Enhancement of Carotenoid Bioaccessibility from ...

Food Biophysics (2017) 12:172–185 DOI 10.1007/s11483-017-9474-7

ORIGINAL ARTICLE

Enhancement of Carotenoid Bioaccessibility from Tomatoes Using Excipient Emulsions: Influence of Particle Size Qian Li 1 & Ti Li 1 & Chengmei Liu 1 & Taotao Dai 1 & Ruojie Zhang 2 & Zipei Zhang 2 & David Julian McClemnets 2

Received: 5 January 2017 / Accepted: 14 February 2017 / Published online: 27 February 2017 # Springer Science+Business Media New York 2017

Abstract The effect of excipient emulsions with different lipid droplet sizes on carotenoid bioaccessibility from tomatoes was investigated using a simulated gastrointestinal tract (GIT). Excipient emulsions with different surface-weighted mean droplet diameters were fabricated: d32 = 0.15 μm (small), 0.40 μm (medium), and 22.3 μm (large). Changes in particle size, microstructure, ζ-potential, and carotenoid bioaccessibility were measured when tomato-emulsion mixtures that had received different thermal and mixing treatments were passed through the GIT model. Carotenoid bioaccessibility decreased with increasing initial droplet size (small ≥ medium > large), which was attributed to two effects. First, smaller droplets extracted carotenoids from tomato tissue more efficiently. Second, smaller droplets were digested faster leading to more rapid mixed micelle formation, thereby increasing carotenoid solubilization in intestinal fluids. Carotenoid bioaccessibility was higher from boiled than raw tomatoes because thermal disruption of the plant tissue facilitated carotenoid release. Carotenoid bioaccessibility was higher when tomatoes were boiled with emulsions than when they were boiled alone and then added to emulsions. In conclusion, excipient emulsions are highly effective at increasing carotenoid bioaccessibility Chengmei Liu and David Julian McClemnets contributed equally to this manuscript. * Chengmei Liu [email protected] * David Julian McClemnets [email protected] 1

State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, Jiangxi, People’s Republic of China

2

Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA

from tomatoes, but lipid droplet size must be optimized to ensure high efficacy. Keywords Olive oil excipient nanoemulsions . Tomato carotenoids . Carotenoids bioaccessibility . In vitro gastrointestinal digestion

Introduction Epidemiological studies suggest that consumption of certain fruits and vegetables may decrease the risk of chronic diseases, such as cancer and cardiovascular disease[1, 2]. The positive correlation between fruit and vegetable intake and disease prevention has been attributed to the presence of bioactive nutraceuticals in these products, such as carotenoids and other phytochemicals [3, 4]. Carotenoids are natural fatsoluble pigments synthesized by plants that are found at relatively high levels in numerous fruits and vegetables [5, 6]. Tomatoes are one of the major dietary sources of carotenoids in the human diet because of their relatively high carotenoid content and their high level of global consumption [5]. Lycopene, β-carotene, and lutein are the three major carotenoids in tomato [7]. Studies have reported that consumption of raw tomatoes gave a protective effect against cancers of the digestive tract [8]. Other studies indicated that low serum lycopene concentrations were correlated with an increased risk of pancreatic and bladder cancers [9, 10]. Conversely, high dietary intakes and serum concentrations of lycopene appear to protect against cervical intraepithelial neoplasia [11]. Some studies suggest that the combination of carotenoids found in whole tomato powder is more effective in disease prevention than lycopene alone [12, 13]. Thus, the combination of nutraceuticals obtained from whole foods may be more beneficial to human health than those of single isolated

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nutraceuticals in supplements. However, the oral bioavailability of carotenoids in plant tissues is often relatively low due to limited release from the plant tissues, poor water-solubility, and chemical instability [14]. Carotenoid absorption occurs through a series of steps: (i) carotenoids are released from the food matrix during processing and digestion; (ii) they enter the lipid phase of any coingested food product, (iii) they are released from the lipid phase when it is digested; (iv) they are incorporated into mixed micelles within the intestine; (v) the carotenoidloaded micelles are absorbed by epithelium cells; (vi) the carotenoids are packaged into chylomicrons within the epithelium cells; (vii) the carotenoid-loaded chylomicrons are expelled from the epithelium cells into the lymphatic system, where they are transported to the systemic circulation [5, 15]. Many studies have reported that ingestion of carotenoids with digestible lipids is crucial for the proper absorption of carotenoids because of their low solubility in gastrointestinal fluids [16–18]. For example, the presence of rapeseed oil during cooking of carrot pulp and the presence of sunflower oil during the cooking of leafy green vegetables have been shown to increase the release of β-carotene during digestion [19, 20]. More generally, it has been shown that careful design of food matrix composition and structure can be used to increase the bioavailability of nutraceuticals by altering their bioaccessibility, absorption, or transformation profiles [21, 22]. This insight has led to the concept of Bexcipient foods^ that are specifically designed to increase the bioavailability of nutraceuticals in other foods (such as fruits and vegetables). Indeed, recent studies have shown that excipient emulsions can be used to increase the bioavailability of carotenoids from various types of plant-based foods, including peppers, carrots, and mangoes [23–25]. In these examples, the improvement in overall bioavailability was mainly attributed to an increase in carotenoid bioaccessibility due to two physicochemical effects. First, the lipid droplets in the emulsions acted as a non-polar solvent that could extract carotenoids from the plant tissue. Second, the rapid digestion of the lipid droplets in the small intestine led to the rapid formation of mixed micelles capable of solubilizing and transporting the carotenoids. Studies have indicated that the ability of excipient emulsions to increase carotenoid bioaccessibility from plant tissues was more effective than that of a similar quantity of bulk oil [14], which highlights the importance of the structure of the lipid phase. In a recent study, we found that olive oil excipient emulsions increased the bioaccessibility of carotenoids from tomatoes by extracting carotenoids from tomato tissues and forming mixed micelles that solubilized the carotenoids [26]. Olive oil is rich in relatively long chain fatty acids (mainly C18:1), which have previously been shown to be highly effective at increasing carotenoid bioaccessibility [27]. In the current study, the influence of lipid droplet size in the excipient

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emulsions on carotenoid bioaccessibility from tomatoes was examined. In addition, we investigated two different cooking methods to provide a better understanding of the mechanisms by which olive oil emulsions boost the bioaccessibility of carotenoids in tomatoes. Our hypothesis was that lipid droplet size and cooking method would impact the bioaccessibility of carotenoids by altering their release from tomatoes and/or their solubilization within lipid droplet or mixed micelles. The information obtained from this study may be helpful in the design of excipient foods that can improve the potential health benefits of nutraceuticals in plant-based foods, such as fruits and vegetables.

Materials and Methods Materials and Chemicals Raw tomato was purchased from a local supermarket. Whey protein isolate (WPI) was obtained from Davisco Foods International Inc. (Le Sueur, MN, USA). As stated by the manufacturer, the protein content was 97.6% (dry basis). Olive oil was purchased from a commercial food supplier (Bertolli, Mizkan America Inc., USA). The manufacturer reported that the saturated, monounsaturated, and polyunsaturated fat contents of this product were approximately 14.3, 71.4, and 14.3%, respectively. β-carotene, mucin from porcine stomach, pepsin from porcine gastric mucosa (250 units/ mg), porcine lipase (100–400 units/mg), and porcine bile extract were purchased from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO, USA). All other chemicals used in this paper were purchased from either Sigma-Aldrich or Fisher Scientific. All solvents and reagents were of analytical grade. Double distilled water from a water purification system (Nanopure Infinity, Barnstaeas International, Dubuque, IA, USA) was used for the preparation of all solutions. Methods Emulsion Preparation Olive oil-in-water emulsions were prepared by homogenizing 8 wt% olive oil with 92 wt% aqueous phase (1 wt% WPI, pH 7.0, 10 mM phosphate buffer). Excipient emulsions with different particle sizes (small, medium, or large) were prepared using different homogenization procedures. Emulsions containing large-sized droplets were simply prepared by blending olive oil and aqueous phase together using a highspeed blender (M133/1281–0, Biospec Products, Inc., ESGC, Switzerland). Emulsions containing medium-sized droplets were formed by passing emulsions containing large-sized droplets through a microfluidizer at 7000 psi for 3 passes (M110Y, Microfluidics, Newton, MA). Emulsions containing

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small-sized lipid droplets were formed by passing large-sized emulsion through the microfluidizer at 12000 psi for 3 passes.

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modification of that described in our previous studies [27, 28]. All experiments were carried out at 37 °C using solutions that had been heated to this temperature prior to mixing.

Tomato-Emulsion Sample Preparation Tomatoes were cut into small disks that were approximately 10 mm high and 10 mm wide. These tomato pieces were then used to prepare the following samples. Raw tomato - emulsion mixture (Raw-TE): Raw tomato disks were mixed with a half equal mass of buffer solution (pH 7.0) and then placed in a household blender for 2 min to break down the tomato structure. The resulting tomato puree was then mixed with excipient emulsion (8 wt% oil, different droplet size) to make the final oil content of the mixture to be 2 wt%. The resulting samples were given different designations according to the particle size of the excipient emulsion added: Raw-TE-Small, Raw-TE-Medium, and Raw-TELarge. Boiled tomato - emulsion mixture (Boiled-TE/Boiled-TE*): The boiled tomatoes were prepared using two protocols: (1) Boiled-TE: The raw tomato-emulsion mixture prepared previously was put in a 100 mL beaker, and then was heated by a hot plate with a probe in the mixtures at 100 °C for 10 min. After heating, selected samples were immediately placed in an ice-water bath. These samples were designated: Boiled-TE-Small, Boiled -TEMedium, and Boiled-TE-Large; (2) Boiled-TE*: Raw tomato disks were mixed with a half mass of phosphate buffer solution and then placed in a household blender for 2 min to break down the tomato structure. Afterwards, the mixtures were heated for 10 min at 100 °C. After heating, selected samples were immediately placed in an ice-water bath and then diluted with excipient emulsion (8 wt% oil, different droplet size) to make the final oil content of mixture to be 2 wt% (Boiled-TE*). These samples were designated: Boiled-TE*-Small, Boiled -TE*-Medium, and BoiledTE*-Large. These two treatments were used to take into account that tomato may be consumed raw (e.g., in salads) or they may be cooked (e.g., in soups or in sauces). Raw and boiled tomato samples were also prepared using buffer solution (rather than excipient emulsions) as controls. These samples allowed us to determine the influence of the lipid droplets on the bioaccessibility of the carotenoids. In Vitro Digestion Model Mixtures of tomato with emulsion or phosphate buffer (control) were passed through a GIT model designed to mimic the mouth, stomach, and small intestine phases, which was a slight

Initial System An aliquot (20 mL) of tomato/emulsion or tomato/buffer mixture was placed into a glass beaker in an incubated shaker (Innova Incubator Shaker, model 4080, New Brunswick Scientific, New Brunswick, NJ, USA). Mouth Phase Twenty milliliters of simulated saliva fluid (SSF) containing 30.0 mg/mL mucin was mixed with the initial system. After being adjusted to pH 6.8, the mixture was incubated in the incubator shaker for 2 min to mimic agitation in the mouth. Stomach Phase Twenty milliliters of the sample resulting from the mouth phase was mixed with 20 mL of simulated gastric fluid (containing 3.2 mg/mL pepsin), then the solution was adjusted to pH 2.5. This mixture was incubated in the incubator shaker for 2 h to mimic stomach conditions. Small Intestine Phase Thirty grams of sample from the stomach phase were poured into a 100 mL glass beaker and then the solution was adjusted to pH 7.00. One and a half milliliters of simulated intestinal fluid were added to the reaction vessel, followed by 3.5 mL of bile salt solution with constant stirring. The pH of the reaction system was adjusted back to 7.00. Two and a half milliliters of lipase solution were then added to the sample, and an automatic titration unit (Metrohm, USA, Inc.) was used to monitor the pH and maintain it at pH 7.00 by titrating 0.25 N NaOH solution into the reaction vessel for 2 h at 37 °C. The percentage of Free Fatty Acids (FFA) released was calculated using the following eq. (1): ! " V NaOH $ C NaOH $ M Oil FFAð%Þ ¼ 100 $ ð1Þ W Oil $ 2 Here, VNaOH is the volume of NaOH solution required to neutralize the FFAs produced at digestion time (L), CNaOH is the molarity of the NaOH solution used to titrate the sample (mol/L), Moil is the molecular weight of the oil (g/mol), and Woil is the total mass of lipid initially present in the incubation cell (g). Particle Size and Charge Measurements The particle size distribution and ζ-potential of the particles in the samples were measured as they passed through the various stages of the GIT model. Reliable data could not be obtained for the lipid droplets in the presence of tomato because the large plant tissue fragments dominated the light-scattering signal. For this reason, we also measured the microstructure of the samples using microscopy (next section). The particle size

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distribution of the samples was determined using static light scattering (Mastersizer 2000, Malvern Instruments Ltd., Malvern, Worcestershire, UK). Samples were diluted in aqueous solutions and stirred in the dispersion unit with a speed of 1200 rpm to ensure homogeneity. Phosphate buffer (10 mM, pH 7.0) was used to dilute initial, mouth, and small intestine samples, whereas pH 2.5-adjusted distilled water was used to dilute stomach samples. Average particle sizes are reported as the surface-weighted mean diameter (d32). The refractive indices used in the calculations of the particle sizes were 1.47 and 1.33 for the particles and surrounding liquid, respectively. In practice, lipid droplets and tomato tissue have different refractive indices. However, for particles (such as tomato fragments) that are relatively large compared to the wavelength of light, the particle size distribution is not strongly influence by the refractive index of the particles. Consequently, the use of a single refractive index is appropriate for analyzing the mixed systems. The ζ-potential of emulsions was measured using an electrophoresis instrument (Zetasizer Nano ZS series, Malvern Instruments Ltd.). Prior to analysis, initial, mouth, and small intestine samples were diluted with 10 mM phosphate buffer (pH 7.0), whereas stomach samples were diluted with pH 2.5adjusted distilled water. Microstructure Measurements The microstructures of samples were measured after exposure to the various stages of the GIT model using either optical or confocal scanning laser microscopy with a 20× objective lens or a 60× oil immersion objective lens (Nikon D-Eclipse C1 80i, Nikon, Melville, NY, USA) [23]. Before analysis, 2 mL samples were mixed with 0.1 mL of Nile Red solution (1 mg/ mL ethanol) to dye the oil phase. The excitation and emission spectra for Nile Red were 543 and 605 nm, respectively. An aliquot of sample was placed on a microscope slide, covered by a coverslip, and then microstructure images were acquired using image analysis software (NIS-Elements, Nikon). Bioaccessibility The bioaccessibility of carotenoids were determined after each sample had been subjected to the full in vitro digestion process using a method described previously [23]. The bioaccessibility was taken to be the fraction of carotenoids present within the small intestine digesta that were solubilized within the mixed micelle phase. After the small intestinal stage, digesta samples were collected and centrifuged at 12000 rpm and 4 °C for 60 min, which resulted in samples that contained sediment at the bottom and a clear supernatant above. The supernatant was collected and assumed to be the Bmicelle^ fraction, in which the carotenoids were solubilized. Aliquots of 5 mL of digesta or micelle fraction were extracted with 5 mL hexane/ acetone (1:1, v/v) mixture, vortexed and centrifuged at

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2000 rpm for 6 min at room temperature. The supernatant layer was collected in a second tube. The extraction process was repeated twice. The combined organic fractions were mixed with saturated sodium chloride solution, and the mixture was shaken vigorously. After the supernatant hexane layer was collected, the lower phase was extracted again with hexane/acetone mixture. Then the combined supernatant hexane phases were measured at 450 nm using a spectrophotometer (Ultrospec 3000 pro, GE Health Sciences, USA). A cuvette containing pure hexane was used as a reference cell. The concentration of carotenoids extracted from a sample was determined from a calibration curve of absorbance versus β-carotene concentration in hexane. The bioaccessibility of carotenoids was then calculated using the following Eq. (2): ! " C Micelle Bioaccessibility ¼ 100 $ ð2Þ C Digesta Where, Cmicelle and Cdigesta are the concentrations of carotenoids in the micelle fraction and in the overall sample (raw digesta) after the simulated intestinal digestion experiment, respectively. Statistical Analysis Experiments were carried out using three freshly prepared samples. The results are reported as averages and standard deviations, and the differences among treatments were calculated on the basis of the analysis of variance (ANOVA) and Duncan test with a confidence level of 95%. These analyses were carried out using statistical analysis software (SPSS, IBM Corp., Armonk, NY, USA).

Results and Discussion Initial Characteristics of the Excipient Emulsions Excipient emulsions with three different mean particle diameters were prepared: small (d 32 ≈ 0.15 μm); medium (d32 ≈ 0.40 μm); and large (d32 ≈ 22.3 μm). The small and medium emulsions had monomodal particle size distributions, whereas the large emulsions had a bimodal distribution with a minor peak around 5 μm and a major peak around 10–100 μm (data not shown). The electrical potential (ζ-potential) of the droplets in the excipient emulsions was highly negative, its magnitude decreased with increasing mean particle diameter: −43.6, −38.7 and −32.8 mV for small, medium, and large emulsions, respectively. These results are in agreement with those reported in another recent study by Zhang, Zhang, Zou, Xiao, Zhang, Decker and McClements [14]. The high negative charge of the excipient emulsions is due to the presence of an adsorbed layer of anionic whey protein molecules at the

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lipid droplet surfaces, since the solution pH was above the protein’s isoelectric point, pI ≈ 5 [23]. The observed reduction in ζ-potential with increasing droplet size may be because the concentration of non-adsorbed proteins in the emulsions increased as the droplet surface area decreased. These nonadsorbed proteins increase the ionic strength of the aqueous phase [29], which will decrease the magnitude of the droplet ζ-potential [30]. Impact of Mixing with Tomatoes and Heating on Emulsion Properties Initially, the impact of mixing procedure and thermal processing on the properties of mixtures of excipient emulsions and tomatoes was determined (Figs. 1 to 4). After the homogenized tomato (raw or boiled) was mixed with different excipient emulsions, the mean particle diameter of the overall mixtures was much greater than that of the original emulsions (p < 0.05) (Fig. 4). This effect can be mainly attributed to the presence of relatively large tomato fragments (Figs. 1 and 3), which would dominate the overall light scattering pattern. The mean particle diameters (d32) of raw tomato/emulsion mixtures were 41.7, 43.7, and 56.3 μm for small, medium, and large emulsions, respectively. The particle size distribution

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data showed that all Raw-TE samples had a bimodal distribution with a minor peak of small particles and a major peak of large particles (Fig. 1a). The smaller particles are likely to be the lipid droplets, whereas the larger particles are likely to be tomato tissue fragments. The optical and confocal microscopy images confirmed that there were some small lipid droplets and some large tomato tissue fragments in these samples (Figs. 2 and 3). Thermal processing of the tomatoes also had an impact on their particle size and microstructure, as did the order of thermal processing relative to emulsion addition: emulsions added before boiling (Boiled-TE) or emulsions added after boiling (Boiled-TE*). The initial particle sizes of the Boiled-TE and Boiled-TE* samples were much larger than those of the RawTE samples (Fig. 4). This increase in particle size after thermal processing is probably because there was some loosening and swelling of the tomato tissues caused by boiling [31, 32]. In addition, some components released from the tomato tissues during heating (such as acids, minerals, or biopolymers) may have promoted lipid droplet aggregation [23]. Finally, heating itself may have promoted aggregation instability in the lipid droplets, since it has previously been shown that wheyprotein coated droplets aggregate when heated above their thermal denaturation temperature [33, 34]. Indeed, the confocal

Fig. 1 Influence of gastrointestinal stage on the particle size distribution of raw/boiled tomato samples with small particle emulsion. a Raw-TE- Small; b Boiled-TE-Small; c Boiled-TE*- Small. Similar results were obtained for the other samples (data not shown)

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Fig. 2 Changes in the microstructure of raw/boiled tomato-excipient emulsion mixtures as they passed through the simulated GIT as observed by confocal fluorescent microscopy using a lipid dye. a Raw-TE mixtures; b Boiled-TE mixtures; c Boiled-TE* mixtures

microscopy images indicated that extensive droplet aggregation occurred in the excipient emulsions that were boiled (Fig. 2b).

The overall appearance of the initial tomato-excipient emulsion mixtures was highly dependent on droplet size and

Fig. 3 Microstructures of tomato/emulsion mixtures after they were exposed to different regions of a simulated GIT by optical and confocal fluorescent microscopy. a Raw-TE-mixtures; b Boiled-TE-mixtures. Optical microscopy images show the microstructure of the tomato tissue,

where confocal microscopy images show the location of the lipid phase. Boiled-TE*-samples showed similar results with Raw-TE-mixtures (data not shown)

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Fig. 4 Influence of passage through a simulated GIT on the mean particle diameter (d32) of samples containing raw/boiled tomato and excipient emulsions with different particle size. a Raw-TE mixtures; b Boiled-TE mixtures; c Boiled-TE* mixtures. Samples designated with different letters (a, b, c, d) were significantly different (Duncan, p < 0.05) when

compared between different GIT regions (same sample). Samples designated with different upper case letters (A, B) were significantly different (Duncan, p < 0.05) when compared between different samples (same GIT region)

thermal processing (Fig. 5). The emulsions containing smaller droplets had a more uniform and turbid appearance than those containing larger droplets, which can be attributed to less gravitational separation and greater light scattering for smaller droplets. Moreover, a layer of oil was observed on top of the excipient emulsions containing large droplets after heating (Boiled-TE-Large), which suggests that they were highly susceptible to droplet coalescence. Interestingly, the samples where the tomato and small excipient emulsions were heated together (Boiled-TE) had a much more intense orange color than the samples where the emulsions were unheated (RawTE and Boiled-TE*). This result suggests that the lipid droplets in the small emulsions acted as non-polar solvents that extracted the carotenoids from the tomato tissue more effectively at elevated temperatures. The solubility and diffusion of hydrophobic molecules in emulsions tends to increase with increasing temperature [35], which may account for this observation. The transfer of carotenoids

from plant tissue to emulsion droplets would be a fruitful area for future studies. The particles in the initial mixed systems were less negative than the lipid droplets in the initial excipient emulsions (Fig. 6). For example, the ζ-potentials of Raw-TE-Small, −Medium, and -Large were −32.7, −34.3, and −32.0 mV, respectively. This effect may be because tomato fragments had a lower negative charge than the lipid droplets, or because there were some components released from the tomato tissues (such as acids, minerals, or biopolymers) that altered the droplet surface charge or the solution ionic strength [23].

Potential GIT Fate of Tomato Samples The impact of the initial size of the droplets in the excipient emulsions on the potential GIT fate of the tomato samples was assessed using a static in vitro digestion model.

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Fig. 5 Macroscopic appearance of raw/boiled tomato samples with small and large particle size emulsion at the initial stage of digestion. a Tomato mixed with small emulsion; b Tomato mixed with large emulsion. For BoiledTE-Large sample, there formed an oil layer on the surface of samples

Mouth After incubating in the mouth stage, the mean particle diameters of all the samples remained relatively large (Fig. 4), which suggested that the tomato tissue fragments remained intact. Confocal microscopy indicated that the lipid particles in the mouth phase were larger than those in the initial samples (Fig. 2), which suggested that some droplet flocculation or coalescence had occurred. Droplet aggregation may have been due to bridging or depletion effects associated with the presence of mucin in the simulated saliva [36, 37]. After exposure to oral conditions, the ζ-potential of all the samples remained highly anionic, but there was a slight decrease in the magnitude of the negative charge compared to the initial values (Fig. 6). The pH of the simulated saliva was similar to that of the initial emulsions, and would therefore not be expected to have a major impact on particle charge. Instead, it is likely that the observed change in particle charge is due to electrostatic screening effects associated with the mineral ions in the simulated saliva [30] and/or due to the interaction of the mucin molecules with the lipid droplet surfaces [38]. Stomach Samples collected at the end of the oral phase were incubated in simulated gastric fluids, and then their particle size, microstructure, and charge were measured. For the samples containing boiled tomatoes (Boiled-TE and Boiled-TE*), there was a

significant decrease in mean particle diameter compared to the mouth phase (p < 0.05) (Fig. 4). This decrease may at least be partly attributed to the disintegration of the tomato tissue fragments under the highly acidic conditions in the stomach with continuous stirring. In the case of the Boiled-TE samples there also appeared to be some breakup of the droplet flocs when the samples moved from the mouth to the stomach (Figs. 2 and 3). In the case of the samples containing raw tomatoes there was actually an increase in the mean particle diameter when they moved from the mouth to the stomach phases (Fig. 4a). The confocal microscopy images suggest that this increase was due to enhanced lipid droplet aggregation in the gastric fluids (Fig. 2a). This type of aggregation may have occurred due to the change in pH and ionic strength of the aqueous phase surrounding the oil droplets [36]. The ζ-potential of all the samples significantly decreased and became close to zero after incubation in simulated gastric fluids (p < 0.05). This effect can be attributed to the low pH and high ionic strength of the simulated stomach phase. Presumably, adsorption of anionic mucin molecules onto the surfaces of the cationic protein-coated lipid droplets led to charge neutralization at acidic gastric pH[39]. In addition, the decreasing of charge can also be attributed to the hydrolysis of the protein molecules adsorbed to the oil droplet surfaces, which may alter their interfacial composition and properties [40]. The relatively low charge on the droplets would also account for the fact that the lipid droplets in many of the emulsions were highly aggregated, which can be attributed to a weak electrostatic repulsion between them.

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Fig. 6 Influence of passage through a simulated GIT on the electrical characteristics of the particles (ζ-potential) of samples containing raw/ boiled tomato and excipient emulsions. a Raw-TE mixtures; b BoiledTE mixtures; c Boiled-TE* mixtures. Samples designated with different letters (a, b, c, d) were significantly different (Duncan, p < 0.05) when

compared between different GIT regions (same sample). Samples designated with different upper case letters (A, B) were significantly different (Duncan, p < 0.05) when compared between different samples (same GIT region)

Small Intestine

The magnitude of the ζ-potential on the particles in all of the samples became highly negative after exposure to simulated small intestinal conditions (Fig. 6). This effect has also been reported in other studies using emulsion-based delivery system [38, 42], where it was attributed to the presence of colloidal particles comprised of various types of anionic species, such as bile salts, phospholipids, free fatty acids, proteins, and peptides. There was no significant difference in the magnitude of the negative charges on the particles in the samples containing excipient emulsions after the small intestine phase (Fig. 6). This suggests that the interfacial composition of the colloidal particles in the different systems was fairly similar.

After exposure to the small intestine phase, the particle size, microstructure, and charge of the samples were measured. The mean particle diameter of all samples significantly increased when they moved from stomach to small intestine conditions (Fig. 4). Previous studies have also reported this effect, which was attributed to the formation of a complex mixture of colloidal particles that contribute to the light scattering pattern, such as non-digested lipid droplets, micelles, vesicles, insoluble calcium soaps, liquid crystals, and plant tissue fragments [41]. The optical microscopy images confirmed that there were still intact tomato tissue fragments in the small intestine phase (Fig. 3) and the confocal microscopy images indicated that various kinds of lipid-rich structures were also present (Fig. 2). Interestingly, numerous large non-digested lipid particles were observed in the Boiled-TE-Large samples at the end of the small intestine phase (Figs. 2 and 3), which may have been because the rate of lipid digestion was relatively slow in this system (see later).

Lipid Digestion A pH-stat method was used to determine the effects of initial droplet size and processing method on the rate and extent of lipid digestion in the samples. The FFA release profiles depended strongly on initial lipid droplet size and thermal

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processing conditions. For all samples, there was a relatively rapid increase in FFAs released during the first 10 or 20 min of digestion, followed by a more gradual increase at longer times (Fig. 7). However, the initial rate of lipid digestion decreased in the following order: small emulsion ≥ medium emulsion > large emulsion. These differences can be attributed to variations in the specific surface areas of the lipid droplets entering the small intestine (rather than in the initial emulsions). Presumably, the individual lipid droplets reaching the small intestine phase were larger for the large emulsion than for the small and medium emulsions. Smaller droplets are digested faster than larger ones due to the higher surface area of lipids exposed to the digestive enzymes. Similar results have been reported in other studies by Zhang, Zhang, Zou, Xiao, Zhang, Decker and McClements [14] and Zou, Zheng, Liu, Liu, Xiao and McClements [41]. The nature of the thermal processing step also had an impact on the rate and extent of lipid digestion. Boiled-TE and Boiled-TE* samples had a lower rate of lipid digestion than the corresponding Raw-TE samples. This effect may be due to boiling increased the size of the lipid droplets in the

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emulsions, and therefore reduced the surface area available for lipase to adsorb. In addition, boiling may have stimulated the release of components (such as acids, pectin, or minerals) from the tomato tissues that promoted droplet aggregation, which also reduced the available surface area. It is noteworthy that numerous non-digested lipid droplets were observed in the Boiled-TE-Large samples using confocal microscopy (Fig. 2b), which is in agreement with the relatively low extent of lipid digestion in this system by the end of the small intestine phase. Carotenoid Bioaccessibility In this section, the influence of initial droplet size in the excipient emulsions on carotenoid bioaccessibility in tomatoes with or without heat-treatment was investigated. The results showed that mixing the tomatoes with an excipient emulsion significantly increased carotenoid bioaccessibility (BA ≈ 15– 51%) compared to the control (BA ≈ 10%). This effect can be attributed to two main factors. First, the lipid droplets act as a non-polar solvent that can extract carotenoids from the plant

Fig. 7 Percentage of free fatty acids released in tomato/emulsion mixtures under simulated small intestine conditions as measured using a pH-stat method. a Raw-TE samples; b Boiled-TE samples; c Boiled-TE*-samples

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tissue. Second, the lipid droplets act as a source of FFAs and monoacylglycerols that increase the solubilization capacity of the mixed micelle phase in the small intestine. Previous studies have reported that the bioaccessibility of lipophilic compounds in fruits and vegetables can be increased by coingesting them with lipids [19, 43]. However, most of these studies have used either bulk oils or emulsion-based foods (such as dressings) rather than specially designed excipient emulsions. Carotenoid bioaccessibility was higher when small or medium excipient emulsions were used rather than large excipient emulsions (Fig. 8). No significant difference was observed between the bioaccessibility of small and medium emulsions. For example, the carotenoid bioaccessibilities for Raw-TESmall and Raw-TE-Medium were 26.5% and 24.7%, respectively, whereas the corresponding value for Raw-TE-Large was 18.4%. As mentioned earlier, there are two main factors impacting carotenoid bioaccessibility from plant-based sources: (i) release from plant tissues; and, (ii) solubilization within mixed micelles. It has previously been reported that carotenoids are present in a crystalline form or are associated with proteins embedded in chromoplasts, which limits their release during digestion [19, 44]. However, these structures may be degraded during the thermal processing, mechanical treatment, mastication, and digestion of tomato-based products.[45] Mechanical treatments physically breakdown plant tissue structures, thereby releasing carotenoids and other cellular materials [46]. Thermal processing may lead to softening, swelling and dissociation of plant tissues [31]. Mastication physically disrupts plant tissues to form smaller fragments [47]. Passage through the GIT may cause further changes in plant tissue structure due to alterations in pH, ionic

strength, and enzyme activity. These alterations in tomato structure should facilitate the ability of lipid droplets to penetrate into the plant tissue and extract the carotenoids. One would expect that the lipid droplets have to be below some critical size to allow them to easily move through the plant tissues and come into proximity to the carotenoids. This would partly account for the ability of small lipid droplets to extract more carotenoids from the tomato tissue during the boiling stage than the large lipid droplets (Fig. 5). In addition, smaller lipid droplets are digested more rapidly than larger ones in the small intestine phase (Fig. 7), which would lead to faster generation of mixed micelles capable of solubilizing the carotenoids released from the lipid phase.

60 Small Emulsion

Bioaccessibility (%)

50

Ab

Ab

Medium Emulsion

Ab

Large Emulsion

Ac

40 30

Bb

Aa ABa

Bab Ba

20 10 0 Raw-TE

Boiled-TE

Boiled-TE*

Processing method

Fig. 8 Bioaccessibility of carotenoids released under simulated GIT conditions from samples containing raw/boiled tomatoes and excipient emulsions. Samples designated with different letters (a, b, c) were significantly different (Duncan, p < 0.05) when compared between different treatments (some emulsion). Samples designated with different upper case letters (A, B) were significantly different (Duncan, p < 0.05) when compared between different particle size emulsion (same treatment)

Fig. 9 Optical and confocal microscopy images of boiled tomato puree containing carotenoids crystalline

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Interestingly, the bioaccessibility of carotenoids from boiled tomatoes was higher than that from raw tomatoes (Fig. 8). In plant tissue, carotenoids are compartmentalized in plastids (chloroplasts and chromoplasts), which are themselves contained within fibrous cell walls [44]. Boiling may have promoted partial disruption of the cell wall structure, which facilitated droplet penetration and carotenoid release. A number of other studies have reported that weakening of fibrous plant cell walls using heat treatments substantially increases carotenoid release [48–50]. For example, a human feeding study showed that β-carotene absorption from a single portion of carrot was much higher when it was cooked than when it was raw [51]. The nature of the thermal process also had an impact on the carotenoids bioaccessibility, i.e., the excipient emulsion was added to the samples before (Boiled-TE) or after (Boiled-TE*) boiling the tomatoes. From Fig. 8, the carotenoid bioaccessibility of Boiled-TE samples was higher than that of BoiledTE* samples. This effect may be because boiling promoted the transfer of carotenoids from the tomato tissue clusters into the lipid droplets. The solubility and diffusion of hydrophobic bioactive agents is known to increase with increasing temperature, which may account for this effect. In addition, For Boiled-TE* samples, numerous Fig. 10 Macroscopic appearance of tomato/emulsion mixtures after they were exposed to different regions of a simulated GIT. a Raw-TE-Small; b Raw-TELarge; c Boiled-TE-Small; d Boiled-TE-Large. Samples with medium emulsion showed similar results with samples containing small emulsion and Boiled-TE* samples showed similar results with Raw-TE samples

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small orange clusters were observed in the optical microscopy images (Fig. 9a). These orange clusters had an intrinsic fluorescence signal that could be detected by confocal microscopy (Fig. 9b). We speculated that these clusters were carotenoid-containing fragments released from the tomato tissues during boiling. For the Boiled-TE-Large samples, a thin layer of oil formed on the surface of the tomato/emulsion mixtures during boiling, with the thickness of the oil layer increasing with boiling time (data not shown). This suggests that the large lipid droplets were prone to coalescence and oiling off during heating. The oil layer remained visible on top of the samples in the mouth and stomach stages of the model GIT (Fig. 10d), and may partly account for the slower digestion rate of these samples (Fig. 7). This phenomenon is likely to have reduced the transfer of carotenoids from the tomato tissues into the oil phase of the emulsions, which would also account for the relatively low carotenoid bioaccessibility for the Boiled-TE- Large emulsions. Conversely, the Boiled-TE samples containing small and medium excipient emulsions had no visible oil layer on the top throughout the whole GIT (Fig. 10c), which indicated that the smaller droplets in these emulsion were more stable to boiling. As a result, these samples had a relatively high bioaccessibility (Fig. 8).

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Conclusions This study showed that olive oil excipient emulsions significantly increased the bioaccessibility of carotenoids originally present within raw or cooked tomatoes. Excipient emulsions initially containing small oil droplets (d32 < 0.5 μm) were more effective at increasing carotenoid bioaccessibility than those containing larger droplets. This effect was attributed to the influence of droplet size on the tissue penetration and digestion rates of the lipid droplets. The results indicate that specially designed excipient emulsions are effective at enhancing the bioaccessibility of carotenoids from plant-based foods, and highlight the importance of controlling lipid droplet size. The bioaccessibility of the carotenoids was found to be higher for boiled tomatoes than for raw ones, since heating partially disrupts the cell wall structure and promotes the penetration of lipid droplets and the release of carotenoids. Finally, the point when the excipient emulsions are mixed with the tomatoes was also shown to be important, e.g., before or after thermal processing. Carotenoid bioaccessibility was higher when tomato samples were heated in the presence of excipient emulsions than when the emulsions were added after heating. Overall, this study shows that excipient emulsions can be used to boost the bioaccessibility of hydrophobic nutraceuticals from natural plant-based foods. In the food industry, it may be possible to develop a range of products based on this principle, such as specially designed dressings to pour on raw tomatoes in salads, or specially designed creamy sauces to cook tomato-based foods (such as soups, ketchup or pasta sauces).

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Acknowledgements We thank the following financial support: BNational Natural Science Foundation of China^ (31460394). This material was partly based upon work supported by the Cooperative State Research, Extension, Education Service, USDA, Massachusetts Agricultural Experiment Station (MAS00491) and USDA, NRI Grants (2013-03795).

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