Comparative study of the bioaccessibility of the colorless carotenoids phytoene and phytofluene in powders and pulps of tomato: microstructural analysis and effect of addition of sunflower oil

1 COMPARATIVE STUDY OF THE COLORLESS CAROTENOIDS 1 PHYTOENE AND PHYTOFLUENE BIOACCESSIBILITY IN POWDERS AND 2 PULP OF TOMATO: MICROSTRUCTURAL ANALYSIS AND EFFECT OF 3 ADDITION OF SUNFLOWER OIL 4 5 Paula Mapelli-Brahm, Carla M. Stinco, Antonio J. Meléndez-Martínez 6 7 Food Colour & Quality Lab., Area of Nutrition & Food Science, Universidad de 8 Sevilla, Seville, Spain. 9 10 Abstract 11 The objective was to assess the potential bioavailability of phytoene (PT) and 12 phytofluene (PTF) from tomato powders used as raw materials for supplements as 13 compared to the pulp of a common and a cherry tomato. PT and PTF are attracting 14 much interest nowadays as they can provide health and cosmetic benefits. PT and PTF 15 levels in the more concentrated powder were up to 1000 times higher than that in the 16 tomatoes. The bioaccessibility from the powders was lower as compared to the tomato 17 fruits and increased markedly when sunflower oil was added. However, the best source 18 of potentially absorbable PT and PTF (0.5 and 2 mg/g respectively) was by far the 19 powder with higher levels of them. This result could be due to the higher carotenoid 20 concentration in the powder, the reduction of the particle sizes, and the rupture of cell 21 structures compared to the pulps. 22 23 Keywords: bioavailability, chromoplasts, colorless carotenoids, digestion, matrix effect, 24 plastids. 25 2 1. INTRODUCTION 26 Carotenoids are lipophilic compounds responsible for the colouring in many fruits 27 and vegetables that constitute everyday consumer foods. It is widely acknowledged that 28 the proper intake of carotenoids can provide health benefits for humans. 1,2 In contrast to 29 the other major bioavailable carotenoids in humans, the colourless phytoene (PT) and 30 phytofluene (PTF) have been little studied in the context of the diet nutrition and health, 31 although they are eliciting a great interest in the carotenoid field as evidence is 32 accumulating that they may provide health and cosmetic benefits. 3 These carotenoids 33 seem to have anti-inflammatory and anti-oxidant properties among others and their 34 consumption may be related to the reduction of the risk of developing certain cancers 35 and other diseases. 4–6 Importantly, PT and PTF are present in a wide variety of fruits 36 and vegetables, as tomato and tomato-based food products, 7,8 carrots, citrus9, apricots 37 and several other common and exotic foods. 6 It has been estimated that the daily per 38 capita intake of PT and PTF combined represents 16% of the total dietary intake of 39 carotenoids in Luxembourg. 10 However, in addition to intake, carotenoid bioavailability 40 must also be considered when it comes to gain insight into their biological effects.11 41 One of the key factors in the bioavailability of lipophilic compounds like carotenoids is 42 the release from the food matrix and their incorporation into mixed micelles. The 43 bioaccessibility, i.e. the percentage of carotenoid content in a food that is incorporated 44 into mixed micelles and is available for the absorption, can be evaluated by in vitro 45 models. It is accepted that the carotenoid bioaccessibility data obtained with in vitro 46 models are good to predict the bioavailability of these compounds in humans and so the 47 in vitro models seems to be a good choice for previous studies and comparisons 48 between different matrices. 12 49 3 The processing and storage of food are important processes to take into account 50 since they can lead to chemical degradation or physical losses of the carotenoids while 51 they can also increase their bioaccessibility, and therefore their bioavailability, as a 52 consequence of the disruption of the cell walls of plant tissues, the dissociation of the 53 nutrient-matrix complexes, or the transformation into more active molecular 54 structures.11 55 Dehydration is a common technique that has been used for a long time to improve 56 the preservation, storage and transportation of foods. Such practice leads to the 57 concentration of the food constituents as a result of the elimination of water. On the 58 other hand, the preparation of powders from fresh foods results in a decrease in particle 59 size and microstructural changes, which are known to affect the release of carotenoids 60 from the matrix and, hence, increase their bioaccessibility. 13 The combination of these 61 two effects, i.e. increase of concentration and enhancement of the release from the 62 matrix can result in a marked increase of the actual amount of carotenoids that can be 63 absorbed and be available to exert their health-promoting biological actions. Due to all 64 these facts, dehydrated and ground foods offers many advantages to be used as raw 65 materials in the functional food, cosmetic or pharmaceutical industries. However, 66 surprisingly, the study of the potential bioavailability of carotenoids from powdered 67 food products has been ignored. More insight in this regard is important not only for 68 their importance as raw materials for other products, but also because dehydrated fruits 69 are increasingly common in supermarkets. 70 In this context, the aim of this study was to assess the bioaccessibility of PT and 71 PTF from two types of tomato powders used as raw materials for the development of 72 other products and to compare it with those from the pulp of a common tomato and a 73 cherry tomato commercially available from retailers. To gain further insight into the 74 4 factors involved in the bioaccessibility of these compounds, the effect of adding 75 sunflower oil as a source of fat to favour bioaccessibility was also studied. Sunflower 76 oil was chosen as a source of fat because it is widely consumed and does not contain 77 detectable amounts of carotenoids14, as the presence of other carotenoids could affect 78 the bioaccessibility of PT and PTF. Additionally, this oil is rich in unsaturated fatty 79 acids, which seem to be related to a greater increase in the carotenes bioaccessibility in 80 comparison with the saturated fatty acids 15 81 Furthermore, the samples were observed under the transmission electron 82 microscope and the epifluorescence microscope to assess differences in the structure 83 and integrity of the plastids and the distribution of the compounds of interests in the 84 different materials under study. 85 86 2. MATERIALS AND METHODS 87 2.1. Chemicals and standards 88 All the solvent used, which were of analytical grade or higher, were purchased from 89 Panreac (Barcelona, Spain) or Sigma-Aldrich (Bornem, Belgium). The digestive 90 enzymes, i.e. pepsin (porcine, 367 units/mg solid, measured as TCA- soluble products 91 using hemoglobin as substrate) and pancreatin (porcine, 8 × USP specifications of 92 amylase, lipase and protease), were also from Sigma-Aldrich. The standards of the 93 colourless carotenoids, i.e. phytoene and phytofluene, were isolated as previously 94 described 16 and their purity (> 95%) were checked by HPLC. The reagents for 95 obtaining the Transmission Electron Microscopy (TEM) images were acquired from 96 Ted Pella (Redding, USA) or Electron Microscopy Sciences (PA, USA). 97 2.2. Samples 98 5 Two types of tomato powders were analysed. One had a higher carotenoid 99 concentration (TPH) and the other a lower concentration (TPL). Tomato powders tested 100 are produced from proprietary, non-GMO tomatoes (courtesy of IBR. LTD), which 101 undergo concentration processes followed by freeze-drying and grinding, resulting in a 102 carotenoid-rich natural tomato powder. Differences in carotenoid contents between the 103 different products derives from the different crops combined into the respective tomato 104 powder grades. The pulps from ripe fruits of a common tomato (Solanum lycopersicum 105 var. daniela, hereafter named as tomato) and of a vine cherry tomato (Solanum 106 lycopersicum var. cerasiforme, hereafter named as cherry) were also evaluated for 107 comparison. The fruits were obtained from a supermarket located in Seville (Spain). 108 2.3. Carotenoid extraction from matrices 109 Whenever possible, the samples were kept on ice and the extraction was carried out 110 in dim light. The extraction method was based on the protocol of Stinco et al. 17 with 111 some minor modifications as follows. An specific amount of each sample (0.1 g for 112 cherries, tomatoes and TPL and 0.005 g for TPH) was weighed into a Eppendorf tube 113 and 750 µL of a mixture of trichloromethane (TCM) : methanol (2:1, v/v) and 250 µL 114 of distilled water was added. This solution was shaken vigorously for c.a. 1 min, 115 sonicated for 5 minutes, and centrifuged at 14,000 rpm (Microfuge 22R, Beckman 116 Coulter, Madrid, Spain) for 5 min, and at 4 °C to promote phase separation. Then, the 117 lower organic phase was transferred to a new Eppendorf tube. The aqueous phase was 118 re-extracted with 500 µL of TCM until colourless of the extraction solvent. The lower 119 phases were combined all together. The combined organic phase was washed with water 120 and a solution of saturated sodium chloride. The extract was concentrated to dryness in 121 a rotating evaporator at 25 °C (Eppendorf Concentrator Plus, Hamburg, Germany) and 122 6 was kept at - 80 °C under nitrogen until the HPLC analysis (for no more than one 123 week). 124 2.4. In vitro gastrointestinal digestion 125 The digestion protocol followed was based on that described by Mapelli et al. 16 126 and is detailed below. Moreover, to study the effect of the addition of fat on the 127 bioaccessibility, the same digestion protocol was carried out with the tomato powder 128 samples but adding 200 µL of sunflower oil to the samples immediately before adding 129 the gastric juice. Taking into account that tomatoes usually have a water content of ca. 130 95%, the amount of oil added corresponded to approximately 5% volume/wet weight. 18 131 2.4.1. Gastric phase 132 A specific amount of each sample was weighed (specifically 0.25 g of the powders 133 samples and 2 g of the cherry and tomato pulp) in a 50 mL Falcon tube. Fifteen 134 millilitres of physiological saline and two millilitres of a pepsin solution (40 mg/mL in 135 0.1 M HCl, prepared the day of usage) were added. The pH was then adjusted to 3. The 136 sample was incubated in a rotating incubator (Max Q5000 shaker, Labware, Madrid, 137 Spain) at 37 °C at 100 strokes per minute (spm) for 1 h to simulate the gastric digestion. 138 2.4.2. Intestinal phase 139 The sample was placed in ice and a mixture of 4 mg/mL of pancreatin and 24 140 mg/mL of bile extract in 0.1 M of NaHCO3 (prepared the day of the analysis) was 141 incorporated into the gastric digesta. The pH was adjusted to 7 and after the sample was 142 brought with physiological saline to a volume of 50 mL. Then, the intestinal phase was 143 simulated by incubation the samples during 2 h at 37 °C and at 100 spm in the rotating 144 incubator. Twelve millilitres of the digesta were pipetted into a Falcon tube and were 145 centrifuged during 1 h at 5000 g and at 20 °C. Six millilitre aliquot of the aqueous 146 micellar phase was taken with a syringe and was filtered (0.2-μm nylon membrane 147 7 filter) in order to isolated the mixed micelles. Then, a 4 mL aliquot was stored in 148 another Falcon tube under a nitrogen atmosphere at -80 °C for the subsequent 149 carotenoid extraction on the following day. 150 2.4.3. Carotenoid extraction from digesta 151 For the carotenoid extraction 4 mL of a mixture of TCM : methanol (2:1, v/v) was 152 added to the 4 mL aliquot of the isolated mixed micelles solution. After stirring for 1 153 min and centrifuging at 4000 rpm at 4 °C for 5 min, the lower phase with carotenoids 154 was transferred to a new 50 mL Falcon tube. The extraction was repeated with 4 mL of 155 TCM until the colour exhaustion of the extraction solvent. The lower phases were 156 combined all together and then this combined phase was washed with water and a 157 solution of saturated sodium chloride. Lastly, the organic phase was concentrated to 158 dryness and the extract was kept at – 80 °C under nitrogen until the HPLC analysis. 159 2.5. HPLC analysis 160 Carotenoid concentrations were determined by High Performance Liquid 161 Chromatography (HPLC) by using an Agilent Technologies 1100 system. The 162 separation was carried out by gradient elution with methanol (MeOH), methyl-tert-butyl 163 ether (MTBE), and water according to Stinco et al. 19 with minor modifications. 164 Ammonium acetate was added to the methanol at a concentration of 0.1% (p/v) to avoid 165 carotenoid losses during the analysis. A 3 µm-C30 column (150 × 4.6 mm, YMC 166 America, Inc., Allentown, PA, USA) was used. The column was kept at 30 °C and the 167 flow rate was 1 mL/min. The diode array detector was set at 286 and 350 nm for the 168 detection of phytoene and phytofluene, respectively. To identify the geometrical 169 isomers of PT and PTF, their spectral and chromatographic characteristics were 170 compared with those reported in a previous work. 20 The quantification was carried out 171 by external calibration. 172 8 2.6.Transmission Electronic Microscopy (TEM) 173 The protocol followed to prepare the samples to obtain the images by TEM was 174 similar to that described by Mapelli et al. 21 with slight modifications. For the fruits, a 175 small piece of the pulp was taken in an Eppendorf tube, was centrifuged (microfuge 176 22R, Beckman Coulter, Krefeld, Germany) at 2000 rpm for 1 min at 4 °C and the 177 supernatant was discarded. The sample was fixed with 1 mL of Karnovsky fixative 178 (0.5% glutaraldehyde, 2.5% formaldehyde) for 5 h at room temperature in the dark. 179 Then, the sample was centrifuged at 2000 rpm for 1 min and the upper phase was 180 discarded. The matrix was washed with 0.1 M sodium cacodylate buffer (pH 7.4) three 181 times. The sample was post-fixed with 2% osmium tetraoxide in the buffer for 1 h at 182 25 °C after which it was washed with MilliQ water three times for 20 min at 4 °C. The 183 staining was carried out with 2% uranyl acetate for 2 h at 25 °C. After that, the sample 184 was dehydrated with acetone in series at 25 °C (starting with 50% acetone in ethanol 185 and ending with 100% acetone). Samples were embedded in Spurr resin in series at 186 25 °C (starting with a 3: 1 mixture of acetone: Spurr and ending with 100% Spurr). The 187 sample was polymerized for 13 and a half hours in an oven at 70 °C. To select the areas 188 of interest semi-thin sections of approximate 350 nm were observed under a 189 microscope. Subsequently, ultrathin sections (70 nm) were cut with an ultramicrotom 190 (Leica UC7, Wetzlar, Germany). As a sample holder, 200-mesh carbon-coated copper 191 grids were used. The images were taken in a Zeiss Libra 120 microscope (Oberkochen, 192 Germany) equipped with a digital SSCCD camera. The same protocol was followed for 193 the powders but, before the post-fixation, cacodylate buffer with 3% agarose was added 194 to the sample. 195 2.7. Epifluorescence Microscopy 196 9 PTF can be detected by fluorescence microscopy as it emits light at approximately 197 510 nm when is excited with near-UV light. 6 The procedure and equipment used to 198 obtain the images were the same as recently described by Mapelli et al. 21 199 2.8. Statistical analysis and calculations 200 All samples were extracted and digested in triplicate. The samples were digested in 201 order to determine the bioaccessibility and the amount of carotenoid per gram of 202 product that is potentially absorbable. Although there is still not a consensus to name 203 this latter parameter, in this paper it will be referred to as carotenoid bioaccessible 204 content (CBC). CBC were calculated by determining the absolute carotenoid levels in 205 the micellar fractions. Bioaccessibility was calculated as the percentage of carotenoids 206 content that remained in the micellar aqueous fraction after centrifugation in relation to 207 the respective initial content in the food matrices. 208 One-way analysis of variance (ANOVA) were performed in order to detect 209 statistically significant differences among samples and, when it was necessary, 210 Bonferroni post hoc test were used to determine between which samples were these 211 differences occurred. For the statistical analysis the IBM SPSS Statistics 20® software 212 (SPSS Inc., 2012) was used. 213 214 3. RESULTS AND DISCUSSIONS 215 3.1. Carotenoid profile and content 216 The main isomer of PT in all the samples was the 15-cis-PT, the predominant one 217 in most carotenogenic organisms. 22 The percentage of the 15-cis isomer of PT in 218 relation to the total content of PT was as follows: 100% for tomato and cherry, 98.5% 219 for TPL, and 95.7% for TPH. 220 16 Thus, the increases in the bioaccessibility due to the addition of 5% oil ranged between 347 ca. 3.1 and 3.2-fold times for PT and between 3.5 and 4.2-fold times for PTF. Similarly, 348 Schweiggert et al. observed that the bioaccessibility of lycopene from tomato pulp 349 increased 3.3-fold times when sunflower oil was added to the sample.40 A significant 350 increase in the bioaccessibility of β-carotene from tomato with the addition of the oil 351 was also found in this study. Also, in agreement with our results, it has been observed 352 that the bioaccessibility of β-carotene and lutein from drumstick leaves powders was 353 greatly increased by adding peanut oil. More specifically, increments of 84.2-fold and 354 of 1.8-fold were found for β-carotene and for lutein, respectively. 41 Similar results were 355 also found with lutein and zeaxanthin from the microalgae Scenedesmus almeriensis 356 when olive oil was added. 18 357 Recently, it has been observed that the increase in the bioaccessibility of 358 carotenoids with the addition of fat depends on the type of fat. 42 Thus, the increase in 359 the bioaccessibility of the carotenes seems to be higher when the added fat is rich in 360 unsaturated fatty acids. This fact agrees with the great increase in the bioaccessibility of 361 PT and PTF observed in this work, since sunflower oil is rich in unsaturated fatty acids. 362 15 Nevertheless, taking into account that the bioaccessibility of β-carotene and lutein has 363 been shown to increase more with oleic acid (main fatty acid of olive oil) than with 364 linoleic acid (main fatty acid of sunflower oil), 43 even higher increases in the 365 bioaccessibility of PT and PTF might be expected if olive oil had been added instead of 366 sunflower oil to the powder samples. 367 The clear increases in the bioaccessibility of the carotenoids observed when 368 sunflower oil was added is related to the fact that fat is needed to form the micelles 369 required for the transport of carotenoids from the matrix to the enterocytes. 44 370 Physiologically, fat also stimulates digestive secretions that favour the formation of 371 17 micelles. 45 In other words, when fat is added, there is more “micellar material” for the 372 incorporation and transport of carotenoids. Thus, taking into account that dietary fats 373 can increase the carotenoids bioavailability in humans by increasing absorption, 374 possibly by enhancing micellization in the small intestine,36 it is possible that the 375 addition of sunflower oil led also to an increase in the bioavailability of PT and PTF 376 from the samples. 377 378 Table 2. Bioaccessibility of phytoene and phytofluene. 379 Sample Phytoene Phytofluene TPH 27.43 ± 0.52d 16.37 ± 0.52c TPL 29.59 ± 1.69d 21.32 ± 1.55c TPH + Sunflower oil 86.65 ± 1.77 bc 69.41 ± 1.16b TPL + Sunflower oil 92.09 ± 0.86b 74.90 ± 1.08b Tomato 102.22 ± 0.33a 95.77 ± 0.72a Cherry 82.15 ± 7.13c 79.38 ± 7.00b TPH, Tomato Powder with Higher carotenoid concentration; TPL, Tomato Powder with Lower 380 carotenoid concentration; Tomato, common tomato pulp; Cherry, cherry tomato pulp. Values are the 381 mean ± SD of 3 independent measures. Values within a column with different letters indicate statistically 382 significant differences (P ≤ 0.05). 383 384 3.5. Carotenoid bioaccessible content (CBC) 385 As stated before (Section 3.4.), the bioaccessibility of PT and PTF in the powders 386 were lower than that in tomatoes fruit, however, it is to be considered that 387 bioaccessibility was expressed as a percentage and that it is much more interesting to 388 18 assess which is the actual amount of a carotenoid that is incorporated into micelles and 389 potentially absorbable (CBC). 390 By analysing the results obtained without adding sunflower oil to the samples, it 391 was concluded that the source leading potentially to a higher absorption of PT and PTF 392 was by far TPH, with 2 and 0.5 mg of potentially absorbable PT and PTF per gram of 393 product, respectively (data not shown). As TPH was the sample with the lowest 394 bioaccessibility (Table 1) it can be concluded that this sample has the highest CBC 395 mainly due to its very high carotenoid content. These differences were even more 396 evident when sunflower oil was added to the powders because the bioaccessibility 397 increased considerably as it was discussed in the previous section. As a self-explanatory 398 example, the PT and PTF bioaccessible contents of the sample TPH with oil (the sample 399 with overall highest CBC) were 1149 and 753-fold higher relative to the tomato sample 400 (the one with overall lowest CBC). 401 To know which matrices are better sources of colourless carotenoids from a more 402 realistic point of view, the amount of bioaccessible carotenoid per food ration was 403 compared. The rations chosen were the same chosen by Granado-Lorencio et al., 46 that 404 is, 1 g of powders, 10 g of powders with oil, and 100 g of fruits. Also in this case, TPH 405 was the best source of PT and PTF (Figure 3). In particular, when sunflower oil was 406 added to this sample the difference between their CBC and that of the other samples 407 were astounding (Figure 3). The bioaccessible contents of PT and PTF per ration of 408 TPH with oil was 173 and 60-fold higher respectively than that per ration of a fresh 409 juice (250 mL) of a mutant orange (Pinalate) with a very high concentration of 410 colourless carotenoids, 21 and 18 and 19-fold higher than that per ration of a tomato 411 juice (250 mL), respectively. 16 412 19 With all this, it can be concluded that, in principle, the tomato powders are by far 413 better sources of colourless carotenoids compared to tomato and cherry pulps. Although 414 it is though that carotenoid bioaccessibility is well correlated with bioavailability, 11,36,47 415 further studies in vivo are necessary to confirm that, indeed, tomato powders can lead to 416 higher increases in circulating carotenoids than tomato fruits. As it can be readily 417 observed in Table 3, all the tomato and tomato-based products analysed are better 418 sources of potentially absorbable PT than of PTF. 419 420 421 422 423 Figure 3. Carotenoid bioaccessible content of phytoene and phytofluene (in mg) per 424 ration of food*. TPH, Tomato Powder with Higher carotenoid concentration; TPL, 425 Tomato Powder with Lower carotenoid concentration; Tomato, common tomato pulp; 426 Cherry, cherry tomato pulp; SO, Sunflower Oil. Values are the mean ± SD of 3 427 independent measures. * Ration of food: 1 g powders (TPH and TPL), or 10 g powders 428 with oil, or 100 g fruits (tomato or cherry). 429 430 4. CONCLUSIONS 431 Despite the lower carotenoid bioaccessibility of the powders in comparison with the 432 commercial tomato pulps studied, their bioaccessible content is much higher. Both the 433 disruption of the cell structures that suffered the samples as a result of the dehydration 434 0 10 20 30 40 50 60 70 TPH + SO TPH TPL + SO TPL Cherry Tomato Carotenoid bioaccessible content per ration of food* Phytoene Phytofluene 20 and powdering and the higher concentration of PT and PTF in the powders compared to 435 the pulps may contribute to this. Interestingly, adding sunflower oil to the samples can 436 increase the already high carotenoid bioaccessibility of such concentrated sources of PT 437 and PTF, and hence their bioavailability. With all this, tomato powders, which could be 438 used as ingredients of supplements (or related compounds) or incorporated in a 439 functional food, seem to be a markedly richer source of potentially absorbable 440 colourless carotenoids in comparison with fresh common and cherry tomatoes. 441 442 CONFLICT OF INTEREST 443 Antonio J. Meléndez-Martínez is a member of the advisory board of IBR – 444 Israeli Biotechnology Research, Ltd. (Yavne, Israel). 445 446 ACKNOWLEDGEMENTS 447 This work was partially funded by IBR – Israeli Biotechnology Research, Ltd., 448 Yavne, Israel. The authors also acknowledge funding from the Andalusian Council of 449 Economy, Innovation, Science and Employment (ref. CAROTINCO-P12-AGR-1287) 450 and Carotenoid Network: from microbia and plants to food and health (CaRed), funded 451 by the Spanish Ministry of Economy and Competitiveness (BIO2015-71703-REDT). 452 We thank the technical staff of the services of Biology and Microscopy (SGI, 453 Universidad de Sevilla). 454 REFERENCES 455 1 G. Britton, S. Liaaen-Jensen and H. Pfander, in Carotenoids, eds. G. Britton, S. 456 Liaaen-Jensen and H. Pfander, Birkhäuser, Basel, Switzerland, 2009, vol. 5. 457 21 2 M. Rodriguez-Concepcion, J. Avalos, M. L. Bonet, A. Boronat, L. Gomez-458 Gomez, D. Hornero-Mendez, M. C. Limon, A. J. Meléndez-Martínez, B. 459 Olmedilla-Alonso, A. Palou, J. Ribot, M. J. Rodrigo, L. Zacarias and C. Zhu, 460 Prog. Lipid Res., 2018, 70, 62–93. 461 3 A. J. Meléndez-Martínez, P. Mapelli-Brahm and C. M. Stinco, J. Food Compos. 462 Anal., 2018, 67, 91–103. 463 4 A. Kaulmann and T. Bohn, Nutr. Res., 2014, 34, 907–929. 464 5 E. Bony, F. Boudard, P. Brat, E. Dussossoy, K. Portet, P. 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