Accepted Manuscript
Gypenosides as natural emulsifiers for oil-in-water nanoemulsions loaded with astaxanthin: Insights of formulation, stability and release properties
Zhang Chen, Gaofeng Shu, Noamane Taarji, Colin J. Barrow, Mitsutoshi Nakajima, Nauman Khalid, Marcos A. Neves
PII: S0308-8146(18)30677-0
DOI: https://doi.org/10.1016/j.foodchem.2018.04.054
Reference: FOCH 22749
To appear in: Food Chemistry Received Date: 23 October 2017 Revised Date: 13 April 2018 Accepted Date: 16 April 2018
Please cite this article as: Chen, Z., Shu, G., Taarji, N., Barrow, C.J., Nakajima, M., Khalid, N., Neves, M.A., Gypenosides as natural emulsifiers for oil-in-water nanoemulsions loaded with astaxanthin: Insights of formulation, stability and release properties, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.04.054
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Gypenosides as natural emulsifiers for oil-in-water nanoemulsions loaded with astaxanthin: Insights of formulation, stability and release properties
Zhang Chena, Gaofeng Shua, Noamane Taarjia, Colin J. Barrowb, Mitsutoshi Nakajimaa,d, Nauman Khalidc*, Marcos A. Neves a,d*
a Tsukuba Life Science Innovation Program (T-LSI), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
b Center for Chemistry and Biotechnology, Deakin University, Waurn Ponds, Victoria 3217, Australia
c School of Food and Agricultural Sciences, University of Management and Technology, Lahore 54000, Pakistan
d Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
---
* To whom correspondence should be addressed.
E-mail: [email protected] (Marcos A. Neves), Tel.: +81 29 853 6763 E-mail: [email protected] (Nauman Khalid), Tel.: +61 3 522 78313
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Abstract
The formulation, physicochemical stability and bioaccessibility of astaxanthin (AST) loaded oil-in-water nanoemulsions fabricated using gypenosides (GPs) as natural emulsifiers was investigated and compared with a synthetic emulsifier (Tween 20) that is commonly applied in food industry. GPs were capable of producing nanoemulsions with a small volume mean diameter (d4,3 = 125 ± 2 nm), which was similar to those prepared using Tween 20 (d4,3 = 145 ± 6 nm) under the same high-pressure homogenization conditions. GPs-stabilized nanoemulsions were stable against droplet growth over a range of pH (6-8) and thermal treatments (60-120 oC).
Conversely, instability occurred under acidic (pH 3-5) and high ionic strength (25-100 mM CaCl2) conditions. In comparison with Tween 20, GPs were more effective at inhibiting AST from degradation during 30 days of storage at both 5 and 25 oC.
However, GPs led to lower lipid digestion and AST bioaccessibility from nanoemulsions than did Tween 20.
Keywords: Astaxanthin; Nanoemulsions; Gypenosides; Physicochemical stability;
Bioaccessibility
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1. Introduction
Astaxanthin (AST) is a carotenoid that has beneficial effects on human health and wellness. AST exhibits stronger antioxidant activity than β-carotene and vitamin E, due to the presence of conjugated double bonds and hydroxyl groups in its molecular structure (Wang, Zhao, Guan, Zhang, Dang, 2017, Dong, et al., 2017). The regular consumption of AST also provides other health benefits, such as boosting and modulating of the immune system, reducing the risk of cardiovascular diseases, certain cancers, oxidative stress, inflammation and cataracts (Higuera-Ciapara, Felix-Valenzuela, & Goycoolea, 2006). In addition, natural astaxanthin is a natural and safe alternative to synthetic colorants used in the food industry. However, AST cannot be produced in the human body and is obtained primarily through the consumption of AST-rich food, such as salmon (Sowmya & Sachindra, 2011).
Like other carotenoids, the absorption of AST by the human body from food is low due to its low water solubility. Furthermore, AST is unstable and sensitive to high temperature, pH changes, oxygen and light (Taksima, Limpawattana, & Klaypradit, 2015), which limits the utilization of AST as a nutraceutical ingredient in functional foods and beverages. Extensive efforts have been carried out to improve the stability and delivery properties of AST by entrapping this nutraceutical component into delivery systems that can be dispersed into aqueous-based food and beverage products (Li, Zahi, Yuan, Tian, & Liang, 2015; Liu, McClements, Cao, & Xiao, 2016; Tamjidi, Shahedi, Varshosaz, & Nasirpour, 2014a).
Among these colloidal delivery systems, emulsions and nanoemulsions are good candidates to deliver lipophilic bioactive compounds including AST. Nanoemulsion
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delivery systems provide improved physical stability and high optical clarity (Guttoff, Saberi, & McClements, 2015). Nanoemulsions can be prepared simply by homogenizing an oil phase containing oil-soluble functional compounds with an aqueous phase containing a water-soluble surfactant. Moreover, nanoemulsion-based delivery systems are effective in term of increasing the water-dispersibility and bioavailability for numerous non-polar bioactive compounds, such as carotenoids, and hydrophobic vitamins (Mayer, Weiss, & McClements, 2013; Ozturk, Argin, Ozilgen,
& McClements, 2015).
Oil-in-water (O/W) nanoemulsions are thermodynamically unstable systems that contain small oil droplets (d < 200 nm) dispersed in aqueous phase. Selection of the correct emulsifier is crucial for producing a stable nanoemulsion. An emulsifier stabilizes an emulsion by protruding its polar group into the water phase and non-polar groups into the oil phase, then forms an interfacial layer to provide protection against droplet aggregation or coalescence (McClements, 2015). There are numerous types of food-grade emulsifier that are used to produce emulsion-based products in the food industry, such as polysaccharides (e.g., pectin and gum arabic), proteins (e.g., sodium caseinate and whey protein isolate), phospholipids (e.g., lecithin) and small molecule surfactants (e.g., Tweens) (Ozturk, Argin, Ozilgen, &
McClements, 2014; Shariffa, Tan, Abas, Mirhosseini, Nehdi, & Tan, 2016). Small molecule surfactants produce emulsions with relatively small droplet size, due to their relatively high adsorption kinetics (Mao, Xu, Yang, Yuan, Gao, & Zhao, 2009). Small molecule synthetic emulsifiers, such as the Tween series, have been used in fabricating nanoemulsion-based delivery system for AST (Affandi, Julianto, &
Majeed, 2012; Kim, Hyun, Yun, Lee, & Byun, 2012; Tamjidi, Shahedi, Varshosaz, &
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Nasirpour, 2014a). However, consumers are increasingly demanding the use of natural ingredients, including natural replacements for synthetic emulsifiers in emulsion-based foods and beverages.
In this manuscript we investigate the ability of gypenosides (GPs) to act as emulsifiers in the formulation of AST-enriched nanoemulsions. GPs are the major bioactive components isolated from the Gynostemma pentaphyllum, and are saponins with established beneficial effects, including lowering the serum lipid and cholesterol levels, antitumor, immune-modulatory functions, anti-inflammatory and anti-oxidative activities (Lu, Chen, Lai, Yang, Weng, Ma, et al., 2010; Lüthje, Lokman, Sandström, Östenson, & Brauner, 2015). To the best of our knowledge, there are no studies have reporting about the utilization of GPs as natural emulsifiers to formulate nanoemulsion-based delivery system for nutritional components, such as AST. These new nanoemulsion systems are expected to have dual health benefit, due to both the encapsulated AST and the emulsifiers themselves (GPs).
Thus, the aim of our present study was to fabricate a nanoemulsion-based delivery system for AST using GPs as natural emulsifiers via high-pressure homogenization methods. The properties and stability of GPs-stabilized nanoemulsions was also compared with those for a synthetic emulsifier (Tween 20) that is widely used in commercial food application. Moreover, the in vitro small intestinal digestion behavior and bioaccessibility of AST-enriched nanoemulsions were also investigated.
2. Materials and methods
2.1 Materials
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Gypenosides (SJGLD161207, saponins 98%) were purchased from Xi’an Season Biotechnology Co., Ltd (Shanxi, China). Zanthin® astaxanthin complex (purity 10%) was procured from Valensa International (Eustis, FL, USA). Standard astaxanthin (purity > 97%), pancreatin from porcine pancreas (P7545) and bile extract porcine (B8631) were purchased from Sigma-Aldrich (St. Louis, MO). The following chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan):
refined soybean oil, Tween 20, HPLC grade methanol and dichloromethane. All other chemicals used in the present work were of analytical grade. Milli-Q water was used to prepare all the solutions and nanoemulsions.
2.2 Formulation of AST-loaded nanoemulsions
The oil phase containing AST was prepared by dissolving 2% (w/w) Zanthin®
astaxanthin complex in refined soybean oil (passed through a 0.45 μm filter). The aqueous phase was prepared by dissolving 1% (w/w) either gypenosides or Tween 20 in Milli-Q water containing 0.02% (w/w) sodium azide as antimicrobial agent. The O/W nanoemulsions encapsulated with AST were producing by using a two-step homogenization method, according to our previous study (Shu, Khalid, Zhao, Neves, Kobayashi, & Nakajima, 2016). Briefly, coarse emulsions were prepared in the first step by mixing the oil phase (5%, w/w) and the aqueous phase (95%, w/w) using a rotor-stator homogenizer (Polytron, PT-3000 Kinematica-AG, Littace, Switzerland) for 5 min at 8000 rpm min-1. The final nanoemulsions were then obtained in the second step by homogenizing the coarse emulsions via a high-pressure homogenizer (NanoVater, NV200, Yoshida Kikai, Nagoya, Japan) at 100 MPa for 4 passes.
2.3 Stability assessment of AST-loaded nanoemulsions
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2.3.1 Effect of pH
The nanoemulsions were prepared at different pH (3-8) by adjusting the samples with 0.1 M HCl or NaOH. Samples were stored at refrigerator (5 oC) overnight prior to analysis.
2.3.2 Effect of ionic strength
The nanoemulsions were added with the same volume of CaCl2 solutions to form the samples with desired final salt concentrations (0-100 mM). Samples were stored at refrigerator (5 oC) overnight prior to analysis.
2.3.3 Effect of thermal treatment
The nanoemulsions were transferred to a glass test tube with screw cape and then placed in a water bath or autoclaving machine (KTS-2346, ALP Co., Ltd, Tokyo, Japan) setting at different temperatures (60-120 oC). After heating for 30 min, the samples were cooled down to room temperature and then were used to measure their droplet size and AST concentration. The samples without thermal treatment (25 oC) were used as control.
2.3.4 Storage stability
The nanoemulsions (neutral pH, no salt added) were transferred to sealed glass bottles and placed in dark at 5 oC or 25 oC for a period of 30 days. The droplet size and AST concentration of the samples were determined. The AST encapsulation efficiency (EE) in samples was calculated according to equation (1), as follows:
Where Ct is the AST concentration of the nanoemulsions stored at different storage time, and Co is the concentration of AST in the freshly prepared samples.
2.4 Measurement of droplet size, size distribution
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The droplet size and size distribution of nanoemulsions were measured using a laser diffraction particle size analyzer (LS 13 320, Beckman Coulter, Brea, USA). The refractive indexes were set at 1.471 and 1.333 for the soybean oil and water, respectively. The droplet size of the samples was expressed as volume mean diameter (d4,3). All measurements were conducted in triplicates.
2.5 Measurement of ζ-potential
The electric charge (ζ-potential) of the nanoemulsions droplet was determined using a ζ-potential analyzer (Zetasizer Nano ZS, Malvern Instruments Ltd., Worcestershire, UK). Before analysis, samples were diluted 100 times with phosphate buffer solution (5 mM) at the same pH as the samples tested. The refractive indexes were set at 1.471 and 1.333 for the soybean oil and water, respectively. All measurements were conducted in triplicate.
2.6 Measurement of AST concentration in nanoemulsions
The concentration of AST in nanoemulsion system was measured using a UV–visible spectroscopy method described in a previous study (Qian, Decker, Xiao, &
McClements, 2012), with minor modification. In the solvent extraction approach, the nanoemulsion (200 μL) were diluted into 10 mL using the organic solvent containing dichloromethane and methanol (2:1, v/v) to isolate the AST from the samples. The resulting transplant solutions were transferred to a cuvette, and then their absorbance were measured using a UV/VIS/NIR spectrophotometer (V-570, JASCO Co., Hachioji, Japan) at 480 nm, with the pure dichloromethane and methanol solution as a blank. The concentration of AST in nanoemulsion system was calculated by using a standard curve (R2 = 0.9952).
2.7 In vitro small intestinal digestion
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An in vitro small intestinal digestion model used in our study was a slight modification of those reported previously (Ahmed, Li, McClements, & Xiao, 2012;
Zangenberg, Müllertz, Kristensen, & Hovgaard, 2001). Before passing through the digestion model, nanoemulsions were diluted 5 times with phosphate buffer (5 mM, pH 7) to form the samples containing 1 % oil. The mixture (30 g) were placed in a clean beak, and then heated in a water bath (37 oC, 10 min) under magnetic stirring at 100 rpm min-1. Then, 4 mL bile extract (46.8 mg mL-1) dissolved in phosphate buffer (5 mM, pH 7) and 1 mL of CaCl2 (110 mg mL-1) dissolvedin Milli-Q water were added into the sample. The mixture was then adjusted to pH 7, if needed. An aliquot (2.5 mL) of freshly prepared pancreatin suspension (24 mg mL-1) dispersed in phosphate buffer (5 mM, pH 7) was incorporated into the resulting mixture. From this point, NaOH solution (0.5 M) was manually titrated into the reaction system to maintain it at pH 7. The digestion process was performed for 2 h. The volume of added NaOH (0.5 M) versus digestion time was recorded and then was used to calculate the percentage of free fatty acids (FFA) released from lipid digestion using the equation (2), as follows:
where VNaOH (t) is the volume (L) of NaOH (0.5 mM) added to samples at a specific digestion time (min), MNaOH is the molarity of NaOH used (M), Moil is the molecular weight of the refined soybean oil (g/mol), and Woil is the total amount (g) of the oil initially present in the reaction cell.
2.8 Evaluation of the AST bioaccessibility
After full digestion, 10 mL of raw digesta was collected and centrifuged at 12,500 rpm for 60 min using a MX-307 centrifuge (Tomy Digital Biology Co., Ltd.,
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Tokyo, Japan). After centrifugation, the supernatant fraction (middle phase) was collected and then passed through a syringe filter (0.45 μm). The obtained clear solution was assumed to be the micelles phase that solubilized the AST. The extraction of AST from raw digesta phase and micelles phase was using dichloromethane-methanol (2:1, v/v) as an organic solvent. An aliquot (1 mL) of samples were mixed with 4 mL organic phase, and then centrifuged at 10,000 rpm (20 min). The transparent organic phase (lower part) was collected and quantified the concentration of AST using the UV–visible spectroscopy method described in Section 2.7. The bioaccessibility and chemical stability of AST were calculated according to the equation (3) and (4), respectively, as follows:
(3)
where CMicelles and CDigesta are the AST concentrations in micelles phase and raw digesta phase, respertively. CInitial is the AST concentration calculated based on the initial amount added.
2.9 Statistical analysis
All experiments were carried out at least 2 times and the results were reported as average and standard deviations. Analysis of variance (ANOVA) tests were used to analyze characterization data at a confidence level of 95%. Least significant difference (LSD) was calculated using Statistix 8.1 software (Tallahassee, USA) according to the method described by Steel, Torrie, and Dickey (1997).
3. Results and discussion
3.1 Properties of initial nanoemulsions
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We examined the properties of AST-encapsulated nanoemulsions prepared by GPs and Tween 20, including droplet size, size distribution, ζ-potential and AST concentration (Fig. 1 and Table. S1). The fresh nanoemulsions stabilized by GPs and Tween 20 exhibited monomodal droplet size distributions with relatively small and similar droplet sizes: d4,3 = 125 ± 2 and 145 ± 6 nm, respectively. These results indicated that both GPs and Tween 20 were capable of producing small droplet under the high-pressure homogenization conditions used in our study. The reason for this phenomenon is probably the fast adsorption of these two small-molecule emulsifiers onto the droplets during homogenization, leading to droplet breakup enhancing, facilitating the formulation of an emulsion containing small-sized droplets (Mao, Xu, Yang, Yuan, Gao, & Zhao, 2009).
The surface charge of oil droplets affects the stability of an emulsion (McClements, 2015). As seen from Table S1, both types of emulsifier fabricated oil droplets had a negative charge, with Tween 20 giving a significantly (p < 0.05) lower magnitude of ζ-potential than GPs. Tween 20 as a nonionic emulsifier should produce droplets with no charge at neutral pH. The observed moderate negative charge in the Tween 20 system is likely related to the adsorption of OH− species from the water, or impurities such as free fatty acids present in the oil or surfactant (McClements, 2015).
Saponin-based emulsifiers (such as quillaja saponins) tend to formulate nanoemulsions containing oil droplets with a relatively high negative charge at neutral pH, which is attributed to the presence of carboxylic acid groups associated with their hydrophilic head (Ozturk & McClements, 2016; Y. Yang, Leser, Sher, & McClements, 2013). The carboxylic acid groups are also found to be present in the molecule
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structure of some types of GPs (Z. Yang, Chen, & Hu, 2007). Thus, we presumed this groups gave GPs an anionic character observed in our study.
The concentrations of AST in GPs- and Tween 20-stabilized nanoemulsions were 92.7
± 1.7 and 95.4 ± 2.1 μg/mL (Table. S1), which are close to the theoretical concentration (≈ 100 μg/mL). This suggested that the high-pressure homogenization process applied in our study had some impacts on the encapsulation efficiency of AST, regardless of emulsifier type. However, other studies have shown that severe loss of AST occurred during preparation due to free radicals created by the high-pressure homogenization process (Anarjan, Tan, Nehdi, & Ling, 2012; Liu, McClements, Cao,
& Xiao, 2016). In our study the presence of some tocopherol antioxidants in the AST mixture (Zanthin®) may have protected AST from degradation (Tamjidi, Shahedi, Varshosaz, & Nasirpour, 2014b).
3.2 Influence of pH on the stability of nanoemulsions
The physical stability of nanoemulsions, prepared using either GPs or Tween 20 as emulsifier, was investigated as a function of storage pH. The nanoemulsions were treated at different pH values from 3 to 8. Nanoemulsions stabilized by Tween 20 were stable against droplet aggregation across the pH range studied, with droplet size remaining constant and no sign of phase separation (Fig. 2a). There was almost no change in d4,3 for GPs-coated droplets at pH 6 to 8, suggesting nanoemulsions formed with GPs were sable against pH above 6. However, there was an increase in the droplet size of GPs-stabilized nanoemulsions with decreasing pH, until they became highly unstable with visible oiling off occurring at pH 3 (Fig. 2a). Therefore, the results indicated that the nanoemulsions stabilized by GPs had a narrower range of pH
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stability (pH 6-8) than those stabilized by Tween 20 (pH 3-8). GPs would be expected to form a thin protective layer around the oil droplet, due to their relatively low molecular weight. As a result, the steric repulsion generated by this emulsifier would not be expected to be strong enough to resist the attractive forces (such as Van der Waals attraction) between the nanoemulsions. Thus, we believe that GPs stabilize the oil droplets from aggregation mainly through electrostatic repulsion, which is due to the presence of carboxylic acid groups (pKa ≈ 3.25) (Ozturk & McClements, 2016).
The carboxylic acid group are negatively charged (COO-) and exposing at high pH (pH >> pKa). Conversely, these groups are uncharged (COOH) at low pH (pH <<
pKa). A decrease in the magnitude of electric charge with decreasing pH in GPs-stabilized nanoemulsions was observed experimentally (Fig. 2b). Previous studies have reported that ζ-potential > 30 (absolute value) will provide good emulsion stability (Jacobs, Kayser, & Müller, 2000; Tan, Yussof, Abas, Mirhosseini, Nehdi, & Tan, 2016). The GPs-coated droplets had relatively low ζ-potential under acidic conditions (pH < 6). Thus, at low pH, the electric repulsion was not strong enough to prevent coalescence, thereby leading to droplet aggregation and oil phase separation. The high stability of Tween 20-coated droplets across this entire pH range studied was due to the steric repulsion generated by Tween 20, which is relatively insensitive to pH (McClements, 2015).
3.3 Influence of ionic strength on the stability of nanoemulsions
The influence of ionic strength (0-100 mM CaCl2) on the physical stability of nanoemulsions prepared using GPs and Tween 20 was examined. The Tween 20-stabilized nanoemulsions exhibited good stability in the presence of CaCl2 up to 100 mM with little increase in d4,3 and no evidence of phase separation (Fig. 3). This was attributed to the steric repulsive forces between Tween 20-coated droplets that
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were strong enough to keep the droplet constant even at the highest CaCl2
concentration. Similar results were reported previously (Teo, Goh, Wen, Oey, Ko, Kwak, et al., 2016). Conversely, nanoemulsions prepared using GPs were highly prone to salt-induced coalescence, as severe phase separation (oiling off) occurred even at the lowest CaCl2 concentration (25 mM) studied (Fig. 3). This instability is likely due to the screening effect of Ca2+ ions on the electric charge between GPs-coated droplets (McClements, 2015). In the presence of salt, the electrostatic repulsion between droplets became weak, thereby promoting droplets coalescence oiling off.
3.4 Influence of thermal treatment on the stability of nanoemulsions
The effect of thermal treatment on the stability of AST-loaded nanoemulsions was evaluated according to the changes in droplet size and AST retention after incubation at high temperatures (60, 90 and 120 oC) for 30 min. There was no distinct change in droplet size of GPs-stabilized nanoemulsions after all thermal treatments (Fig. 4a), indicating they were physically stable at elevated temperatures. The strong electrostatic repulsion between GPs-stabilized droplets are responsible for their excellent thermal stability. In comparison, the nanoemulsions fabricated by Tween 20 were only stable at 60 oC, without notable increase in their droplet size. However, their droplet size significantly (p < 0.05) increased at 90 oC, and the nanoemulsions became highly unstable to phase separation with a reddish oil layer being observed at the top of the tube after heating at 120 oC. This result is consistent with a recent study indicating that Tween-20 fabricated nanoemulsions containing corn oil droplets were unstable to droplet coalescence when incubated at 90 oC for 15 min (Teo, et al., 2016).
The instability of Tween-20 stabilized nanoemulsions at 90 and 120 oC is not
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surprising, since these temperatures are well above the cloud point of Tween 20, which is around 76 oC (Yuan, Gao, Zhao, & Mao, 2008). A nonionic emulsifier such as Tween 20 can lose its ability to stabilize an emulsion at temperatures approached the cloud point (McClements, 2015), resulting in droplet aggregation and coalescence.
Nanoemulsions stabilized by GPs and Tween 20 showed similar chemical stability (Fig. 4b). There was little change in AST retention after treated at 60 oC, whereas severe loss of AST occurred as the temperature increased to 90 and 120 oC. The results indicate that temperature had significant impact on the short-term thermal stability of nanoemulsions, with AST degradation increasing as temperature increased.
Rao, Sarada, and Ravishankar (2007) reported that the rate of AST degradation in gingelly or palm oil was much faster at higher temperatures (120 and 150 oC) than at lower temperatures (70 and 90 oC) during 8 h of treatment.
3.5 Storage stability of nanoemulsions
Fig. 5a shows the physical stabilities of GPs- and Tween 20-stabilized nanoemulsions over 30 days storage at 5 and 25 oC. During storage the droplets remained constant in all nanoemulsions, indicating that they were physical stable against droplet growth, regardless of temperature and emulsifier type tested. It is well established that nanoemulsions made from long-chain triglycerides are relatively physically stable due to their small droplet size and slow rate of Ostwald ripening (Wooster, Golding, &
Sanguansri, 2008). In addition, the protective role of these two emulsifiers was also presumably responsible for the production of AST-loaded nanoemulsions with high physical stability at a relatively low storage temperature.
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Although all the AST-loaded nanoemulsions were physical stable during storage, the chemical stability was highly dependent on the storage temperature, as well as on the emulsifier type. In general, the nanoemulsions displayed a slower rate of AST degradation during storage at 5 oC than at 25 oC for both types of emulsifier (Fig. 5b).
This result suggested that chemical stability of AST could be improved by storing the nanoemulsions at a relatively low temperature. The lower loss of AST in nanoemulsions during storage at lower temperature was in agreement with previous studies reporting that long-term chemical stability of AST in nanodispersions increased with reducing storage temperature, due to the thermal instability of AST (Anarjan & Tan, 2013). At both incubation temperatures, the rate of AST degradation was lower in the nanoemulsions stabilized by GPs than for those stabilized by Tween 20 (Fig. 5b). The GPs-stabilized nanoemulsions showed relatively high chemical stability, with only a small AST loss of 5.6% after 30 days of storage at 4 oC. In contrast, a large loss of AST (47.3%) was observed in Tween-20 stabilized nanoemulsions under the same storage conditions. Gypenosides has antioxidant and free radical scavenging properties (JP Saleeby, 2006; Shang, Liu, Zhu, Zhao, Feng, Wang, et al., 2006), which could account for GPs being more effective than Tween 20 at protecting the encapsulated AST from degradation over time.
3.6 Influence of emulsifier type on release properties of AST-loaded nanoemulsions 3.6.1 Lipid digestion
The emulsifier type used to stabilize the nanoemulsions clearly had a major impact on the rate and extent of lipid digestion (Fig 6a). For Tween 20-stabilized nanoemulsions, a sharp increase in the percentage of free fatty acid (FFA) release was observed within the initial 5 min, followed by a more gradual increase over time, until a relatively
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constant plateau was reached. In comparison, nanoemulsions containing GPs-stabilized lipid droplets were digested more slowly, and the total amount of released FFA at the end of digestion process was also significantly (p < 0.05) lower.
There are two potential mechanisms to account for the slow rate of lipid digestion for GPs-coated droplets. Firstly, large amount of droplet flocculation and/or coalescence was visibly observed in GPs-stabilized nanoemulsions before adding the lipase (data not shown), which could be attributed to shielding effects of the calcium ions present in the digestion phase as described in Section 3.3. Droplet aggregation result in a decrease in exposed surface area, thereby making it is more difficult for lipase to access the undigested lipid in flocculated droplets (Zhang, Zhang, Zhang, Decker, &
McClements, 2015). Secondly, GPs possess inhibitory activity toward porcine pancreatic lipase (Su, Wang, Ma, Liu, Rahman, Gao, et al., 2016), which may further decrease the release rate of FFA during digestion.
3.6.2 Bioaccessibility and chemical stability of AST
The bioaccessibility of AST in a nanoemulsion is defined as the fraction of AST that is successfully incorporated within the mixed micelles formed during the digestion process. In general, the mixed micelles are formed from bile extracts (phospholipids and bile salts) and lipid digestion products (FFA and monoacylglycerols). Fig. 6b shows the influence of emulsifier type on the bioaccessibility and chemical stability of AST after the nanoemulsions were digested. The bioaccessibility of AST incorporated in GPs-stabilized nanoemulsions was around 20% and was significantly (p <0.05) lower than that stabilized by Tween-20 (≈ 40%), which suggested that the emulsifier type played an important role in the solubilization of AST in the micelle phase generated at the end of the digestion period. The decrease in AST bioaccessibility in GPs-stabilized nanoemulsions, compared those with Tween 20, can be attributed to a
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number of factors: (i) The lower amount of FFA released resulted in less formulation of mixed micelles present in the digestion fluid that are capable of solubilizing the released AST; (ii) GPs-coated droplets were not fully digested, therefore, more AST remained in the undigested oil, instead of releasing into the digestion phase; and (iii) GPs molecules could inhibit micellar formation (Su, et al., 2016), which may decrease the amount of AST solubilized within the mixed micelles. Fig. 6b also shows that there was almost no loss of AST in all nanoemulsions, indicating that the encapsulated AST did not degrade during digestion. This is consistent with a previous study showing that β-carotene in a nanoemulsion-based delivery system was chemically stable when exposed to the gastrointestinal phase, over a relatively short 3 h digestion time (Yi, Li, Zhong, & Yokoyama, 2014). The current work used a relatively simple digestion mode to evaluate the influence of emulsifier type on AST bioaccessibility. A more detailed investigation using both in vitro and in vivo models would provide more detailed bioaccessibility information.
4. Conclusions
The current work demonstrates the successfully encapsulation of AST within nanoemulsion delivery systems produced using high-pressure homogenization. GPs were used as natural emulsifiers and compared with Tween 20, a synthetic surfactant widely used in the food industry. The results showed that GPs were able to produce nanoemulsions with a small droplet size (d4,3=125 ± 2 nm), and had similar size distribution to those produced using Tween 20. The high-press homogenization conditions applied in our study had little impact on the encapsulation efficiency of AST for both GPs and Tween 20, which is attributed to the tocopherols present in the AST extract. GPs-stabilized nanoemulsion droplets had large negative charge at neutral pH, and the magnitude of the charge decreased with decreasing pH, due to the
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carboxylic acid groups on the GPs. Acidic conditions or addition of CaCl2 led to physical instability of nanoemulsions prepared using GPs. However, unlike Tween 20, GPs was able to protect the oil droplets from coalescence during thermal processing at elevated temperatures (60-120 oC) for 30 min. However, the rate of AST degradation increased with increasing temperature.
All nanoemulsions showed excellent stability against droplet coalescence when stored at 5 oC and 25 oC for at least 30 days, regardless of emulsifier type. However, the retention of AST in the nanoemulsions was highly dependent on storage temperature, as well as emulsifier type. The loss of AST in both nanoemulsions was slowed by incubating them at lower temperatures. GPs provided better protection against AST degradation than did Tween 20, probably due to the free radical scavenging ability of GPs. During in vitro digestion, the GPs-stabilized nanoemulsions exhibited a lower rate and extent of lipid digestion than those stabilized by Tween 20. As a result, a relatively low percentage of AST was incorporated within the micelles phase when GPs was used as an emulsifier to stabilize the AST-loaded nanoemulsions. AST was chemically stable against digestion conditions over the short time of the digestion process. Overall, our findings provide new information that GPs are an effective natural emulsifier that may be suitable for some food and pharmaceutical emulsion purposes.
Conflict of interest
The authors declare that there is no conflict of interest relating to the publication of
this article.
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Figure captions
Fig. 1: Effect of emulsifier type on droplet size distribution of nanoemulsions loaded with AST. ( ) denotes GPs and ( ) denotes Tween 20.
Fig. 2: Effect of pH on the physical stability of AST-loaded nanoemulsions. (a) d4,3 and (b) ζ-potential of the nanoemulsions. ( ) denotes GPs and ( ) denotes Tween 20.
d4,3 and ζ-potential were not measured for samples with oil phase separation.
Fig. 3: Effect of CaCl2 concentration on the physical stability of AST-loaded nanoemulsions. ( ) denotes GPs and ( ) denotes Tween 20. d4,3 was not measured for samples with oil phase separation
Fig. 4: Effect of thermal treatment on the physiochemical stability of AST-loaded
nanoemulsions. (a) physical stability and (b) chemical stability. ( ) denotes GPs and ( ) denotes Tween 20. d4,3 and AST concentration were not measured for samples with oil phase separation.
Fig. 5: Effect of emulsifier type on the physiochemical stability of AST-loaded
nanoemulsions during 30 days of storage at 5 and 25 oC. (a) physical stability and (b) chemical stability. ( ) denotes GPs (5 oC), ( ) denotes GPs (25 oC), ( ) denotes Tween 20 (5 oC) and ( ) denotes Tween 20 (25 oC).
Fig. 6: Effect of emulsifier type on in vitro digestion behavior of AST-loaded nanoemulsions. (a) Effect of emulsifier type on free fatty acids (FFA) profiles. ( ) denotes GPs and ( ) denotes Tween 20. (b) Effect of emulsifier type on bioaccessibility and chemical stability of AST-loaded nanoemulsions. ( ) denotes AST bioaccessibility and ( ) denotes AST stability.
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Fig. 1
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Fig. 2
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Fig. 3
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Fig. 4
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Fig. 5
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Fig. 6
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Highlights
GPs was used as natural emulsifier for stabilizing O/W emulsions
AST was successfully encapsulated in gypenosides stabilized emulsions
Environmental factors influenced the stability of O/W nanoemulsions
The nanoemulsions exhibited good physical stability at 5 and 25 oC
GPs led to lower lipid digestion and AST bioaccessibility from nanoemulsions
