by the TCA method. The observation was similar to those reported for SPI-encapsulated curcumin; the protein components in SPI are more vulnerable to gastric protein hydrolysis (SGI) than to SIF (Chen et al., 2015). The same is true for green pea protein (Deshpande & Ddamodaran, 1989). In addition, the release of TCA-soluble nitrogen during the SGF digestion was much higher in the LSPH/CUR complex than in the LSPH. A similar phenomenon (pepsin digestibility) in simulated SGI has also been described for a few key food allergens when they interacted with green tea catechins (Tagliazucchi, Verzelloni, & Conte, 2005; Tantoush et al., 2012). But, some contrasting observations have also been made, showing adverse and insignificant influences for the digestibility of many proteins, such as β-lactoglobulin, and legume protein (Carbonaro, Grant, Cappelloni, & Pusztai, 2000; Stojadinovic et al., 2013; Venkatachalam & Sathe, 2003). Consequently, the effect of phenolic-protein complexes on the digestibility of proteins is multifaceted and associated with the nature of both the phenolic compounds and proteins.
significantly enhanced the stability of curcumin. Interestingly, it can be observed that, after 120 min of digestion without the addition of proteases, only about 15% of free curcumin was transferred to the aqueous phase, rendering it bioaccessible whereas, in the LSPH/CUR complex, it was about 72%, suggesting that state of curcumin (free or bound) affects the digestive stability of curcumin (Fig. 4B). The stability enhancement of curcumin by the protease activity during digestion might be largely due to the curcumin-protein interaction or complexation. In the presence of protease, curcumin in the LSPH/CUR complex did not decrease significantly during the digestion, whereas a significant amount of free curcumin survived the whole digestion from 15% to 83%. In the previous study reported by Chen et al. (2015) on the digestive stability of SPI-encapsulated curcumin, it was reported that presence of enzyme during the digestion enhanced curcumin stability due to the interaction between curcumin and protein or complexation. It is seen that the bioaccessibility of curcumin is greatly governed by the state of curcumin (free and bound) and the presence of enzymes (Fig. 4A). In the absence of enzymes, < 1% of curcumin was shifted to the aqueous phase as free curcumin exhibiting exceptionally low bioaccessibility, compared to approximately 67% of the curcumin encapsulated in LSPH (Fig. 4A). With digestion by enzymes, bioaccessibility of free curcumin increased up to 22% and, if the degradation of curcumin is considered into account, it may reach about 95%. Hence, in the presence of protease, the optimum bioaccessibility for free curcumin would be around 25%, whereas for curcumin encapsulated in LSPH it was significantly improved from 67% to 95% (Fig. 4A).
3.5. Antioxidant activity and metal chelating property The antioxidant potentials of LSPH and LSPH/CUR complex were studied by ABTS, DPPH, and FRAP assays. Metal binding activity was measured to study the inhibitory effect on radical generation. Ascorbic acid was used as a control. Fig. 5a and 5b show the DPPH, ABTS+ radical-scavenging, and FRAP reducing power activities of LSPH and LSPH/CUR complex. Results indicated that the antioxidant activities in terms of free radical-scavenging, hydrogen donation, as well as
3.4.2. Protein in vitro digestibility The bioaccessibility of curcumin in the encapsulated LSPH could be related to the enzymatic hydrolysis of the proteins during digestion. In our study, we saw that LSPH complexation with curcumin affected the digestibility of the proteins during the sequential simulated digestion 5
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Fig. 4. A) Percentage of curcumin remaining in the aqueous phase for free curcumin and LSPH/CUR complex after the whole simulated digestion of 120 min in the absence or presence of the protease. B) Percentage of curcumin in the whole digest of free curcumin and LSPH/CUR complex after the digestion of 120 min in the absence or presence of the protease. Data are presented as means of triplicate measurement on a separate sample. C) Release kinetics of TCA-soluble nitrogen during the sequential SGF and SIF digestion of LPSH and LPSH-curcumin complex.
influenced by its concentration, molecular size, ionic strength, solubility, temperature, and pH. Foaming contributes to the aeration and whipping property of an amphoteric molecule associated with the two-phase interface. Many studies report that protein molecules significantly help in the uniform distribution of air cells which provide a continuous inter-molecular cohesiveness and elasticity to the air bubble in the structure of foods (Hou et al., 2017). We determined the emulsifying and foaming activity of LSPH and LSPH/CUR complex to study the effect of complexation on the performance of the Lepidium sativum protein hydrolysate (10–30 kDa). Table 1 represents the emulsifying and foaming activity of both LSPH and LSPH/CUR complex, indicating no significant variation in the functional attributes of LSPH after complexation with curcumin. This study reveals that the LSPH/CUR complex was able to form emulsion and foam, and therefore it is possible to use the complex as a food and pharmaceutical ingredient and in novel formulation development in order to enhance acceptability. Our results are in agreement with the previous work reported by Tapal and Tiku (2012), showing no significant change in the emulsifying and foaming capacity of SPI-
reduction, are concentration-dependent. Antioxidant activity of proteins is contributed by Trp, Tyr, Phe, and His amino acids which have hydrogen donor groups, e.g. indole, phenyl and imidazole, or by Met, Cys, and Pro, amino acids, which are the endogenous protein antioxidants (Levine, Mosoni, Berlett, & Stadtman, 1996; Zhang, Wang, & Xu, 2008). Curcumin has a phenolic hydroxyl group that has the ability to scavenge radicals through H-atom donating (Tapal & Tiku, 2012). The antioxidant activity of LSPH/CUR complex is higher than that of LSPH due to the presence of curcumin in the LSPH/CUR complex. 3.6. Foaming and emulsion capacity Emulsion capacity (m2/g) measures the ability of protein and another amphoteric molecule to form a stable emulsion within a prescribed period of time. Hou et al. (2017) reported that hydrophilic-lipophilic properties of protein molecules significantly affect the emulsion property by lowering the tension at the oil-water interfaces and they control diffusion and aggregation of oil droplets by forming an adsorption layer. The diffusion of a protein molecule is prominently 6
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Fig. 5. (a) ABTS and DPPH activities; (b) FRAP antioxidant activity.
Declaration of Competing Interest
curcumin complexation at neural pH compared to the SPI.
None declared.
4. Conclusion This study has shown that the LSPH (10–30 kDa) fraction can undergo a compact complexation with curcumin at pH 3.0 that significantly improves its solubility (in the aqueous phase), stability and even bioaccessibility. The functional attributes of the LSPH/CUR complex, especially the foam and emulsion forming capacities, were promising for its applications in food product formulations. Furthermore, the antioxidant activity of LSPH increased after complexation with curcumin. The present work reveals that the LSPH complex can be formulated as a functional food and it can act as a useful vehicle for a lipophilic bioactive molecule. Nevertheless, the denatured proteins in LSPH might significantly influence the bioavailability of encapsulated bioactive ingredients. Therefore, further studies should be carried out to evaluate the efficacy of the curcumin-LSPH complex for in-vivo digestion models.
Acknowledgment The authors are thankful for the Basic Scientific Research Fellowship in Sciences by the University Grants Commission, Government of India, for providing financial assistance (Grant number: 2812/UGC-SAP) during this investigation.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125091.
Table 1 Foaming and emulsion capacities of LSPH and LSPH/CUR complex. Concentration of protein (mg/ml)
5 10 15
Foaming capacity (%)
Emulsion capacity
10–30 kDa fraction of LSPH
10–30 kDa fraction of LSPH-curcumin complex
10–30 kDa fraction of LSPH
10–30 kDa fraction of LSPH-curcumin complex
27.43 ± 2a 31.20 ± 1b 32.30 ± 1bc
23.30 ± 2a 26.40 ± 2ab 26.40 ± 2b
0.88 ± 0.02a 0.93 ± 0.01b 0.98 ± 0.03c
0.75 ± 0.1a 0.87 ± 0.2b 0.94 ± 0.2c
All data are expressed as means ± standard deviation. Means with different superscript letters in a column differ significantly (P < 0.05). 7
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