The type and quantity of lipids present during digestion influence the in vitro bioaccessibility of lycopene from raw tomato pulp

Food Research International 45 (2012) 250–255

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The type and quantity of lipids present during digestion influence the in vitro bioaccessibility of lycopene from raw tomato pulp Ines J.P. Colle, Sandy Van Buggenhout, Lien Lemmens, Ann M. Van Loey, Marc E. Hendrickx ⁎ Laboratory of Food Technology and Leuven Food Science and Nutrition Research Centre (LFoRCe), Department of Microbial and Molecular Systems (M²S). Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, Box 2457, B-3001 Leuven, Belgium

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Article history: Received 11 August 2011 Accepted 25 October 2011 Keywords: Lycopene Lipids Tomato In vitro bioaccessibility

a b s t r a c t This study elucidates the impact of the type and quantity of lipids, added upon digestion of raw tomato pulp, on the bioaccessibility of lycopene. Lycopene bioaccessibility was studied by measuring the micellarization during in vitro digestion. Coconut oil, palm oil, cocoa butter, olive oil, sunflower oil and fish oil were selected because of their distinctly different fatty acid composition. Upon adding 5% of lipid to raw tomato pulp, all tested lipids significantly improved the lycopene bioaccessibility. The largest increase in lycopene bioaccessibility was noticed after supplying 5% of sunflower oil, followed by olive oil and cocoa butter (not all differences were significant). A slightly smaller increase was observed when fish oil, coconut oil and palm oil were used. In addition, the effect of different quantities (0–10%) of coconut oil, olive oil and fish oil was examined. Over the entire concentration range, increasing the amount of coconut oil increased the lycopene bioaccessibility, while the highest bioaccessibility was found using 1 and 2% of respectively fish oil and olive oil. Moreover, depending on the amount of added lipid, the type of lipid resulting in the highest lycopene bioaccessibility differed. The results obtained clearly indicate that lycopene bioaccessibility depends both on the type and on the quantity of the lipid present during in vitro digestion of raw tomato pulp. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Lycopene is one of the dietary carotenoids relevant for human health. Carotenoids such as β-carotene and β-cryptoxanthin serve as a source of vitamin A (Fernández-García et al., in press). However, since lycopene is devoid of provitamin A activity, its promising implications for human health must be attributed to other mechanisms of actions. Due to its polyene nature consisting of 11 conjugated double bounds, lycopene is found to be an effective antioxidant (Conn, Schalch, & Truscott, 1991; Di Mascio, Kaiser, & Sies, 1989). Moreover, Erdman, Ford, and Lindshield (2009) reviewed that lycopene or lycopene containing products have the ability to decrease cancer cell growth and inflammation, increase gap junctional communication, inhibit androgen/estrogen signaling, induce detoxification enzymes, decrease cell surface adhesion and intima-media thickness, decrease serum cholesterol and decrease C-reactive protein. All these mechanisms of action can contribute to reduce the risk of chronic diseases (Bramley, 2000; Rao & Agarwal, 1999) such as cancers (Giovannucci, 1999) and cardiovascular diseases (Willcox, Catignani, & Lazarus, 2003). Humans are unable to synthesize lycopene de novo and therefore depend on their diet to supply this compound. The main dietary source Abbreviations: TG, triglyceride; MG, monoglyceride. ⁎ Corresponding author at: Kasteelpark Arenberg 22, Box 2457, B-3001 Leuven, Belgium. Tel.: + 32 16 32 15 85; fax: + 32 16 32 19 60. E-mail address: [email protected] (M.E. Hendrickx). 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.10.041

of lycopene are tomatoes and tomato-based products (Bramley, 2000). In this regard, bioavailability of lycopene is of importance. Carotenoid bioavailability includes (i) release of carotenoids from the food matrix and conversion into a potentially absorbable form (=bioaccessibility), (ii) absorption, (iii) metabolism, (iv) transport and tissue distribution, and (v) bioactivity (Faulks & Southon, 2005; Fernández-García et al., in press). Carotenoid uptake follows the same fate as lipids and they need to be incorporated into micelles to be absorbable (Borel, 2003). Next to bile salts and biliary phospholipids, mixed micelles contain free fatty acids and monoglycerids (MGs) resulting from hydrolysis of triglycerides (TGs) (Yonekura & Nagao, 2007). Therefore ingestion of fat along with carotenoids is thought to be crucial for the absorption of carotenoids (van het Hof, West, Weststrate, & Hautvast, 2000). The importance of dietary fat for the bioaccessibility and bioavailability of β-carotene is already well established. In vitro, it was demonstrated that the presence of rapeseed oil during cooking of carrot pulp and the presence of sunflower oil during cooking of green leafy vegetables increased the release of β-carotene during digestion (Hedrén, Diaz, & Svanberg, 2002; Hedrén, Mulokozi, & Svanberg, 2002). Similarly, the micellarization of β-carotene was found to increase by the addition of olive oil to carrot samples during cooking (Hornero-Méndez & Mínguez-Mosquera, 2007) and by the presence of a minimal amount of lipid in a salad puree (Huo, Ferruzzi, Schwartz, & Failla, 2007). In vivo, Brown et al. (2004) observed that the appearance of β-carotene in plasma chylomicrons was higher after the ingestion of salads with full fat than with reduced fat salad dressing. In addition, higher plasma

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β-carotene levels were found after intake of a high fat diet compared with a low fat diet (Dimitrov et al., 1988) and after consuming salad with avocado oil compared with without oil (Unlu, Bohn, Clinton, & Schwartz, 2005). For lycopene, some recent studies indicated that lipids can positively influence its bioaccessibility and bioavailability. After the in vitro digestion of a salad puree (Huo et al., 2007), a meal containing courgette, red pepper and spinach (O'Connell, Ryan, O'Sullivan, Aherne-Bruce, & O'Brien, 2008) and a tomato soup (Colle et al., 2011), a significantly enhanced micellarization of lycopene was found in the presence of oil. Likewise, in human subjects, the lycopene bioavailability was improved by adding avocado oil to a salad (Unlu et al., 2005) and by using full fat salad dressing instead of reduced fat salad dressing (Brown et al., 2004). What is less clear, is the amount of dietary fat needed for optimal carotenoid bioavailability. Only a small amount of fat is needed to ensure uptake. The minimum amount of fat required depends, however, on the physicochemical characteristics of the fat-soluble compounds (Roodenburg, Leenen, van het Hof, Weststrate, & Tijburg, 2000). Only one study (Huo et al., 2007) suggested that the bioaccessibility of carotenoids increases more by the consumption of long chain-triglycerides than when short/medium chain triglycerides are ingested. Furthermore, controversial results have been published on the effect of the fatty acid degree of saturation on the carotenoid bioavailability (Clark, Yao, She, & Furr, 2000; Hu, Jandacek, & White, 2000; Huo et al., 2007; Lee, Thurnham, & Chopra, 2000). Therefore, the present study was designed to elucidate the effect of the type and quantity of lipids, added upon digestion of raw tomato pulp, on the bioaccessibility of lycopene. A tomato pulp containing 5% of coconut oil, palm oil, cocoa butter, olive oil, sunflower oil or fish oil was prepared to investigate the effect of the type of lipids. Moreover, different amounts (0–10%) of coconut oil, olive oil and fish oil were added to evaluate the effect of the quantity of the lipids. Lycopene bioaccessibility was studied by quantifying the amount of lycopene that was transferred from the food matrix to the aqueous micellar phase during in vitro digestion. 2. Materials and methods 2.1. Materials A number of oils and fats with different fatty acid composition were selected. Coconut oil, palm oil, olive oil, sunflower oil and fish oil were donated by Vandemoortele (Gent, Belgium). Cocoa butter was a kind gift of Barry Callebaut (Lebbeke-Wieze, Belgium). The fatty acid composition of the lipids is listed in Table 1. None of the lipids contained lycopene (analysed using RP-HPLC) (data not shown). Red ripe tomatoes (var. Patrona) were harvested in Spain in 2010. They Table 1 Fatty acid composition of lipids used. Fatty acid (%)

Coconut oil

Palm oil

Cocoa butter

Olive oil

Sunflower oil

Fish oil

C8 C10 C12 C14 C16 C18 C18:1 C18:2 C18:3 C20 C20:1 C20:5 C22 C22:6 Others

7.0 6.0 46.0 19.0 9.0 3.0 7.5 2.0 nd nd nd nd nd nd 0.5

nd nd 0.2 1.0 43.0 4.5 40.4 9.0 0.1 0.4 0.1 nd 0.1 nd 1.2

nd nd nd 0.1 24.9 37.8 33.3 2.7 1.2 nd nd nd nd nd nd

nd nd 0.0 0.0 11.4 2.9 74.4 8.9 0.6 0.5 0.3 nd 0.2 nd 0.8

nd nd 0.0 0.1 6.0 4.0 28.6 60.0 0.3 0.1 0.1 nd 0.8 nd nd

nd nd nd 5.6 15.4 3.3 13.9 1.6 1.0 1.8 5.3 9.4 1.4 13.5 27.8

nd: not detected.

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were washed, dried, quartered, frozen in liquid nitrogen and stored at −40 °C. Upon use, a tomato pulp free from seeds and skin was prepared. Hereto, the tomato quarters were thawed, mixed (3 times 5 s) (Büchi Mixer B-400, Flawil, Switzerland) and sieved (pore diameter 1 mm) in the absence of added lipids. All chemicals and reagents used were of analytical or HPLC grade. 2.2. In vitro digestion procedure To determine the lycopene bioaccessibility, a two step (gastric and small intestinal) in vitro digestion procedure (Colle, Van Buggenhout, Van Loey, & Hendrickx, 2010) was carried out. Immediately prior the in vitro digestion, a certain amount (0–10%) of lipid was added to freshly prepared tomato pulp so that the total sample weight was 5.0 g (tomato pulp and lipid). This was gently mixed with 5.0 ml stomach electrolyte solution (0.30% NaCl, 0.11% KCl, 0.15% CaCl2 2H2O, 0.05% KHPO4, 0.07% MgCl2 6H2O in water) and 5.0 ml NaCl/ ascorbic acid solution (0.9% NaCl, 1% ascorbic acid) in a brown falcon tube. The pH of this mixture was adjusted to pH 4 ± 0.05 with 1 M HCl or 1 M NaHCO3. Next, 5.0 ml pepsin solution (0.52% porcine pepsin in electrolyte solution) was added and the headspaces of the tubes were flushed with nitrogen. The samples were incubated at 37 °C during 30 min, while shaking end-over-end. Subsequently, the pH was lowered to pH 2 ± 0.05. The headspaces were flushed again with nitrogen prior to continuing incubation for 30 min at 37 °C. To mimic the small intestinal digestion, the pH was raised to pH 6.9 ± 0.05 and a solution of pancreatin, lipase and bile salts (3 ml) (0.4% porcine pancreatin, 0.2% porcine pancreas lipase, 2.5% bile extract, 0.5% pyrogallol and 1% α-tocopherol in water) was added. Once again, the headspaces of the tubes were flushed and the samples were incubated during 2 h at 37 °C. Following exposure to the digestive conditions, the aqueous micellar phase was separated from the undigested oil droplets and from the undigested tomato pulp by centrifugation (L7 Ultracentrifuge, Beckman, Palo Alto, Calif., U.S.A.) at 165,000 g during 1 h and 5 min at 4 °C. The aqueous fraction was collected and filtered (Chromfil PET filters, 0.20 μm pore size, 25 mm diameter) to remove any crystalline lycopene or lipid. For each sample, the in vitro digestion procedure was carried out in quadruple. The lycopene content in the aqueous phase was determined as described below. 2.3. Lycopene quantification The total lycopene content in the tomato pulp and in the micellar phase after digestion was determined spectrophotometrically following extraction. The extraction procedure was based on the method described by Sadler, Davis, and Dezman (1990). The micellar phase or 2.0 g tomato pulp was stirred with 0.5 g NaCl and 50 ml extraction solvent during 20 min at 4 °C. The extraction solvent consisted of hexane, acetone, ethanol (50:25:25 v:v:v) and butylated hydroxytoluene (0.1%). Next, reagent grade water (15 ml) was added and stirring was continued for 10 min. The hexane layer (25 ml), containing lycopene, was separated from the polar phase in a separation funnel, collected and filtered (Chromfil PET filters, 0.20 μm pore size, 25 mm diameter). The extraction procedure was performed under subdued light to prevent lycopene degradation. Immediately after extraction, the absorbance of lycopene was measured at 472 nm using hexane as a reference. The total lycopene concentration was calculated using the Beer–Lambert law:

C¼

A  104 E1% 1 cm  l

With C the total lycopene concentration (μg/ml), A the absorbance at 472 nm, E11%cm the extinction coefficient of lycopene in

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hexane (=3450 (g/100 ml)− 1 cm− 1) (Hart & Scott, 1995) and l the path length (=1 cm). 2.4. Data analysis Differences in lycopene bioaccessibility were analyzed by Tukey's Studentized Range Test (statistical software package SAS, version 9.2, Cary, N.C., U.S.A.). The level of significance was set at P b 0.05. 3. Results and discussion

Bioaccessible total lycopene content (µg/g tomato pulp)

In a first experiment, the influence of the type of added lipid on the micellarization of lycopene was investigated. Hereto, 5% of coconut oil, palm oil, cocoa butter, olive oil, sunflower oil and fish oil were added to freshly prepared tomato pulp. The selected lipids clearly differed in fatty acid composition (Table 1). The total lycopene content of the different batches of tomato pulp ranged from 40.2 ± 0.8 to 53.9 ± 3.8 μg/g. Fig. 1 shows the bioaccessible total lycopene content of the tomato samples to which 5% of lipid was added. To get a better idea of the bioaccessible fraction of lycopene, lycopene bioaccessibility was also expressed relative to the lycopene present in the sample. In this way, the differences in lycopene content of each batch of prepared tomato pulp was taken into account. The relative lycopene bioaccessibility is depicted in Fig. 2. In plain tomato pulp, the amount of lycopene incorporated in micelles was very low, being 1.0 ± 0.2 μg/g or 1.9 ± 0.5%. Gupta, Kopec, Schwartz, and Balasubramaniam (2011) reported even less than 0.5% micellarization of lycopene from raw tomato juice. All tested lipids significantly increased the lycopene bioaccessibility after adding 5% to the tomato pulp. This was expected since lipids play a role in the release of carotenoids from plant matrices, in the transfer to the fat phase of the gastrointestinal contents and in the solubilization in micelles (Brown et al., 2004). Previous studies already revealed that the presence of oil during cooking vegetables can positively influence the carotenoid bioaccessibility (Colle et al., 2011; Hedrén, Diaz, & Svanberg, 2002; Hedrén, Mulokozi, & Svanberg, 2002; Hornero-Méndez & MínguezMosquera, 2007). Also co-digestion of raw vegetables with lipids resulted in an enhanced carotenoid bioaccessibility or bioavailability compared to digestion in the absence of lipids (Brown et al., 2004; Huo et al., 2007; O'Connell et al., 2008; Unlu et al., 2005). With regard to lipid type, the largest increase in lycopene bioaccessibility was noticed after adding 5% of sunflower oil, followed by olive oil and cocoa butter (not all differences were significant). A slightly smaller increase was observed when fish oil, coconut oil and palm oil were used (Figs. 1 and 2). This difference might be related to the fatty acid composition of the applied lipids. The largest bioaccessibility was obtained with lipids containing a large fraction of C18, C18:1 and/or C18:2 fatty acids while lipids with a considerable amount of C12 and C16 fatty acids resulted in a lower lycopene bioaccessibility. 7

A

6 AB

5 BC

4 3 2

B

CD D E

1 0

Fig. 1. Bioaccessible total lycopene content (mean ± standard deviation) (μg/ g tomato pulp) in tomato pulp containing no or 5% (w/w) added lipids. Significant differences (P b 0.05) are indicated with different letters.

Bioaccessible lycopene fraction (%)

252

14 A

12 10

AB BCD

8

CD

6 4

ABC D

E

2 0

Fig. 2. Relative bioaccessible total lycopene content (mean ± standard deviation) (%) in tomato pulp containing no or 5% (w/w) added lipids. Significant differences (P b 0.05) are indicated with different letters.

This is consistent with the study of Huo et al. (2007) who found that micellarization of lycopene from a salad puree significantly increased with increasing acyl chain length of TGs added (2.5% v/w). Similarly, the chylomicron β-carotene response was remarkably higher when β-carotene was ingested with long chain TGs instead of medium chain TGs (Borel et al., 1998). To explain this, it is important to understand that the core of micelles consists of MGs and free fatty acids resulting from lipid hydrolysis. Also, carotenoids are accommodated in the micelle core (Yonekura & Nagoa, 2007). Furthermore, mixed micellar species present in the aqueous phase are in equilibrium with multilamellar and unilamellar vesicles (Porter & Charman, 2001). When the lipid load is low, on the one hand, long chain TGs are almost completely digested and the long chain free fatty acids and MGs result in effective swelling of the mixed micellar species. On the other hand, medium chain TGs will be completely digested and their lipid hydrolysis products will be completely incorporated in mixed micelles without forming vesicles. However, micellar species formed by medium chain fatty acids and MGs reflect in poor swelling of the micelles, which is accompanied with relatively poor solubilizing capacity, possibly leading to lycopene precipitation (Porter et al., 2004). This poor swelling of micelles might explain why limited lycopene bioaccessibility was observed for lipids containing many C12 and C16 fatty acids. It is important to note that when the lipid load is high, the picture can totally change. Because long chain TGs are hydrolysed to a smaller extent than medium chain TGs (Li, Hu, & Mcclements, 2011), medium chain TGs can still be completely digested at high lipid load while the digestion of long chain TGs will be incomplete. Consequently, medium chain TGs might outperform long chain TGs in increasing lycopene bioaccessibility because carotenoids will rather stay in the undigested oil phase than to be solubilized in mixed micelles (Malaki Nik, Corredig, & Wright, 2010). Furthermore, Figs. 1 and 2 show that the lycopene bioaccessibility was not significantly different after adding olive oil or sunflower oil. This suggests that the in vitro transfer of lycopene from chyme to mixed micelles was not affected by the degree of unsaturation in the added TGs, which is in line with the results of Huo et al. (2007). Also, the in vivo study of Lee et al. (2000) supports this observation, since they found similar lycopene plasma levels in humans after consuming tomato products together with olive oil or together with sunflower oil. In contrast, the absorption of lycopene into the mesenteric lymph duct of rats was larger when an olive oil emulsion (65% C18:1) was directly infused into the duodenum of rats compared with a corn oil emulsion (58% C18:2) (Clark et al., 2000). Moreover, the appearance of β-carotene in chylomicrons and in each VLDL subfraction was lower after β-carotene was ingested with a meal containing sunflower oil (69% C18:2) than after ingestion with beef tallow (45% C18:1) (Hu et al., 2000).

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6 5 4 B

D E

0 0

1

2

5

10

Amount of added lipids (%)

Bioaccessible total lycopene content (µg/g tomato pulp)

B 6

A A

5 4

C

2 D

0 0

1

2

5

10

Amount of added lipids (%)

C Bioaccessible total lycopene content (µg/g tomato pulp)

Bioaccessible lycopene fraction (%)

8

A

6 B

4 2

6 5 4 A

3

AB BC

2

C

D

0 0

1

B

C D

0 0

1

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5

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Amount of added lipids (%)

14 A

12 10

B

8

B C

6 4 2

D

0 0

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Amount of added lipids (%)

14 12 10 8 6

A

AB

4 2

BC

C

5

10

D

0 0

1

2

Amount of added lipids (%)

B

3

1

10

C

C

2

1

12

A

3

1

14

B

Bioaccessible lycopene fraction (%)

Bioaccessible total lycopene content (µg/g tomato pulp)

A

A

Bioaccessible lycopene fraction (%)

Interesting from Figs. 1 and 2 is the rather low lycopene bioaccessibility obtain after supplementing fish oil, which might be due to the considerable amount of C20:5 and C22:6 fatty acyl chains in fish oil (Table 1). Long chain polyunsaturated fatty acids esterified in TGs are hydrolysed more slowly in vitro by porcine pancreatic lipase (Carlier, Bernard, & Caselli, 1991; Ikeda et al., 1995; Mu & Hoy, 2004) resulting in less hydrolytic products compulsory for micelle formation. In the second experiment, the influence of the quantity of added lipid was investigated. Three lipids were selected: one with mostly medium chain fatty acids (coconut oil), one with mainly long chain fatty acids (olive oil) and fish oil with its characteristic C20:5 and C22:6 fatty acids. Prior to the bioaccessibility measurement, 0, 1, 2, 5 and 10% of these oils were added to freshly prepared tomato pulp. Fig. 3 shows the bioaccessible total lycopene content as a function of the amount of added lipids. The relative lycopene bioaccessibility is presented in Fig. 4. The results show that the lycopene bioaccessibility increased with increasing amount of coconut oil added to tomato pulp. For olive oil and fish oil, the highest lycopene bioaccessibility was obtained after adding respectively 2% and 1% of oil. This is in agreement with the results of Huo et al. (2007). In this study, 0.25 to 2.5% of coconut oil and

253

2

5

10

Amount of added lipids (%) Fig. 3. Bioaccessible total lycopene content (mean ± standard deviation) (μg/ g tomato pulp) in tomato pulp containing different amounts of coconut oil (A), olive oil (B) and fish oil (C). Significant differences (P b 0.05) are indicated with different letters.

Fig. 4. Relative bioaccessible total lycopene content (mean ± standard deviation) (%) in tomato pulp containing different amounts of coconut oil (A), olive oil (B) and fish oil (C). Significant differences (P b 0.05) are indicated with different letters.

canola oil was added to salad puree. On the one hand, they observed that lycopene micellarization was not significantly different when 0.25 up to 1.0% coconut oil was added but increased markedly when the salad puree contained 2.5%. On the other hand, the micellarization of lycopene was maximal after the addition of 1.0% of canola oil. The lycopene bioaccessibility from an oleoresin was also affected negatively by an increase of the amount of sunflower oil (FernándezGarcía, Mínguez-Mosquera, & Pérez-Gálvez, 2007). The results in Figs. 3 and 4 clearly indicate that the lycopene bioaccessibility depends both on the type and the quantity of the lipid supplemented upon digestion of raw tomato pulp. When 1 up to 5% of lipid was added, the highest bioaccessibility was obtained with olive oil. However, this was no longer the case after the addition of 10% of lipid, for which the supplementation of coconut oil resulted in the highest lycopene bioaccessibility. The latter observation confirms the hypothesis explained above. When the lipid load was low (1–5%), lipid hydrolysis was complete (no large oil droplets observed after ultracentrifugation). The more swollen micelles formed from long chain fatty acids and MG's coming from olive oil resulted in a higher lycopene solubilization capacity compared with the poorly swollen

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micelles constructed from the hydrolysis products of medium chain TGs, as found in coconut oil. However, when the lipid load was high (10%), complete digestion of medium chain TGs (coconut oil) could lead to vesicle formation with high lycopene solubilization capacity while the hydrolysis of long chain TGs was incomplete (large oil droplets observed after ultracentrifugation), leaving a co-existing lipid phase with high affinity for lycopene (Porter et al., 2004). It is important to keep in mind that throughout the presented study the in vitro digestion was carried out with fixed amounts of bile and digestive enzymes. It is unclear whether the ratio lipid:bile or lipid:lipase was a limiting factor for micellarization. It is known that in vivo intake of dietary lipids stimulates secretions from the gall bladder and pancreas (Porter & Charman, 2001). 4. Conclusion The addition of 5% lipid, prior to the in vitro digestion of raw tomato pulp, significantly increased the lycopene bioaccessibility. The increase was more pronounced when cocoa butter, olive oil and sunflower oil were used compared with tomato pulp supplemented with fish oil, coconut oil or palm oil. Differences were explained by the fatty acid composition of the lipid. The incorporation of lycopene into micelles was affected by the length of fatty acyl chains in TGs while the degree of unsaturation was shown not to be a determining factor. Adding different amounts (0–10%) of coconut oil, olive oil and fish oil to tomato pulp demonstrated that not only the type of lipid but also the quantity influenced the lycopene bioaccessibility. Over the entire concentration range, increasing the amount of coconut oil increased the lycopene bioaccessibility, while the highest bioaccessibility was found using 1 and 2% of respectively fish oil and olive oil. Interestingly, the best performing lipid, regarding lycopene micellarization, depended on the amount added. Although valuable, the results of the current study should be handled with care since they result from a static in vitro experiment with fixed amounts of digestive enzymes. There are several potential events where dietary lipids could influence carotenoid absorption. Firstly, lipids cause alterations in gastric transit (Porter & Charman, 2001). Secondly, they provide a hydrophobic pool in which carotenoids can dissolve (Borel, 2003), improving the release of carotenoids from the matrix (Brown et al., 2004). Moreover, lipids high in polyunsaturated fatty acids might promote carotenoid oxidation in the chyme resulting in less carotenoid available for absorption (Clark et al., 2000). In addition, the secretion of biliary fluid is stimulated upon increased consumption of lipids enhancing lipid digestion (Porter & Charman, 2001) and facilitating micelle formation (Borel, 2003). Furthermore, polyunsaturated fatty acids have higher affinity than more saturated fatty acids for the fatty acid binding protein and are thus more rapidly transported within the mucosal cell (Hu et al., 2000). Therefore, the findings of the current study should be confirmed by human studies. Acknowledgements The authors would like to thank Margot De Haes for the technical assistance and Microbial and Molecular Systems (Kulak, Kortrijk, Belgium) for the fatty acid analysis of the fish oil. This research was financially supported by the Agency for Innovation by Science and Technology in Flanders (IWT-Vlaanderen). S.V.B is a postdoctoral researcher funded by the Research Foundation-Flanders (FWO). References Borel, P. (2003). Factors affecting intestinal absorption of highly lipophilic food microconstituents (fat-soluble vitamins, carotenoids and phytosterols). Clinical Chemistry and Laboratory Medicine, 41(8), 979–994. Borel, P., Tyssandier, V., Mekki, N., Grolier, P., Rochette, Y., exandre-Gouabau, M. C., et al. (1998). Chylomicron beta-carotene and retinyl palmitate responses are

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