Jia-Run
Han
a,
Lu-Ping
Gu
c,
Ruo-Jie
Zhang
d,
Wen-Hui
Shang
a,
Jia-Nan
Yan
a,
David Julian
McClements
d,
Hai-Tao
Wu
*ab,
Bei-Wei
Zhu
*ab and
Hang
Xiao
*d
aSchool of Food Science and Technology, Dalian Polytechnic University, Dalian Liaoning 116034, China. E-mail: zhubeiwei@163.com; wht205@163.com; Fax: +86-411-86318655; Fax: +86-411-86318655; Tel: +86-411-86318655 Tel: +86-411-86318731
bNational Engineering Research Center of Seafood, Dalian Liaoning 116034, China
cKey Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China
dDepartment of Food Science, University of Massachusetts, Amherst, Massachusetts 01003, USA. E-mail: hangxiao@foodsci.umass.edu; Fax: +1 (413) 545-1262; Tel: +1 (413) 545-2281
First published on 19th December 2018
Emulsion-based delivery systems were structured by using scallop gonad protein isolates (SGPIs) as novel food-grade emulsifiers. The effects of carrier oil, including the long chain triglycerides (LCT) and medium chain triglycerides (MCT), on the bioaccessibility and cellular uptake of β-carotene (BC) were investigated. Both LCT and MCT delivery systems remained stable at pH 7–8 but aggregated at lower pH values (3–6) according to the results of light scattering and microscopy measurements. LCT droplets fabricated within SGPIs were digested and released more slowly than MCT droplets during the simulated gastrointestinal tract digestion. The LCT emulsion showed higher BC bioaccessibility (65.5%) than the MCT emulsion (23.1%) as a result of the greater solubilization of BC in mixed micelles fabricated from long-chain fatty acids. Moreover, the LCT emulsion produced higher cellular uptake of BC as compared with the MCT emulsion in intestinal epithelial cells. These results demonstrated that SGPIs could be used as novel food-grade emulsifiers to protect lipophilic bioactive compounds in emulsion-based delivery systems, in which LCT is more suitable to encapsulate and deliver BC than MCT.
Owing to the growing consumer demands for “healthy” food products, natural emulsifiers are widely considered in formulating emulsions as compared with synthetic surfactants or polymers.9 Protein is a kind of natural emulsifier with the advantages of a strong surface activity, low cell toxicity and good stability.10 Nowadays, protein-based emulsifiers applied in the food industry are derived from milk, soybean and eggs.11 Many reports have indicated that these emulsion-based delivery systems can be utilized to enhance the bioaccessibility of BC.12,13 However, due to the dietary preferences and territory restrictions, food manufacturers and consumers are looking for some alternative marine protein sources in the human diet. Based on some literature studies, several marine proteins have already been explored as food-grade emulsifiers.14,15 For instance, shrimp (Penaeus vannamei) heads with a high content of protein have been used to stabilize palm oil-in-water food emulsions.14 Silver carp (Hypophthalmichthys molitrix) protein isolate has displayed certain emulsifying capacity and foaming capacity.15 Nevertheless, the research on shellfish, especially scallop proteins, as an emulsifier to deliver BC is still relatively rare.
Scallop (Patinopecten yessoensis) is an important bivalve extensively distributed in Eastern Asia. In China, scallop production from aquatic breeding has increased to 1.86 million tons in 2016 (FAO 2017). The demand for scallop processing is increasing with the continuous expansion of scallop farming. Scallop gonads are the main edible byproducts with a high level of protein during the processing of the P. yessoensis adductor, and are regarded as good sources to develop a protein matrix. Our previous studies have shown that protein isolates from scallop gonads (SGPIs) provide high nutritional value and good emulsifying properties when compared with crude scallop gonad or soybean protein isolates. SGPIs contain a mixture of proteins, such as vitellogenin, actin, and a little myosin. These proteins have both hydrophobic and hydrophilic regions on their surfaces,16 and these regions can easily consume oil droplets forming an interfacial coating.10 As a consequence, it is necessary to study the effect of SGPIs on emulsion-based delivery systems for BC.
It has been confirmed that the bioaccessibility and bioavailability of hydrophobic bioactive compounds can be improved by combining them with digestible lipids, which is impacted by the lipid nature.17 The digested triacyglycerols form monoacylglycerols and free fatty acids (FFA), which are combined with phospholipids and bile acids forming mixed micelles to solubilise and transport the BC molecules to epithelium cells.18 After absorption, the FFA and monoacylglycerols are recombined together into triacylglycerols, which can be incorporated along with BC into lipoproteins (chylomicrons). Therefore, the type of triacylglycerols will show an important effect on the SGPI-BC emulsion delivery system.
In the current study, protein isolates were prepared from scallop gonads and further used as emulsifiers. Emulsion-based delivery systems adopting SGPIs as food-grade emulsifiers were structured by using long chain triglycerides (LCT) and medium chain triglycerides (MCT) as the carrier oils. A simulated gastrointestinal tract (GIT) and Caco-2 cells model were adopted to investigate the impact of carrier oil on the bioaccessibility and cellular uptake of BC. The knowledge gained from the present study will provide fundamental information for establishing SGPI-based delivery systems to encapsulate BC for further application.
Scallop gonad protein isolates (SGPIs) were prepared from defatted scallop gonad by the isoelectric precipitation process.19 Briefly, distilled water was added to defatted scallop gonads at a ratio of 1:20 (w/v) and adjusted to pH 11.5 using NaOH solution, and then the mixture was centrifuged at 2000g for 20 min. The resulting supernatant was adjusted to pH 3.8 with 0.5 M HCl and centrifuged at 10000g for 30 min. Thereafter, the sediment was washed twice with distilled water, neutralised using NaOH solution, and dialyzed for 48 h. Finally, the sediment was freeze-dried and referred to as SGPIs. The proximate composition of SGPIs was found to be 86.39 ± 1.2 wt% protein, 0.17 ± 0.02 wt% lipid, 3.27 ± 0.28 wt% ash, 6.27 ± 0.31 wt% moisture and 1.9 ± 0.08 wt% carbohydrate.
The concentration of BC was calculated according to the standard curve: Y = 0.0723X + 0.0445. The BC concentration in the entire digesta phase was also determined using the same method. The transformation and bioaccessibility of BC were estimated using the following formulas:26
Here, Cmicelle, Cdigesta and Cinitial are the BC concentrations in the micelle fraction, entire digesta phase, and initial emulsions before digestion, respectively.
Here, Acontrol is the absorbance of the cells incubated with DMEM only, Asample is the absorbance of the cells incubated with samples, and Ablank is the absorbance of cell-free wells.
The particle size distributions and mean particle diameters of both LCT and MCT-based BC emulsions are shown in Fig. 1A, B and Table 1, respectively. In the range of pH 3.0–4.0, both emulsions exhibited physical instability. The emulsions had obviously broad particle size distribution coinciding with visible phase separation after microfluidisation (Fig. 1A and B). Mean particle diameters higher than 10 μm were observed (Table 1). Furthermore, with the increase in pH value, the mean particle diameters of both emulsions significantly decreased (p < 0.05) and tended to be flat when the pH reached 6.0, and the particle size distribution at pH 7.0–8.0 showed narrow single peaks within the mean particle size less than 0.35 μm. As shown in Fig. 1C, both emulsions exhibited fairly similar ζ potential-pH profiles altering from moderately positive changes to notably negative charges at pH 3.0–8.0. The zero-charge point was determined to be approximately pH 3.8. Meanwhile, the ζ-potential magnitude of LCT emulsions was always higher than that of MCT when the pH reached above 4.0, and the ζ-potential of emulsions for LCT and MCT was less than −20 mV at pH 7.0–8.0. Furthermore, the oil droplets seemed to be evenly spread throughout the BC emulsions at pH 7.0–8.0 as revealed from the optical and confocal microscopy images, while an extensive droplet flocculation occurred in both systems at pH 3.0–6.0 (Fig. 2). These results suggest that both LCT and MCT emulsions stabilized by SGPIs exhibited good stability under pH 7.0–8.0, and LCT emulsions have a relatively large particle diameter and ζ-potential magnitude than MCT emulsions.
Fig. 2 Effect of carrier oil on the microstructures of BC emulsions stabilized by SGPIs under different pH conditions: (A) LCT and (B) MCT (scale bar is 1 μm). |
Samples | Mean particle diameter (μm) | |||||
---|---|---|---|---|---|---|
pH 3 | pH 4 | pH 5 | pH 6 | pH 7 | pH 8 | |
Data with different letters in the same row are significantly different (p < 0.05). | ||||||
LCT-emulsion | 19.8 ± 1.28a | 25.42 ± 5.79a | 2.58 ± 0.06b | 0.45 ± 0.03c | 0.34 ± 0.03c | 0.31 ± 0.01c |
MCT-emulsion | 10.99 ± 0.18a | 23.94 ± 2.92b | 1.72 ± 0.05c | 0.39 ± 0.02d | 0.30 ± 0.06d | 0.26 ± 0.02d |
The stability of the emulsion at different pH values is attributed to the electrostatic repulsion between the lipid droplets coated with protein.13 Similar consequences were found in whey protein isolate (WPI) stabilized emulsion. It has been suggested that the WPI-soybean oil emulsion exhibited the greatest phase separation resistance and the emulsions led to more stability at pH 7 than at pH 3 as the WPI possessed a pI at 4.6.28 In contrast, the egg white protein (EWP)-stabilized BC emulsion showed good stability under acidic conditions as the EWP had pI at approximately 6.1 and 10.7 for ovotransferrin and lysozyme, respectively.29 The SGPIs had pI at approximately 3.8, and the oil droplets’ surface potential was supposed to be rather low at pH 3.0–4.0. The charge on the droplet surface was reduced by partial protonation of the fatty acids, which led to the gradual replacement of combined repulsive interactions by the attractive colloidal interaction resulting in the physical destabilization of emulsions.30,31 However, when the pH was far from the pI, relatively stable emulsions could be observed with mean particle diameters less than 0.35 μm at pH 7.0–8.0. Moreover, the LCT emulsions have a relatively large particle diameter and ζ-potential magnitude than MCT emulsions. The reason for this phenomenon may be due to the changes in the dispersed phase viscosity of the emulsions.32 Generally, the efficiency of droplet crushing in a microfluidizer increases as the viscosity of the dispersed phase decreases. LCT has a distinctly higher viscosity and contains more anionic impurities than MCT.33 Therefore, the droplet breakup of the LCT emulsion becomes less efficient during microfluidisation, which results in the formation of larger droplets and with more negative charge.
Fig. 4 Microstructure of SGPI-stabilized BC emulsions with different carrier lipids: (A) LCT; (B) MCT after they were exposed to different stages of a GIT. |
Samples | Mean particle diameter (μm) | |||
---|---|---|---|---|
Initial | Mouth | Stomach | Intestine | |
Data with different letters in the same column are significantly different (p < 0.05). | ||||
LCT-emulsion | 0.35 ± 0.01a | 1.92 ± 0.51a | 15.69 ± 2.26a | 3.36 ± 0.25a |
MCT-emulsion | 0.31 ± 0.05a | 0.84 ± 0.48b | 12.77 ± 1.63a | 10.81 ± 0.72b |
After getting through the mouth and stomach stage, the particle diameter distributions of both BC emulsions became bimodal, accompanied by a comparative rise in mean particle size (Fig. 3A, B and Table 2). Extensive oil droplet accumulation was also observed after exposure to the oral and stomach stages (Fig. 4), which indicated that some flocculation had happened, especially in the LCT emulsions. Meanwhile, there was a palpable decline in the magnitude of negative charge on both the oil droplets after the exposure to the simulated oral phase, with the surface potential of −17.20 and −13.47 mV for LCT and MCT, respectively. Subsequently, a further decrease in ζ-potential was observed when the emulsions were passed through the simulated stomach phase with the surface potential of −1.4 and 0.5 mV for LCT and MCT, respectively (Fig. 3C).
The MCT emulsions exhibited relatively larger particles than LCT emulsions after simulating the small intestinal stage, and the particle size distribution was broad bimodal in MCT emulsions (Fig. 3A, B and Table 2). Moreover, the LCT emulsion had more negative charges than the MCT emulsion (Fig. 3C). During the small intestinal phase, although the aggregated oil droplet breakup has occurred, the MCT emulsions also contained some relatively large particles (Fig. 4), which is in accordance with the light scattering measurements (Fig. 3B). Overall, all these results indicate that both LCT and MCT-based BC emulsions stabilized by SGPIs showed exactly similar behavior when they were subjected to the simulated digestion model while the systems containing LCT may have more anionic fatty acids at the surface of the particle than those containing MCT.
It has been reported that emulsions stabilized by proteins are likely to accumulate during the stomach phase on account of impaired electrostatic repulsion, hydrolysis of adsorbed proteins, and bridging flocculation or depletion induced by mucin.35,36 Our results draw a similar conclusion as well. Meanwhile, a significant reduction in the negative charge after the exposure to the gastric phase is noted, and the ζ-potential is nearly close to zero. This phenomenon is probably due to the fact that some of the proteins may have been displaced and digested, and at the same time, some of the anionic substances (for example mucin) are also adsorbed on the surface of the protein-coated lipid droplet.37 Furthermore, our results coincided with those of the previous studies which have interpreted that the MCT emulsion results in the generation of larger particles than the LCT emulsion after small intestine digestion by using Tween 20 and WPI as the emulsifier.38,39 The high surface negative charge of the particles in both emulsions was observed in the small intestine stage, which can be ascribed to the chemisorption of anionic colloidal particles on their surface, for instance phospholipids, bile salts, free fatty acids, and peptides.35 Interestingly, LCT emulsions had pronouncedly higher negative charges than MCT after digestion, which indicated that the fatty acids formed by digestion of LCT were accumulated at the surface of the particles, whereas the fatty acids produced by digestion of MCT were still retained in the aqueous phase.38 Actually, it is hard to confirm the accurate nature of these particles after digestion because of the particle diversity including micelles, undigested protein aggregates, undigested lipid droplets, and insoluble calcium salts. The entire particles conduce to the integral signal for calculating the ζ-potential.
It has been illuminated that long-chain fatty acid lipids are digested more slowly than medium-chain fatty acid lipids.40 For instance, LCT (i.e., corn oil) includes appreciably long-chain fatty acids (i.e., C16, C18 and C20), while MCT includes comparatively shorter chain ones (i.e., C8 and C10). The relatively low amount of digested LCT is possibly on account of the fact that long chain fatty acids are prone to aggregation at the surface of oil–water, and consequently lipase was restricted access to the surface of the droplets.41 In contrast, the digestive products of MCT-emulsion have high water affinity making the lipase more liable to close to lipid surfaces.42 Interestingly, the release of the total amount of FFA reached 110% at the end of 2 h of simulated GIT digestion in the MCT-emulsions. The final amount of FFA released was greater than 100% in the MCT emulsion which is attributed to some other components, such as proteins (SGPIs) in the digestion models, which were hydrolyzed and dedicated to the pH-stat process. Moreover, some monoacylglycerols might have been transformed into FFA and glycerol,43 which was not considered in the calculations. Our results are also in accordance with other latest reports, which have demonstrated a faster digestion of MCT when they are merged into Tween 20, β-lactoglobulin and modified starch-stabilized BC emulsions in comparison with LCT, reaching 123%, 113% and 120% after 2 h, respectively.24,33,43 As a result, MCT possessing a high specific surface area are easily released from the small intestine leading to quick lipid digestion.
Our results clearly showed that the transformation, i.e., the fraction of BC retaining in its original form after passing through the GIT, was distinctly higher in the LCT-emulsion (45.2%) than in the MCT-emulsions (36.3%) stabilized by SGPIs (Fig. 5B). These results suggest that a greater fraction of the BC in the LCT emulsion was not transformed at the small intestine stage, which may be due to the fact that BC is not subject to degradation by trapping into the interior of the micelle fractions. Thus, encapsulation of BC in the LCT emulsion stabilized by SGPIs gave better protection against chemical degradation. In spite of the fact that the triacylglycerols in both LCT and MCT emulsions were nearly completely digested, the bioaccessibility of BC was significantly distinct. The determined bioaccessibility of BC was approximately 23.1% in the MCT-emulsion, while it was about 65.5% in the LCT-emulsion (Fig. 5B). BC is a highly hydrophobic linear rod-like structure molecule, which favorably passed through the entire micelle core to successfully combine with the surfactant micelles.24 The long-chain fatty acids are more likely to form micelles with a larger solubilisation ability on account of the larger sizes of their hydrophobic core in comparison with medium-chain fatty acids. Our studies are consistent with some research studies showing that BC shows a higher bioaccessibility in Tween 80 stabilized emulsion systems when LCT is utilized as a carrier oil rather than MCT.44 Furthermore, the unsaturation degree of fatty acids can decide the GIT fate of BC and its bioaccessibility.45 Corn oil contains huge amounts of monounsaturated and polyunsaturated long chain fatty acids, which are presumed to be beneficial for BC bioaccessibility. In short, LCT is a more effective carrier oil for ensuring a comparatively high BC bioaccessibility than MCT in the SGPI stabilized BC emulsion system since MCT does not form large enough mixed micelle fractions to dissolve BC.
It is supposed that the mixed micelle fractions solubilize the BC and subsequently transport it to the surface of the Caco-2 cells.47 Therefore, a Caco-2 cell model was utilized to simulate the uptake of the dissolved BC by epithelial cells. After incubation with the samples (DT = 20, the final BC concentration was 0.05 μg mL−1) for 24 h, the content of BC that had accumulated in cells was measured. The cellular uptake of BC in THF/DMSO (1:1, v/v) suspension was 112.0 pmol mg−1 protein, whereas the cellular uptake of BC in the LCT and MCT emulsions stabilized by SGPIs showed a 1.9- and 1.5-fold increase, respectively (Fig. 6B). These results are consistent with those of previous studies which indicated that the cellular uptake values of whey protein isolates, sodium caseinate, and soybean protein isolate loaded BC emulsions were 687, 891, and 452 pmol mg−1 protein, respectively, which were significantly higher than that of the THF/DMSO-BC control (246 pmol mg−1 protein).48 The potential reason for the difference between our results and previously published results is that previous studies mainly focused on the emulsions that were directly applied to the Caco-2 cell model without GIT digestion, while the emulsion of our study was applied to the Caco-2 cell model after in vitro GIT digestion. The LCT resulted in a higher absorption of the BC than MCT in the SGPI-stabilized emulsion, which is similar to the effect of oil type on VE bioaccessibility as reported previously.17 Actually, there are several reasons for explaining this phenomenon. In the first place, the property of the mixed micelle fractions formed is known to lie in the type of fatty acid, which may have affected the BC transport to the cell surface. The mixed micelle fractions include liquid crystalline phases, vesicles, and micelles which may change in their dimensions and structure, consequently altering their absorption characteristics. Secondly, some fatty acids have the ability to enhance the permeability of the cell membrane, which would change the absorption of the hydrophobic bioactive component. As a result, for disparate kinds of lipid digestion products generated by LCT and MCT, Caco-2 cells may have absorbed different amounts of BC. Thirdly, the intracellular processing and absorption of hydrophobic substances like BC were also influenced by the chain length of fatty acids. Medium chain fatty acids are generally diffused through the epithelial cells rather than being shipped to the endoplasmic reticulum like long chain fatty acids, which are then transformed into triacylglycerols for secreting into the lymphatic system.17,49 As a result, more BC in the LCT emulsion was absorbed by the epithelial cells than that in the MCT emulsion.
In our study, a comparatively simple HPLC technique was utilized to measure the amount of BC. In our future research, it would be beneficial to utilize more comprehensive analytical techniques, for instance HPLC-MS/MS, to offer more detailed information with regard to the changes in the BC chemical structure throughout the GIT and Caco-2 cells.
BC | β-Carotene |
LCT | Long chain triglyceride |
MCT | Medium chain triglyceride |
SGPIs | Scallop gonad protein isolate |
GIT | Gastrointestinal tract |
MTT | 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide |
FITC | Fluorescein isothiocyanate |
DMSO | Dimethyl sulfoxide |
This journal is © The Royal Society of Chemistry 2019 |