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Preliminary study into the factors modulating β-carotene micelle formation in dispersions using an in vitro digestion model
Food Hydrocolloids 26 (2012) 427e433
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Preliminary study into the factors modulating b-carotene micelle formation in dispersions using an in vitro digestion model Pan Wang a, Hai-Jie Liu a, Xue-Ying Mei a, Mitsutoshi Nakajima b, Li-Jun Yin a, b, * a
Key Laboratory of Functional Dairy Science of Beijing and Ministry of Education, College of Food Science and Nutritional Engineering, China Agricultural University, Qinghua East Road, Beijing, 100083, China b Graduate School of Life Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 August 2010 Accepted 24 November 2010
b-Carotene is an active compound associated with prevention of heart disease, cancer and cataracts. For absorption in vivo, b-carotene must be incorporated in mixed micelles. Micelle formulation varies widely and depends on various factors. The aim of this study was to identify and study the main factors governing the bioaccessibility of b-carotene incorporated into dispersions, using an in vitro digestion model. b-Carotene dispersions were prepared by high-pressure homogenization or by combining emulsification and evaporation. The average particle sizes of the dispersions obtained ranged from 45 to 18315 nm. Results show that the concentration of b-carotene, bile extract and pancreatic lipase, pH, and the particle size of the dispersions significantly affected the transfer of b-carotene from dispersions into micelles. The transfer of b-carotene was inversely related to the particle size and the concentration of bile extract and was highest at pH 6 and 0.4 mg/mL pancreatic lipase. Bile salt played different roles depending on the particle sizes of the dispersion. When the mean diameter of b-carotene particle was below 100 nm, the addition of bile extract and pancreatic lipase did not significantly affect bioaccessibility of b-carotene passing through in vitro digestion model. At larger particle sizes, the transfer efficiency of b-carotene increased with bile extract concentration. The outcomes suggest that there is potential to improve the bioavailability of b-carotene by micronizing lipid droplets. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: b-Carotene dispersions Micelle Bioaccessibility In vitro digestion Particle size
1. Introduction Carotenoids are one of the most important classes of natural pigments because of their wide distribution in plant tissues, structural diversity, and numerous functions. In addition to provitamin A activity, carotenoids have recently been implicated in protection against or prevention of serious human health disorders such as cancer, heart disease, macular degeneration, and cataracts (Castenmiller & West, 1998; Iwase, 2002). Amongst the carotenoid pigments, b-carotene can provide the highest vitamin A activity. Studies of b-carotene absorption in humans have been carried out for many years. Generally, it appears that b-carotene absorption is not very efficient and is highly variable. The high hydrophobicity of b-carotene is reported to cause low solubility in aqueous systems and consequent poor uptake in the body, which limits its use in food formulations. b-Carotene absorption through the lymphatic system mainly occurs by the disruption of the food matrix, the
* Corresponding author. Tel./fax: þ86 10 62737424. E-mail address: [email protected] (M. Nakajima). 0268-005X/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2010.11.018
release of b-carotene, emulsification into lipid droplets in the stomach, and incorporation into mixed micelles. After micelle formation, b-carotene is absorbed by the lymphatic system in the small intestine (Thakkar, Maziya-Dixon, Dixon, & Failla, 2007; Van Het Hof, West, Weststrat, & Hautvast, 2000; Yonekura & Nagao, 2007). Because b-carotene cannot be absorbed directly from the food matrix, the transfer from the food matrix to micelles, which can be absorbed, is probably the essential step for its absorption. It has therefore been presumed that the transfer efficiency might directly affect the adsorption of b-carotene (Tyssandier, Lyan, & Borel, 2001). It had been reported that the bioavailability of b-carotene is affected by many factors, i.e. the state of the food matrix, food processing and the conditions in the gastrointestinal (GI) tract (Castenmiller & West, 1998). Yonekura and Nagao (2007) reported that the bioavailability of carotenoids from supplements is usually higher than of those embedded in the matrix of fruits and vegetables, because of the higher degree of food matrix disruption in the former. The bioavailability of carotenoids without processing is regarded as less than 10% from raw vegetables (Ribeiro, Chu, Ichikawa, & Nakajima, 2008). Food processing, including heat,
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mechanical and enzymatic treatments, may facilitate the disruption of the cell wall and organelles, releasing carotenoids from the food matrix and promoting their dispersion in the GI tract (Yonekura & Nagao, 2007). The rate and extent of lipid digestion within the stomach and small intestine may depend on how fast the GI tract can break down the surrounding matrix because the lipids may have to be exposed before they can be digested by lipases (Chen, Remondetto, & Subirade, 2006). Thus, the bioaccessibility of b-carotene, when regarded as a type of nonsaponifiable lipid, will be determined by the state of food matrix. Recent progress in nanotechnology has also gained considerable attention for improving the water solubility and bioavailability of lipophilic bioactive compounds. Ultra fine particles can reduce the inherent limitations of slow and incomplete dissolution of functional lipids due to their larger surface area and higher dissolution pressure (Horter & Dressman, 2001). In previous studies, we prepared b-carotene lipid droplets with a minimum size of approximately 50 nm, which exhibited improved solubility and stability (Yin, Chu, Kobayashi, & Nakajima, 2009). Today, much is known about how b-carotene in natural food matrices moves into emulsified lipid droplets in the diet. However, in processed food matrices, the factors affecting the transfer of b-carotene from dispersions into micelles have only been partially investigated and little is known about the metabolism and absorption of these bcarotene dispersions in humans. The aims of this study were to identify how the size characteristics of b-carotene dispersions and the conditions in a simulated digestion affect the bioaccessibility of b-carotene and to assess the effect of the physiological variations in these factors on the transfer efficiency. Information on the bioaccessibility of b-carotene in dispersions is expected to contribute to the development of active compounds in dispersions with satisfactory control and release properties.
2. Materials and methods 2.1. Materials The emulsifier, decaglycerol monolaurate (ML750), was supplied by Sakamoto Yakuhin Kogyo Co, Ltd (Osaka, Japan). bCarotene was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Soybean oil was purchased from a local supermarket and used without further purification. Standard b-carotene (Type II, 95%) was purchased from SigmaeAldrich Chemical Company (St Louis, MO, USA). The digestive juice was prepared using porcine bile extract (batch 058K0066, EC 232-369-0, Sigma) and porcine pancreatic lipase, type II (batch 096K0747, EC 232-6199, Sigma). HPLC-grade acetonitrile was purchased from CNW Technology GmbH (Germany). HPLC-grade ethanol absolute was purchased from the Tianjin Fine Chemical Engineering Division (Tianjin, China). Deionized water, purified by a Milli-Q Organex system (Millipore, Bedford, MA), was used for preparation of the aqueous phase. All chemicals were of analytical grade. 2.2. Preparation of b-carotene dispersions
b-Carotene dispersions were prepared by modifying the method described by Yin, Kobayashi, and Nakajima (2008). b-Carotene (0.25 wt%) was dissolved in soybean oil (40 C) as the dispersed phase. The organic solution was poured into the continuous phase containing 1% (w/w) ML750 in 100 mM NaHCO3 buffer (pH 7) to give an organic-aqueous phase volume ratio of 1:9. The premix was homogenized using a conventional homogenizer (Ultra TurraxÒ T25 basic, IKA AG, Staufen, Germany) at 5000 rpm for 5 min,
followed by high-pressure homogenization (NS1001L Panda 2K High-Pressure Homogenizer, GEA Niro Soavi, Italy) in a single pass at 140 MPa to obtain a final concentration of 10 wt% soybean oil, 0.025 wt% b-carotene, and 0.9 wt% ML750. Several batches were prepared, using the technique described above, by varying the homogenization pressure (10 MPa and 50 MPa) and b-carotene concentrations (0.01, 0.05 and 0.1 wt%). A sample without homogenization, recorded as 0 MPa, was used as a control. The pH value of prepared dispersions was about 7. To obtain b-carotene dispersions with smaller particle sizes, they were also prepared by the emulsificationeevaporation technique described by Tan and Nakajima (2005). b-Carotene (0.25 wt%) was dissolved and dispersed in hexane. After preparing b-carotene oil-in-water dispersions by homogenizing at either 100 MPa or 140 MPa, hexane was removed by rotary evaporation (SENCO-R Series rotary evaporator, Shanghai Seekic Co., Ltd, Shanghai, China) under reduced pressure (0.25 bar and 40 C). 2.3. Particle size analysis The mean particle sizes of b-carotene dispersions were measured using a laser diffraction particle size analyzer with a lower measuring limit of 40 nm (LS 320, Beckman Coulter, Inc., FL, USA). The experiments were carried out on undiluted suspensions after preparation and all measurements were carried out in triplicate. The particle size of the prepared b-carotene dispersions was described by the surface-weighted mean diameter (d3, 2). Measurement of the mean particle diameter of the nanodispersions was carried out using a dynamic light scattering particle size analyzer with a measuring range from 0.6 nm to 6 mm (Zetasizer Nano ZS, Malvern Instruments Ltd., Worcestershire, UK). A refractive index for b-carotene of 1.47 and water of 1.33 was used in the calculation of particle sizes (Bialek-Bylka, Jazurek, Dedic, Hala, & Skrzypczak, 2003). The particle concentration in the sample was diluted to about 0.005 wt% with Milli-Q water prior to analysis to avoid multiple-scattering affects during measurement. The final particle diameter was calculated from the average of at least three measurements. Measurement of the particle size was also carried out on the supernatant of the nanodispersions after centrifuging the samples at 10 000 g for 30 min. 2.4. In vitro digestion model used to measure the digestion of b-carotene dispersions The in vitro digestion model described by Beyssriat, Decker and McClements (2006) was used to mimic the physical and chemical conditions prevailing in the stomach and duodenum. b-Carotene dispersion was prepared as described above and then stored for approximately 1 h at room temperature. Thirty milliliters of the b-carotene dispersion was acidified to pH 2.0 with 1 M HCL and incubated for 1 h at 37 C with shaking at 95 rpm in an incubator shaker (Model HZQ-X100, Peiying Co., Shanghai, China). After the adjustment of pH, a bile extract and pancreatin mixture of 7.5 mL in 100 mmol/L sodium bicarbonate solution was added. Note that, apart from during the variables experiments (see Section 2.5), the pH of the acidified b-carotene dispersion was raised to 5.3 by adding 0.9 M sodium bicarbonate and the final concentrations of pancreatin and bile extract in the mixture were 0.4 mg/mL and 2.4 mg/mL respectively, in all experiments. Finally, the pH of the b-carotene dispersion was increased to 7.5 by addition of 1 N sodium hydroxide and the samples were incubated in a shaking bath (95 rpm) at 37 C for 2 h to complete the intestinal phase of the in vitro experiments.
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2.5. Experiments on the variables that significantly affect the transfer of b-carotene from dispersions to the aqueous phase In these experiments, the effects of pH, bile extract concentration and pancreatic lipase concentration on the bioaccessibility of b-carotene dispersion were investigated. In each experiment, only one variable was tested, while the other variables were held constant. The experimental design comprised several levels for each variable: (a) for the concentration of bile extract, four levels were used (0, 1.2, 2.4 and 4.8 mg/mL) while the concentration of pancreatin was 0.4 mg/mL, (b) for the concentration of pancreatin, the effect of six levels was investigated (0, 0.1, 0.2, 0.4, 0.6 and 0.8 mg/mL), while the concentration of bile extract was fixed at 2.4 mg/mL, (c) the pH was varied from 2 to 8. 2.6. Determination of the bioaccessibility of b-carotene After in vitro digestion, part of the b-carotene was formulated into water-soluble micelles with the help of the bile extract and pancreatic lipase. The aqueous fraction containing mixed micelles was separated by a method modified from that described by Huo, Ferruzzi, Schwartz, and Failla (2007). Three milliliters of dispersion was taken and isolated by high-speed centrifuge (167 000 g, 35 min, and 10 C) and the b-carotene micelles containing water phase were passed a 0.22 mm membrane. The b-carotene content in micelles was measured by HPLC. The calculation of bioaccessibility is described by Veda, Kamath, Platel, Begum, and Srinivasan (2006). b-Carotene was quantified from the micellar fraction and the residue. The bioaccessibility of b-carotene was expressed as a percentage of this recovered amount as calculated by the following equation: b-carotene recovered in micelle (%) ¼ (b-carotene recovered in the micellar fraction after in vitro digestion)/(b-carotene amount in the original dispersion) 100%. 2.7. Determination of b-carotene concentration 2.7.1. Sample preparation The b-carotene concentration was determined following the method of Tan and Nakajima (2005). Reverse phase solid-phase extraction C18 cartridges (Alltech Associates, Inc., Deerfield, US) were conditioned by washing with 3 mL of methanol and then 5 mL of Milli-Q water prior to use. 0.2 mL of sample was dissolved in 50 g/L aqueous sodium sulfate solution containing 1 mM ethylenediaminetetraacetic acid disodium dihydrate (EDTA). This solution (1 mL) was applied to the conditioned C18 cartridge and the cartridge was continuously washed with deionized water (10 mL), 10% aqueous ethanol (5 mL), followed by elution with acetonitrile/ ethanol (6.5:3.5 v/v, 10 mL). This final elution fraction was collected and used for the determination of b-carotene. 2.7.2. HPLC determination HPLC separation was performed with a Shimadzu liquid chromatography system (SCL-10VAP, Shimadzu Corporation, Kyoto, Japan) equipped with a SPD-10AVvp UVeVIS spectrophotometric detector, a DGU-12A degasser system and a CTO-VAvp column
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oven. Quantitative measurement of b-carotene was carried out at 450 nm. The b-carotene was separated on a 4.6 mm 250 mm, 5 mm silica gel column (Dikma Technologies Inc., Beijing, China) with a mobile phase of acetonitrile/ethanol (6.5:3.5 v/v) at 1.6 mL min1. A 50-mL aliquot of sample was injected. A standard curve (concentration versus peak area) was constructed by linear regression analysis. Injections in triplicate were made at each concentration for standards and samples. The calibration of peak area versus b-carotene concentration was linear in the concentration range 0.05e10.00 mg L1 (R2 ¼ 0.9997, n ¼ 6). 2.8. Statistical analysis All measurements were made in triplicate. Results are expressed as means plus SD. The relationships between two variables were tested for significance by one-way ANOVA. When a significant effect was detected (P < 0.05), means were compared using the post-hoc Duncan test. The statistical analyses were performed using SPSS software version 13.0 for Windows (SPSS Inc., Chicago, IL, USA). 3. Results Several b-carotene dispersions with different particle sizes were produced by high-pressure homogenization or the combination of emulsification and evaporation. Their particle size characteristics are listed in Table 1. The mean particle sizes of dispersions prepared by high-pressure homogenization were between 684 and 1978 nm, with an increase as a result of the various forces induced during homogenization. It has been shown that the emulsificationeevaporation technique can be used to produce b-carotene dispersions with smaller particles in the nanometer range (45 nm at 140 MPa and 60 nm at 100 MPa). The control sample exhibited the largest particle size of 18315 nm. The mean diameters of micelles derived from the two types of b-carotene dispersions are also described in Table 1 and some samples are shown in Fig. 1. 3.1. Effect of bile extract concentration on bioaccessibility Fig. 2 illustrates the effect of bile extract concentration on the transfer of b-carotene from lipid droplets into the aqueous phase. From Fig. 2, it can be seen that absence of bile extract during the intestinal phase of digestion resulted in low detectable levels of bcarotene. It seems that the existence of bile extract might be an important beneficial factor for b-carotene micelle inclusion. Increasing the bile extract concentration significantly increased the bioaccessibility of b-carotene. The quantity of b-carotene in micelles was up to 11.01% when 2.4 mg/mL bile extract was present during digestion and it increased slightly (P < 0.05) at higher concentrations of bile extract. 3.2. Effect of pancreatic lipase concentration on the bioaccessibility Fig. 3 compares the role of pancreatic lipase concentration on the efficiency of b-carotene transfer from lipid droplets into
Table 1 The mean diameters of emulsions, nanodispersions and micelles. Emulsion Pressure (MPa) Mean diameter (nm) AeF a b
140 684 3.5A
Nanodispersion 50 873 1.7B
10 1978 1.7C
0 18315 14.6D
140 49 1.4E
Micelle 100 60 6.3F
140a 59 1.0F
Each value represents the mean SD from three replications. Means within a column with different letters are significantly (P < 0.05) different. Micelles from b-carotene emulsion prepared at 140 MPa. Micelles from b-carotene nanodispersion prepared at 100 MPa.
100b 54 3.0F
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Fig. 3. Percentage of b-carotene transferred from emulsion lipid droplets into the aqueous phase containing mixed micelles, as a function of pancreatic lipase concentration. The emulsion with 0.025 wt% b-carotene was prepared by high-pressure homogenization at 140 MPa and the mean diameter was 684 nm.
Fig. 1. Images of prepared dispersions and solutions containing micelles. No.1, control sample with particle size 18315 nm. No.2, emulsion with particle size 684 nm, prepared by high-pressure homogenization at 140 MPa. No.3, micelles obtained from sample No.2. No.4, nanodispersion with particle size 45 nm, prepared by the homogenizationeevaporation technique at 140 MPa. No. 5, micelles obtained from sample No.4.
micelles. There was a marked difference (P < 0.05) in b-carotene bioaccessibility between presence or absence of pancreatic lipase. At lower concentrations of pancreatic lipase, increasing the concentration provided better availability of b-carotene for transfer into the aqueous fraction. The percentage amounts of b-carotene incorporated in micelles increased significantly (P < 0.05) from 6.7 to 11.42% when the amount of pancreatic lipase added increased to 0.1%. However, further increasing the pancreatic lipase concentration to 0.4% did not markedly change (P > 0.05) the transfer efficiency of b-carotene and excess pancreatic lipase concentration decreased the amount of b-carotene recovered from micelles. The amount of b-carotene incorporated in micelles decreased significantly (P < 0.05) to 8.4% at 0.8 mg/mL pancreatic lipase. 3.3. Effect of pH on bioaccessibility As presented in Fig. 4, the bioaccessibility of b-carotene was significantly (P < 0.05) different when the pH was varied from 2 to 8. Lower transfers at very acidic pH, such as 2 and 3, were observed.
Fig. 2. Percentage of b-carotene transferred from emulsion lipid droplets into the aqueous phase containing mixed micelles, as a function of bile extract concentration. The emulsion with 0.025 wt% b-carotene was prepared by high-pressure homogenization at 140 MPa and the mean diameter was 684 nm.
Higher transfer of b-carotene into micelles occurred at slightly acidic pH conditions ranging from 4 to 6, with the highest bioaccessibility of b-carotene in dispersions occurring at pH 6. Bioaccessibility was impaired markedly (P < 0.05) when the pH was neutral or weakly alkaline. 3.4. Effect of b-carotene dispersion particle size on bioaccessibility To evaluate the effect of b-carotene dispersion particle size on the bioaccessibility of b-carotene, the dispersions were digested with simulated digestion juice. The recovery of b-carotene from the lipid droplets to micelles was assayed, as shown in Fig. 5, which shows that the digestion of b-carotene dispersions with various initial particle sizes could be initiated by adding bile extract and pancreatic lipase. There were significant differences in the formation of b-carotene micelles depending on the various particle sizes of dispersions. In general, the smaller the particle sizes, the higher the efficiency of b-carotene transfer from the dispersion to the aqueous phase. We also prepared b-carotene nanodispersions with particle sizes below 100 nm using the emulsification-evaporation technique described by Tan and Nakajima (2005). The nanodispersions obtained were digested in accordance with the samples above (Fig. 6). Similar to b-carotene dispersions prepared by high-pressure homogenization, b-carotene nanodispersions also showed that smaller particle sizes gave higher efficiency of b-carotene micelle inclusion. However, a similar level of b-carotene was recovered in the aqueous fraction whether or not bile extract and
Fig. 4. Percentage of b-carotene transferred from emulsion lipid droplets to the aqueous phase containing mixed micelles, as a function of pH during intestinal digestion. The emulsion with 0.025 wt% b-carotene was prepared by high-pressure homogenization at 140 MPa and the mean diameter was 684 nm.
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Fig. 5. Percentage of b-carotene transferred from emulsion lipid droplets to the aqueous phase containing mixed micelles, as a function of particle size.
pancreatic lipase was added. When the mean diameter of nanoparticles was decreased to 60 nm and 45 nm, the transfer efficiency of samples during digestion treatment reached 47.5 and 60.6%, respectively, similar to the recovered percentage of samples in the control (absence bile extract and pancreatic lipase during digestion), which were 49.7 and 73.3%, respectively. Further experiments were carried out to detect the effect of bile extract concentration on the bioaccessibility of b-carotene nanodispersions. No significant change in b-carotene micelle inclusion was observed even when the concentration of bile extract was increased 4.8 mg/mL (data not shown). 3.5. Effect of b-carotene concentration on bioaccessibility Dispersions prepared with various b-carotene concentrations were prepared. The transfer of b-carotene into micelles was measured, as shown in Fig. 7, in which the emulsion was prepared at 140 MPa with 0.025 wt % b-carotene and the mean diameter ranged from 600 to 800 nm. From Fig. 7, it was found that the amount of b-carotene partitioned into micelles during digestion was correlated with the concentration in dispersions. However, one-way ANOVA showed that b-carotene concentration in dispersions had a significant effect (P < 0.05) on the concentration of bcarotene that could be transferred into the aqueous phase after digestion. The ratio of b-carotene incorporated into micelles decreased significantly (P < 0.05) when b-carotene concentration increased from 0.025 to 0.05 wt% and then tended to remain stable when b-carotene concentration was further increased.
Fig. 6. Percentage of b-carotene transferred from nanodispersion into the aqueous phase containing mixed micelles, (-) digestion treatment, with 2.4 mg/mL bile extract and 0.4 mg/mL pancreatic lipase in digestive juice. (,) control, with no bile extract and pancreatic lipase. The nanodispersions with 0.025 wt% b-carotene were prepared using the homogenization-evaporation technique at both 100 MPa and 140 MPa, and the mean diameters were 60 and 45 nm, respectively.
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Fig. 7. Percentage of b-carotene transferred from emulsion lipid droplets into the aqueous phase containing mixed micelles, as a function of b-carotene concentration in initial dispersions and in the aqueous phase after digestion.
4. Discussion The main purpose of the present work was to investigate the main factors affecting the transfer of b-carotene from lipid droplets into an aqueous medium. Current knowledge on lipid digestion and on the physicochemical properties of b-carotene has revealed that several factors may affect this transfer. First, we observed a significant increase when the bile extract concentration exceeded a limit between 0 and 2.4 mg/mL and then tended to remain constant (Fig. 2). This suggested that forming b-carotene micelles required the participation of bile extract. Bile salt originating from the liver is a surfactant, which can facilitate the emulsification of lipids by adsorbing to the droplet surfaces to reduce the surface tension (Beyssriat et al., 2006). Therefore, the significant increase in transfer efficiency observed might be attributed to the solubilization of b-carotene in micelles, aided by bile extract when the bile extract concentration reached the critical micellar concentration (CMC) (Tyssandier et al., 2001). A further increase in bile extract concentration would not add to the transfer of b-carotene significantly (P > 0.05). It is possible that at the higher concentration, the surface of b-carotene particles was saturated with bile extract (Chu, Ichikawa, Kanafusa, & Nakajima, 2007). At that point the bile extract concentration is not the key factor controlling transfer but digestion time may play an important role in increasing transfer efficiency. The results show that transfer reached the maximum level when the pancreatic lipase concentration was 0.4 mg/mL which is the closest to the simulated physiological concentration. In the duodenum, the pancreatic lipase complex may bind to the lipid droplet surfaces, where it hydrolyzes triglyceride into free fatty acids (McClements, Deckerd, Park, & Weiss, 2008). The increase of free fatty acid (FFA), in a certain range, will promote the solubility of b-carotene in the aqueous phase, because of its emulsifying properties (Beyssriat et al., 2006). However, further increasing the FFA concentration would acidify the solution system, which might lead to b-carotene degradation and micelle precipitation (Tyssandier et al., 2001). To investigate the effect of pH on the transfer, we chose to vary the pH from acid pH to the weakly alkaline region based on the range that can be expected in the GI tract (Knarreborg, Jensen, & Engberg, 2003). Our results showed that the maximum transfer was obtained at pH 6. Furthermore, the transfer was much lower for the strongly acidic pH (between 2 and 3) and weak alkaline pH (from 7 to 8) than for less acidic pH (between 4 and 5). Because the most efficient pH for pancreatic enzymes is around 6 (McClements et al., 2008), it was supposed that the present results were related to the activity of pancreatic lipase under various pH values. This can be also explained by the fact that at very acidic pH, the micelles,
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which are the only structures that can solubilize b-carotene in the aqueous phase, were precipitated (Tyssandier et al., 2001). It is necessary to construct a reaction system consisting of bcarotene dispersions with various particle sizes to study the effect of particle size on b-carotene transfer. The results obtained from the preparation of b-carotene dispersions showed that, under the conditions of the present study, the intensity of the high shear forces produced during the homogenization process determined the particle size of b-carotene dispersions. As the pressure of the homogenizer was increased, the size of the dispersed droplets decreased, as a result of the various forces induced in the homogenizer. As expected, the results demonstrate that partitioning of bcarotene into micelles during the simulated digestion was enhanced by incubating dispersions having smaller particle sizes with the digestion juice. The micelle formation efficiency of bcarotene was significantly influenced by the initial particle size of the dispersion used (Fig. 5). Indeed, the specific surface area of the lipid droplets is determined by the particle size under the same disperse phase volume fraction (McClements et al., 2008). It is possible that the smaller the mean particle size of the b-carotene dispersion was, the larger the specific surface area exposed. The larger specific surface area was available for pancreatic lipases to attach more easily (Armand et al., 1992) and, therefore, a higher transfer of b-carotene resulted. Because smaller particle sizes could be beneficial to the transfer of b-carotene from dispersion to the aqueous phase, it is reasonable to suppose that further increases in the transfer might be achieved by micronizing the size of the lipid droplets. The current work has shown that much smaller particle sizes of b-carotene dispersions at nano-scaled sizes contributed to a higher efficiency of micelle formation, similar to b-carotene dispersions prepared by highpressure homogenization (Fig. 6). Meanwhile, it is interesting to note that a similar level of b-carotene micelle formation was observed for b-carotene nanodispersions, whether or not bile extract and pancreatic lipase were present, suggesting that the bile extract and pancreatic lipase played different roles in the digestion of the two dispersion systems. It was reported that the average size of bile salt aggregates ranges from 4 nm for bile salt micelles to 60 nm for bile salt vesicles (Hernell, Staggers, & Carey, 1990). The sizes of b-carotene micelles in our work were measured to be between 50 and 60 nm. These results suggest that the initial lipid drops in dispersions might be disrupted to form smaller droplets under the action of bile extract and pancreatic lipase for dispersions with larger particle sizes. However, particles smaller than 60 nm in the b-carotene nanodispersion could potentially be mixed with micelles, which might contribute to the high efficiency of micelle formation in the present investigation. It has been reported that bile salts were able to emulsify bcarotene dissolved in triglycerides but bile salt micelles cannot solubilize pure b-carotene (Acosta, 2008). Furthermore, an increase in the uptake of hydrophobic components through a viable route using nanostructured delivery systems has been also documented (Desai, Labhasetwar, Amidon, & Levy, 1996; Jani, Halbert, Langridge, & Florence, 1990). Therefore, the actual metabolic pathway that bcarotene in nano-sized dispersions undergoes should be investigated to promote the practical application of the b-carotene micronization technique. 5. Conclusion In conclusion, we have identified the effect of the addition of bile extract and pancreatic lipase, pH, b-carotene concentration and dispersion particle size on the bioaccessibility of b-carotene dispersions. Results indicate that the initial particle size of the dispersion significantly influences the bioaccessibility of b-
carotene. The smaller the particle size, the higher the transfer of bcarotene, suggesting that controlling particle size has potential for improving the bioavailability of b-carotene. Meanwhile, it is interesting to note that the transfer efficiency of b-carotene from the two nanodispersions used in the present work was significant higher than in micro-scaled dispersions, and was not affected by the presence of bile salt and pancreatic lipase. Therefore, it is important to consider the level of “deficiency” of this nutrient when assessing the effectiveness of nanoparticle carriers in the future. Acknowledgements This research was carried out with the financial support of National Science Foundation of China (Project No. 20976187 ) and National Keytechnologies R&D Program (No. 2011BAD23B04). The corresponding author thanks the support from Program for New Century Excellent Talents in University (NCET-09-0741). Reference Acosta, E. (2008). 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(2001). Influence of physicochemical properties on dissolution of drugs in the gastrointestinal tract. Advanced Drug Delivery Reviews, 46, 75e87. Huo, T., Ferruzzi, M. G., Schwartz, S. J., & Failla, M. L. (2007). Impact of fatty acyl composition and quantity of triglycerides on bioaccessibility of dietary carotenoids. Agricultural and Food Chemistry, 55, 8950e8957. Iwase, H. (2002). Simultaneous sample preparation for high-performance liquid chromatographic determination of vitamin A and b-carotene in emulsified nutritional supplements after solid-phase extraction. Analytica Chimica Acta, 463, 21e29. Jani, P., Halbert, G. W., Langridge, J., & Florence, A. T. (1990). Nanoparticle uptake by the rat gastrointestinal mucosa: quantitation and particle size dependency. Journal of Pharmacy and Pharmacology, 42, 821e826. Knarreborg, A., Jensen, S., & Engberg, R. M. (2003). Pancreatic lipase activity as influenced by unconjugated bile acids and pH, measured in vitro and in vivo. Journal of Nutritional Biochemistry, 14, 259e265. McClements, D. J., Deckerd, E. A., Park, Y., & Weiss, J. (2008). Designing food structure to control stability, digestion, release and absorption of lipophilic food components. Food Biophysics, 3, 219e228. Ribeiro, S. H., Chu, B. S., Ichikawa, S., & Nakajima, M. (2008). Preparation of nanodispersions containing of b-carotene by solvent displacement method. Food Hydrocolloids, 22, 12e17. Tan, C. P., & Nakajima, M. (2005). b-Carotene nanodispersion: preparation, characterization and stability evaluation. Food Chemistry, 92, 661e671. Thakkar, S. K., Maziya-Dixon, B., Dixon, A. G. O., & Failla, M. (2007). b-Carotene micellarization during in vitro digestion and uptake by caco-2 cells is directly proportional to b-carotene content in different genotypes of Cassava1,2. The Journal of Nutrition, 137, 2229e2233. Tyssandier, V., Lyan, B., & Borel, P. (2001). Main factors governing the transfer of carotenoids from emulsion lipid droplets to micelles. Biochimica et Biophysica Acta, 1533, 285e292. Van Het Hof, K. H., West, C. E., Weststrat, J. A., & Hautvast, J. G. A. J. (2000). Dietary factors that affect the bioavailability of carotenoids. Journal of Nutrient, 130, 503e506.
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Preliminary study into the factors modulating b-carotene micelle formation in dispersions using an in vitro digestion model Pan Wang a, Hai-Jie Liu a, Xue-Ying Mei a, Mitsutoshi Nakajima b, Li-Jun Yin a, b, * a
Key Laboratory of Functional Dairy Science of Beijing and Ministry of Education, College of Food Science and Nutritional Engineering, China Agricultural University, Qinghua East Road, Beijing, 100083, China b Graduate School of Life Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 August 2010 Accepted 24 November 2010
b-Carotene is an active compound associated with prevention of heart disease, cancer and cataracts. For absorption in vivo, b-carotene must be incorporated in mixed micelles. Micelle formulation varies widely and depends on various factors. The aim of this study was to identify and study the main factors governing the bioaccessibility of b-carotene incorporated into dispersions, using an in vitro digestion model. b-Carotene dispersions were prepared by high-pressure homogenization or by combining emulsification and evaporation. The average particle sizes of the dispersions obtained ranged from 45 to 18315 nm. Results show that the concentration of b-carotene, bile extract and pancreatic lipase, pH, and the particle size of the dispersions significantly affected the transfer of b-carotene from dispersions into micelles. The transfer of b-carotene was inversely related to the particle size and the concentration of bile extract and was highest at pH 6 and 0.4 mg/mL pancreatic lipase. Bile salt played different roles depending on the particle sizes of the dispersion. When the mean diameter of b-carotene particle was below 100 nm, the addition of bile extract and pancreatic lipase did not significantly affect bioaccessibility of b-carotene passing through in vitro digestion model. At larger particle sizes, the transfer efficiency of b-carotene increased with bile extract concentration. The outcomes suggest that there is potential to improve the bioavailability of b-carotene by micronizing lipid droplets. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: b-Carotene dispersions Micelle Bioaccessibility In vitro digestion Particle size
1. Introduction Carotenoids are one of the most important classes of natural pigments because of their wide distribution in plant tissues, structural diversity, and numerous functions. In addition to provitamin A activity, carotenoids have recently been implicated in protection against or prevention of serious human health disorders such as cancer, heart disease, macular degeneration, and cataracts (Castenmiller & West, 1998; Iwase, 2002). Amongst the carotenoid pigments, b-carotene can provide the highest vitamin A activity. Studies of b-carotene absorption in humans have been carried out for many years. Generally, it appears that b-carotene absorption is not very efficient and is highly variable. The high hydrophobicity of b-carotene is reported to cause low solubility in aqueous systems and consequent poor uptake in the body, which limits its use in food formulations. b-Carotene absorption through the lymphatic system mainly occurs by the disruption of the food matrix, the
* Corresponding author. Tel./fax: þ86 10 62737424. E-mail address: [email protected] (M. Nakajima). 0268-005X/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2010.11.018
release of b-carotene, emulsification into lipid droplets in the stomach, and incorporation into mixed micelles. After micelle formation, b-carotene is absorbed by the lymphatic system in the small intestine (Thakkar, Maziya-Dixon, Dixon, & Failla, 2007; Van Het Hof, West, Weststrat, & Hautvast, 2000; Yonekura & Nagao, 2007). Because b-carotene cannot be absorbed directly from the food matrix, the transfer from the food matrix to micelles, which can be absorbed, is probably the essential step for its absorption. It has therefore been presumed that the transfer efficiency might directly affect the adsorption of b-carotene (Tyssandier, Lyan, & Borel, 2001). It had been reported that the bioavailability of b-carotene is affected by many factors, i.e. the state of the food matrix, food processing and the conditions in the gastrointestinal (GI) tract (Castenmiller & West, 1998). Yonekura and Nagao (2007) reported that the bioavailability of carotenoids from supplements is usually higher than of those embedded in the matrix of fruits and vegetables, because of the higher degree of food matrix disruption in the former. The bioavailability of carotenoids without processing is regarded as less than 10% from raw vegetables (Ribeiro, Chu, Ichikawa, & Nakajima, 2008). Food processing, including heat,
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mechanical and enzymatic treatments, may facilitate the disruption of the cell wall and organelles, releasing carotenoids from the food matrix and promoting their dispersion in the GI tract (Yonekura & Nagao, 2007). The rate and extent of lipid digestion within the stomach and small intestine may depend on how fast the GI tract can break down the surrounding matrix because the lipids may have to be exposed before they can be digested by lipases (Chen, Remondetto, & Subirade, 2006). Thus, the bioaccessibility of b-carotene, when regarded as a type of nonsaponifiable lipid, will be determined by the state of food matrix. Recent progress in nanotechnology has also gained considerable attention for improving the water solubility and bioavailability of lipophilic bioactive compounds. Ultra fine particles can reduce the inherent limitations of slow and incomplete dissolution of functional lipids due to their larger surface area and higher dissolution pressure (Horter & Dressman, 2001). In previous studies, we prepared b-carotene lipid droplets with a minimum size of approximately 50 nm, which exhibited improved solubility and stability (Yin, Chu, Kobayashi, & Nakajima, 2009). Today, much is known about how b-carotene in natural food matrices moves into emulsified lipid droplets in the diet. However, in processed food matrices, the factors affecting the transfer of b-carotene from dispersions into micelles have only been partially investigated and little is known about the metabolism and absorption of these bcarotene dispersions in humans. The aims of this study were to identify how the size characteristics of b-carotene dispersions and the conditions in a simulated digestion affect the bioaccessibility of b-carotene and to assess the effect of the physiological variations in these factors on the transfer efficiency. Information on the bioaccessibility of b-carotene in dispersions is expected to contribute to the development of active compounds in dispersions with satisfactory control and release properties.
2. Materials and methods 2.1. Materials The emulsifier, decaglycerol monolaurate (ML750), was supplied by Sakamoto Yakuhin Kogyo Co, Ltd (Osaka, Japan). bCarotene was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Soybean oil was purchased from a local supermarket and used without further purification. Standard b-carotene (Type II, 95%) was purchased from SigmaeAldrich Chemical Company (St Louis, MO, USA). The digestive juice was prepared using porcine bile extract (batch 058K0066, EC 232-369-0, Sigma) and porcine pancreatic lipase, type II (batch 096K0747, EC 232-6199, Sigma). HPLC-grade acetonitrile was purchased from CNW Technology GmbH (Germany). HPLC-grade ethanol absolute was purchased from the Tianjin Fine Chemical Engineering Division (Tianjin, China). Deionized water, purified by a Milli-Q Organex system (Millipore, Bedford, MA), was used for preparation of the aqueous phase. All chemicals were of analytical grade. 2.2. Preparation of b-carotene dispersions
b-Carotene dispersions were prepared by modifying the method described by Yin, Kobayashi, and Nakajima (2008). b-Carotene (0.25 wt%) was dissolved in soybean oil (40 C) as the dispersed phase. The organic solution was poured into the continuous phase containing 1% (w/w) ML750 in 100 mM NaHCO3 buffer (pH 7) to give an organic-aqueous phase volume ratio of 1:9. The premix was homogenized using a conventional homogenizer (Ultra TurraxÒ T25 basic, IKA AG, Staufen, Germany) at 5000 rpm for 5 min,
followed by high-pressure homogenization (NS1001L Panda 2K High-Pressure Homogenizer, GEA Niro Soavi, Italy) in a single pass at 140 MPa to obtain a final concentration of 10 wt% soybean oil, 0.025 wt% b-carotene, and 0.9 wt% ML750. Several batches were prepared, using the technique described above, by varying the homogenization pressure (10 MPa and 50 MPa) and b-carotene concentrations (0.01, 0.05 and 0.1 wt%). A sample without homogenization, recorded as 0 MPa, was used as a control. The pH value of prepared dispersions was about 7. To obtain b-carotene dispersions with smaller particle sizes, they were also prepared by the emulsificationeevaporation technique described by Tan and Nakajima (2005). b-Carotene (0.25 wt%) was dissolved and dispersed in hexane. After preparing b-carotene oil-in-water dispersions by homogenizing at either 100 MPa or 140 MPa, hexane was removed by rotary evaporation (SENCO-R Series rotary evaporator, Shanghai Seekic Co., Ltd, Shanghai, China) under reduced pressure (0.25 bar and 40 C). 2.3. Particle size analysis The mean particle sizes of b-carotene dispersions were measured using a laser diffraction particle size analyzer with a lower measuring limit of 40 nm (LS 320, Beckman Coulter, Inc., FL, USA). The experiments were carried out on undiluted suspensions after preparation and all measurements were carried out in triplicate. The particle size of the prepared b-carotene dispersions was described by the surface-weighted mean diameter (d3, 2). Measurement of the mean particle diameter of the nanodispersions was carried out using a dynamic light scattering particle size analyzer with a measuring range from 0.6 nm to 6 mm (Zetasizer Nano ZS, Malvern Instruments Ltd., Worcestershire, UK). A refractive index for b-carotene of 1.47 and water of 1.33 was used in the calculation of particle sizes (Bialek-Bylka, Jazurek, Dedic, Hala, & Skrzypczak, 2003). The particle concentration in the sample was diluted to about 0.005 wt% with Milli-Q water prior to analysis to avoid multiple-scattering affects during measurement. The final particle diameter was calculated from the average of at least three measurements. Measurement of the particle size was also carried out on the supernatant of the nanodispersions after centrifuging the samples at 10 000 g for 30 min. 2.4. In vitro digestion model used to measure the digestion of b-carotene dispersions The in vitro digestion model described by Beyssriat, Decker and McClements (2006) was used to mimic the physical and chemical conditions prevailing in the stomach and duodenum. b-Carotene dispersion was prepared as described above and then stored for approximately 1 h at room temperature. Thirty milliliters of the b-carotene dispersion was acidified to pH 2.0 with 1 M HCL and incubated for 1 h at 37 C with shaking at 95 rpm in an incubator shaker (Model HZQ-X100, Peiying Co., Shanghai, China). After the adjustment of pH, a bile extract and pancreatin mixture of 7.5 mL in 100 mmol/L sodium bicarbonate solution was added. Note that, apart from during the variables experiments (see Section 2.5), the pH of the acidified b-carotene dispersion was raised to 5.3 by adding 0.9 M sodium bicarbonate and the final concentrations of pancreatin and bile extract in the mixture were 0.4 mg/mL and 2.4 mg/mL respectively, in all experiments. Finally, the pH of the b-carotene dispersion was increased to 7.5 by addition of 1 N sodium hydroxide and the samples were incubated in a shaking bath (95 rpm) at 37 C for 2 h to complete the intestinal phase of the in vitro experiments.
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2.5. Experiments on the variables that significantly affect the transfer of b-carotene from dispersions to the aqueous phase In these experiments, the effects of pH, bile extract concentration and pancreatic lipase concentration on the bioaccessibility of b-carotene dispersion were investigated. In each experiment, only one variable was tested, while the other variables were held constant. The experimental design comprised several levels for each variable: (a) for the concentration of bile extract, four levels were used (0, 1.2, 2.4 and 4.8 mg/mL) while the concentration of pancreatin was 0.4 mg/mL, (b) for the concentration of pancreatin, the effect of six levels was investigated (0, 0.1, 0.2, 0.4, 0.6 and 0.8 mg/mL), while the concentration of bile extract was fixed at 2.4 mg/mL, (c) the pH was varied from 2 to 8. 2.6. Determination of the bioaccessibility of b-carotene After in vitro digestion, part of the b-carotene was formulated into water-soluble micelles with the help of the bile extract and pancreatic lipase. The aqueous fraction containing mixed micelles was separated by a method modified from that described by Huo, Ferruzzi, Schwartz, and Failla (2007). Three milliliters of dispersion was taken and isolated by high-speed centrifuge (167 000 g, 35 min, and 10 C) and the b-carotene micelles containing water phase were passed a 0.22 mm membrane. The b-carotene content in micelles was measured by HPLC. The calculation of bioaccessibility is described by Veda, Kamath, Platel, Begum, and Srinivasan (2006). b-Carotene was quantified from the micellar fraction and the residue. The bioaccessibility of b-carotene was expressed as a percentage of this recovered amount as calculated by the following equation: b-carotene recovered in micelle (%) ¼ (b-carotene recovered in the micellar fraction after in vitro digestion)/(b-carotene amount in the original dispersion) 100%. 2.7. Determination of b-carotene concentration 2.7.1. Sample preparation The b-carotene concentration was determined following the method of Tan and Nakajima (2005). Reverse phase solid-phase extraction C18 cartridges (Alltech Associates, Inc., Deerfield, US) were conditioned by washing with 3 mL of methanol and then 5 mL of Milli-Q water prior to use. 0.2 mL of sample was dissolved in 50 g/L aqueous sodium sulfate solution containing 1 mM ethylenediaminetetraacetic acid disodium dihydrate (EDTA). This solution (1 mL) was applied to the conditioned C18 cartridge and the cartridge was continuously washed with deionized water (10 mL), 10% aqueous ethanol (5 mL), followed by elution with acetonitrile/ ethanol (6.5:3.5 v/v, 10 mL). This final elution fraction was collected and used for the determination of b-carotene. 2.7.2. HPLC determination HPLC separation was performed with a Shimadzu liquid chromatography system (SCL-10VAP, Shimadzu Corporation, Kyoto, Japan) equipped with a SPD-10AVvp UVeVIS spectrophotometric detector, a DGU-12A degasser system and a CTO-VAvp column
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oven. Quantitative measurement of b-carotene was carried out at 450 nm. The b-carotene was separated on a 4.6 mm 250 mm, 5 mm silica gel column (Dikma Technologies Inc., Beijing, China) with a mobile phase of acetonitrile/ethanol (6.5:3.5 v/v) at 1.6 mL min1. A 50-mL aliquot of sample was injected. A standard curve (concentration versus peak area) was constructed by linear regression analysis. Injections in triplicate were made at each concentration for standards and samples. The calibration of peak area versus b-carotene concentration was linear in the concentration range 0.05e10.00 mg L1 (R2 ¼ 0.9997, n ¼ 6). 2.8. Statistical analysis All measurements were made in triplicate. Results are expressed as means plus SD. The relationships between two variables were tested for significance by one-way ANOVA. When a significant effect was detected (P < 0.05), means were compared using the post-hoc Duncan test. The statistical analyses were performed using SPSS software version 13.0 for Windows (SPSS Inc., Chicago, IL, USA). 3. Results Several b-carotene dispersions with different particle sizes were produced by high-pressure homogenization or the combination of emulsification and evaporation. Their particle size characteristics are listed in Table 1. The mean particle sizes of dispersions prepared by high-pressure homogenization were between 684 and 1978 nm, with an increase as a result of the various forces induced during homogenization. It has been shown that the emulsificationeevaporation technique can be used to produce b-carotene dispersions with smaller particles in the nanometer range (45 nm at 140 MPa and 60 nm at 100 MPa). The control sample exhibited the largest particle size of 18315 nm. The mean diameters of micelles derived from the two types of b-carotene dispersions are also described in Table 1 and some samples are shown in Fig. 1. 3.1. Effect of bile extract concentration on bioaccessibility Fig. 2 illustrates the effect of bile extract concentration on the transfer of b-carotene from lipid droplets into the aqueous phase. From Fig. 2, it can be seen that absence of bile extract during the intestinal phase of digestion resulted in low detectable levels of bcarotene. It seems that the existence of bile extract might be an important beneficial factor for b-carotene micelle inclusion. Increasing the bile extract concentration significantly increased the bioaccessibility of b-carotene. The quantity of b-carotene in micelles was up to 11.01% when 2.4 mg/mL bile extract was present during digestion and it increased slightly (P < 0.05) at higher concentrations of bile extract. 3.2. Effect of pancreatic lipase concentration on the bioaccessibility Fig. 3 compares the role of pancreatic lipase concentration on the efficiency of b-carotene transfer from lipid droplets into
Table 1 The mean diameters of emulsions, nanodispersions and micelles. Emulsion Pressure (MPa) Mean diameter (nm) AeF a b
140 684 3.5A
Nanodispersion 50 873 1.7B
10 1978 1.7C
0 18315 14.6D
140 49 1.4E
Micelle 100 60 6.3F
140a 59 1.0F
Each value represents the mean SD from three replications. Means within a column with different letters are significantly (P < 0.05) different. Micelles from b-carotene emulsion prepared at 140 MPa. Micelles from b-carotene nanodispersion prepared at 100 MPa.
100b 54 3.0F
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Fig. 3. Percentage of b-carotene transferred from emulsion lipid droplets into the aqueous phase containing mixed micelles, as a function of pancreatic lipase concentration. The emulsion with 0.025 wt% b-carotene was prepared by high-pressure homogenization at 140 MPa and the mean diameter was 684 nm.
Fig. 1. Images of prepared dispersions and solutions containing micelles. No.1, control sample with particle size 18315 nm. No.2, emulsion with particle size 684 nm, prepared by high-pressure homogenization at 140 MPa. No.3, micelles obtained from sample No.2. No.4, nanodispersion with particle size 45 nm, prepared by the homogenizationeevaporation technique at 140 MPa. No. 5, micelles obtained from sample No.4.
micelles. There was a marked difference (P < 0.05) in b-carotene bioaccessibility between presence or absence of pancreatic lipase. At lower concentrations of pancreatic lipase, increasing the concentration provided better availability of b-carotene for transfer into the aqueous fraction. The percentage amounts of b-carotene incorporated in micelles increased significantly (P < 0.05) from 6.7 to 11.42% when the amount of pancreatic lipase added increased to 0.1%. However, further increasing the pancreatic lipase concentration to 0.4% did not markedly change (P > 0.05) the transfer efficiency of b-carotene and excess pancreatic lipase concentration decreased the amount of b-carotene recovered from micelles. The amount of b-carotene incorporated in micelles decreased significantly (P < 0.05) to 8.4% at 0.8 mg/mL pancreatic lipase. 3.3. Effect of pH on bioaccessibility As presented in Fig. 4, the bioaccessibility of b-carotene was significantly (P < 0.05) different when the pH was varied from 2 to 8. Lower transfers at very acidic pH, such as 2 and 3, were observed.
Fig. 2. Percentage of b-carotene transferred from emulsion lipid droplets into the aqueous phase containing mixed micelles, as a function of bile extract concentration. The emulsion with 0.025 wt% b-carotene was prepared by high-pressure homogenization at 140 MPa and the mean diameter was 684 nm.
Higher transfer of b-carotene into micelles occurred at slightly acidic pH conditions ranging from 4 to 6, with the highest bioaccessibility of b-carotene in dispersions occurring at pH 6. Bioaccessibility was impaired markedly (P < 0.05) when the pH was neutral or weakly alkaline. 3.4. Effect of b-carotene dispersion particle size on bioaccessibility To evaluate the effect of b-carotene dispersion particle size on the bioaccessibility of b-carotene, the dispersions were digested with simulated digestion juice. The recovery of b-carotene from the lipid droplets to micelles was assayed, as shown in Fig. 5, which shows that the digestion of b-carotene dispersions with various initial particle sizes could be initiated by adding bile extract and pancreatic lipase. There were significant differences in the formation of b-carotene micelles depending on the various particle sizes of dispersions. In general, the smaller the particle sizes, the higher the efficiency of b-carotene transfer from the dispersion to the aqueous phase. We also prepared b-carotene nanodispersions with particle sizes below 100 nm using the emulsification-evaporation technique described by Tan and Nakajima (2005). The nanodispersions obtained were digested in accordance with the samples above (Fig. 6). Similar to b-carotene dispersions prepared by high-pressure homogenization, b-carotene nanodispersions also showed that smaller particle sizes gave higher efficiency of b-carotene micelle inclusion. However, a similar level of b-carotene was recovered in the aqueous fraction whether or not bile extract and
Fig. 4. Percentage of b-carotene transferred from emulsion lipid droplets to the aqueous phase containing mixed micelles, as a function of pH during intestinal digestion. The emulsion with 0.025 wt% b-carotene was prepared by high-pressure homogenization at 140 MPa and the mean diameter was 684 nm.
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Fig. 5. Percentage of b-carotene transferred from emulsion lipid droplets to the aqueous phase containing mixed micelles, as a function of particle size.
pancreatic lipase was added. When the mean diameter of nanoparticles was decreased to 60 nm and 45 nm, the transfer efficiency of samples during digestion treatment reached 47.5 and 60.6%, respectively, similar to the recovered percentage of samples in the control (absence bile extract and pancreatic lipase during digestion), which were 49.7 and 73.3%, respectively. Further experiments were carried out to detect the effect of bile extract concentration on the bioaccessibility of b-carotene nanodispersions. No significant change in b-carotene micelle inclusion was observed even when the concentration of bile extract was increased 4.8 mg/mL (data not shown). 3.5. Effect of b-carotene concentration on bioaccessibility Dispersions prepared with various b-carotene concentrations were prepared. The transfer of b-carotene into micelles was measured, as shown in Fig. 7, in which the emulsion was prepared at 140 MPa with 0.025 wt % b-carotene and the mean diameter ranged from 600 to 800 nm. From Fig. 7, it was found that the amount of b-carotene partitioned into micelles during digestion was correlated with the concentration in dispersions. However, one-way ANOVA showed that b-carotene concentration in dispersions had a significant effect (P < 0.05) on the concentration of bcarotene that could be transferred into the aqueous phase after digestion. The ratio of b-carotene incorporated into micelles decreased significantly (P < 0.05) when b-carotene concentration increased from 0.025 to 0.05 wt% and then tended to remain stable when b-carotene concentration was further increased.
Fig. 6. Percentage of b-carotene transferred from nanodispersion into the aqueous phase containing mixed micelles, (-) digestion treatment, with 2.4 mg/mL bile extract and 0.4 mg/mL pancreatic lipase in digestive juice. (,) control, with no bile extract and pancreatic lipase. The nanodispersions with 0.025 wt% b-carotene were prepared using the homogenization-evaporation technique at both 100 MPa and 140 MPa, and the mean diameters were 60 and 45 nm, respectively.
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Fig. 7. Percentage of b-carotene transferred from emulsion lipid droplets into the aqueous phase containing mixed micelles, as a function of b-carotene concentration in initial dispersions and in the aqueous phase after digestion.
4. Discussion The main purpose of the present work was to investigate the main factors affecting the transfer of b-carotene from lipid droplets into an aqueous medium. Current knowledge on lipid digestion and on the physicochemical properties of b-carotene has revealed that several factors may affect this transfer. First, we observed a significant increase when the bile extract concentration exceeded a limit between 0 and 2.4 mg/mL and then tended to remain constant (Fig. 2). This suggested that forming b-carotene micelles required the participation of bile extract. Bile salt originating from the liver is a surfactant, which can facilitate the emulsification of lipids by adsorbing to the droplet surfaces to reduce the surface tension (Beyssriat et al., 2006). Therefore, the significant increase in transfer efficiency observed might be attributed to the solubilization of b-carotene in micelles, aided by bile extract when the bile extract concentration reached the critical micellar concentration (CMC) (Tyssandier et al., 2001). A further increase in bile extract concentration would not add to the transfer of b-carotene significantly (P > 0.05). It is possible that at the higher concentration, the surface of b-carotene particles was saturated with bile extract (Chu, Ichikawa, Kanafusa, & Nakajima, 2007). At that point the bile extract concentration is not the key factor controlling transfer but digestion time may play an important role in increasing transfer efficiency. The results show that transfer reached the maximum level when the pancreatic lipase concentration was 0.4 mg/mL which is the closest to the simulated physiological concentration. In the duodenum, the pancreatic lipase complex may bind to the lipid droplet surfaces, where it hydrolyzes triglyceride into free fatty acids (McClements, Deckerd, Park, & Weiss, 2008). The increase of free fatty acid (FFA), in a certain range, will promote the solubility of b-carotene in the aqueous phase, because of its emulsifying properties (Beyssriat et al., 2006). However, further increasing the FFA concentration would acidify the solution system, which might lead to b-carotene degradation and micelle precipitation (Tyssandier et al., 2001). To investigate the effect of pH on the transfer, we chose to vary the pH from acid pH to the weakly alkaline region based on the range that can be expected in the GI tract (Knarreborg, Jensen, & Engberg, 2003). Our results showed that the maximum transfer was obtained at pH 6. Furthermore, the transfer was much lower for the strongly acidic pH (between 2 and 3) and weak alkaline pH (from 7 to 8) than for less acidic pH (between 4 and 5). Because the most efficient pH for pancreatic enzymes is around 6 (McClements et al., 2008), it was supposed that the present results were related to the activity of pancreatic lipase under various pH values. This can be also explained by the fact that at very acidic pH, the micelles,
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which are the only structures that can solubilize b-carotene in the aqueous phase, were precipitated (Tyssandier et al., 2001). It is necessary to construct a reaction system consisting of bcarotene dispersions with various particle sizes to study the effect of particle size on b-carotene transfer. The results obtained from the preparation of b-carotene dispersions showed that, under the conditions of the present study, the intensity of the high shear forces produced during the homogenization process determined the particle size of b-carotene dispersions. As the pressure of the homogenizer was increased, the size of the dispersed droplets decreased, as a result of the various forces induced in the homogenizer. As expected, the results demonstrate that partitioning of bcarotene into micelles during the simulated digestion was enhanced by incubating dispersions having smaller particle sizes with the digestion juice. The micelle formation efficiency of bcarotene was significantly influenced by the initial particle size of the dispersion used (Fig. 5). Indeed, the specific surface area of the lipid droplets is determined by the particle size under the same disperse phase volume fraction (McClements et al., 2008). It is possible that the smaller the mean particle size of the b-carotene dispersion was, the larger the specific surface area exposed. The larger specific surface area was available for pancreatic lipases to attach more easily (Armand et al., 1992) and, therefore, a higher transfer of b-carotene resulted. Because smaller particle sizes could be beneficial to the transfer of b-carotene from dispersion to the aqueous phase, it is reasonable to suppose that further increases in the transfer might be achieved by micronizing the size of the lipid droplets. The current work has shown that much smaller particle sizes of b-carotene dispersions at nano-scaled sizes contributed to a higher efficiency of micelle formation, similar to b-carotene dispersions prepared by highpressure homogenization (Fig. 6). Meanwhile, it is interesting to note that a similar level of b-carotene micelle formation was observed for b-carotene nanodispersions, whether or not bile extract and pancreatic lipase were present, suggesting that the bile extract and pancreatic lipase played different roles in the digestion of the two dispersion systems. It was reported that the average size of bile salt aggregates ranges from 4 nm for bile salt micelles to 60 nm for bile salt vesicles (Hernell, Staggers, & Carey, 1990). The sizes of b-carotene micelles in our work were measured to be between 50 and 60 nm. These results suggest that the initial lipid drops in dispersions might be disrupted to form smaller droplets under the action of bile extract and pancreatic lipase for dispersions with larger particle sizes. However, particles smaller than 60 nm in the b-carotene nanodispersion could potentially be mixed with micelles, which might contribute to the high efficiency of micelle formation in the present investigation. It has been reported that bile salts were able to emulsify bcarotene dissolved in triglycerides but bile salt micelles cannot solubilize pure b-carotene (Acosta, 2008). Furthermore, an increase in the uptake of hydrophobic components through a viable route using nanostructured delivery systems has been also documented (Desai, Labhasetwar, Amidon, & Levy, 1996; Jani, Halbert, Langridge, & Florence, 1990). Therefore, the actual metabolic pathway that bcarotene in nano-sized dispersions undergoes should be investigated to promote the practical application of the b-carotene micronization technique. 5. Conclusion In conclusion, we have identified the effect of the addition of bile extract and pancreatic lipase, pH, b-carotene concentration and dispersion particle size on the bioaccessibility of b-carotene dispersions. Results indicate that the initial particle size of the dispersion significantly influences the bioaccessibility of b-
carotene. The smaller the particle size, the higher the transfer of bcarotene, suggesting that controlling particle size has potential for improving the bioavailability of b-carotene. Meanwhile, it is interesting to note that the transfer efficiency of b-carotene from the two nanodispersions used in the present work was significant higher than in micro-scaled dispersions, and was not affected by the presence of bile salt and pancreatic lipase. Therefore, it is important to consider the level of “deficiency” of this nutrient when assessing the effectiveness of nanoparticle carriers in the future. Acknowledgements This research was carried out with the financial support of National Science Foundation of China (Project No. 20976187 ) and National Keytechnologies R&D Program (No. 2011BAD23B04). The corresponding author thanks the support from Program for New Century Excellent Talents in University (NCET-09-0741). Reference Acosta, E. (2008). 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