Deposition Form and Bioaccessibility of Keto-carotenoids from Mamey

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Deposition Form and Bioaccessibility of Keto-Carotenoids from Mamey Sapote (Pouteria sapota), Red Bell Pepper (Capsicum annuum), and Sockeye Salmon (Oncorhynchus nerka) Filet Tania Chacón-Ordóñez, Patricia Esquivel, Víctor M. Jiménez, Reinhold Carle, and Ralf M. Schweiggert J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b06039 • Publication Date (Web): 18 Feb 2016 Downloaded from http://pubs.acs.org on February 19, 2016

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Journal of Agricultural and Food Chemistry

Deposition Form and Bioaccessibility of Keto-Carotenoids from Mamey Sapote (Pouteria sapota), Red Bell Pepper (Capsicum annuum), and Sockeye Salmon (Oncorhynchus nerka) Filet

Tania Chacón-Ordóñez†, Patricia Esquivel‡, Víctor M. Jiménez§, #, Reinhold Carle†, ┴, Ralf M. Schweiggert†,* †

Institute of Food Science and Biotechnology, Chair Plant Foodstuff Technology and

Analysis, University of Hohenheim, Garbenstrasse 25, D-70599 Stuttgart, Germany. ‡

School of Food Technology, University of Costa Rica, 2060, San José, Costa Rica.

§

CIGRAS, University of Costa Rica, 2060, San José, Costa Rica.

#

Food Security Center, University of Hohenheim, D-70599 Stuttgart, Germany.

┴

Biological Science Department, King Abdulaziz University, P.O. Box 80257, Jeddah 21589,

Saudi Arabia.

*

Corresponding author. (Tel.: +49 711 459 22995; Fax: +49 711 459 24110; Email:

[email protected])

1 ACS Paragon Plus Environment


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ABSTRACT

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The ultrastructure and carotenoid-bearing structures of mamey sapote (Pouteria sapota)

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chromoplasts were elucidated using light and transmission electron microscopy and compared

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to carotenoid deposition forms in red bell pepper (Capsicum annuum) and sockeye salmon

5

(Oncorhynchus nerka). Globular-tubular chromoplasts of sapote contained numerous lipid

6

globules and tubules embodying unique provitamin A keto-carotenoids in a lipid-dissolved

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and presumably liquid-crystalline form, respectively. Bioaccessibility of sapotexanthin and

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cryptocapsin was compared to that of structurally-related keto-carotenoids from red bell

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pepper and salmon. Capsanthin from bell pepper was the most bioaccessible pigment,

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followed by sapotexanthin and cryptocapsin esters from mamey sapote. In contrast,

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astaxanthin from salmon was the least bioaccessible keto-carotenoid. Thermal treatment and

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fat addition consistently enhanced bioaccessibility, except for astaxanthin from naturally

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lipid-rich salmon which remained unaffected. Although the provitamin A keto-carotenoids

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from sapote were highly bioaccessible, their qualitative and quantitative in vivo

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bioavailability and their conversion to vitamin A remains to be confirmed.

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KEYWORDS: keto-carotenoids, β-carotene, sapotexanthin, cryptocapsin, capsanthin,

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astaxanthin, chromoplast, globular, tubular, ultrastructure


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INTRODUCTION

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A diet rich in fruits and vegetables plays an important role in the prevention of numerous

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chronic diseases, such as diabetes, Alzheimer’s disease, cardiovascular disease, and cancer. A

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wide array of health-promoting micronutrients has been proposed to be responsible for the

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health-promoting effects of fruit and vegetable consumption. Among them, carotenoids have

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been suggested to be beneficial for the immune and visual system as well as for human

25

growth and development.1 Since vitamin A deficiency has been reported to cause moderate to

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severe health problems in at least 122 countries, fruits and vegetables rich in provitamin A

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carotenoids represent valuable dietary sources for diminishing this severe issue.2 In order to

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exert potential health benefits, carotenoids need to be released from the food matrix, and

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subsequently absorbed by human body, i.e., they need to be bioavailable. Despite the

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existence of more than 700 different carotenoids, only few have been found in human plasma,

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among them β-carotene, β-cryptoxanthin, lycopene, lutein, α-carotene, and zeaxanthin.3

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Besides a discrimination by the human organism, carotenoid release from the food matrix and

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their subsequent micellization, together considered as “bioaccessibility”, are most often

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limiting factors. Among a large number of factors,4 carotenoid bioaccessibility has been

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shown to be drastically enhanced by the addition of lipids, thermal treatment, and mechanical

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comminution, additionally depending on the initial crystalline, liquid-crystalline, protein-

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bound, or lipid-dissolved deposition form of the carotenoids in the chromoplasts of the

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respective food.5 In contrast to costly and time-consuming human clinical trials for the

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determination of carotenoid bioavailability, their bioaccessibility may be estimated by in vitro

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digestion models.6 When comparing relative results, in vitro studies often yield similar trends

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like in vivo trials, although the exact magnitude of in vivo differences should not be deduced

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from in vitro results.7



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While the bioaccessibility and bioavailability of β-carotene, β-cryptoxanthin, lycopene, and

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some xanthophylls from a large number of fruits have been evaluated in detail,7-10 data on

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keto-carotenoids is widely lacking. Prominent dietary sources of the common keto-

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carotenoids capsanthin and astaxanthin are red bell pepper and salmon, respectively.11,12

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Despite not being precursors for provitamin A, capsanthin and astaxanthin (Figure 1) were

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also suggested to provide health benefits due to their antioxidant activity.13,14 The potential

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provitamin A-active sapotexanthin and several cryptocapsin esters (Figure 1) are less

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frequently encountered. To date, sapotexanthin has only been found in mamey sapote

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(Pouteria sapota (Jacq.) H.E.Moore & Stearn).15 Mamey sapote is a tropical fruit native to

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Central America and Mexico with a soft, red- or orange-colored, and sweet flesh.16 It is

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usually consumed fresh or used to prepare smoothies, purees, cakes, sorbets, and ice creams.

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Noteworthy, neither the qualitative and quantitative in vivo bioavailability nor the in vitro

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bioaccessibility of sapotexanthin or cryptocapsin have been reported yet. In addition, their

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physical deposition form in the chromoplasts of sapote fruits has not been elucidated, despite

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its potential importance for their bioaccessibility and bioavailability. In contrast, a limited

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number of reports on deposition forms, bioaccessibility and bioavailability of capsanthin from

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red peppers and astaxanthin from salmon is available.17-20

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Therefore, the primary goal of the present study was to elucidate the chromoplast

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ultrastructure in mamey sapote fruits by means of light and transmission electron microscopy,

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and to subsequently compare our findings to previously observed chromoplasts from red

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pepper and carotenoid distributions in salmon flesh. The second goal was to compare the

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bioaccessibility of mamey sapote keto-carotenoids to that of red bell pepper and salmon, two

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other known keto-carotenoid sources, using an in vitro digestion model. The test foods were

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digested raw and after cooking them with 1% (w/w) oil in order to determine the effect of

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thermal treatment and lipid addition. Prior to trials on their bioavailability in humans, 4 ACS Paragon Plus Environment

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bioaccessibility of sapotexanthin and cryptocapsin needed to be determined as described in

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this report.

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MATERIALS AND METHODS

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Sample material and preparation. Mamey sapote (Pouteria sapota (Jacq.) H. E. Moore &

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Stearn) fruits (Accession Number #11129) were sampled at the Tropical Agricultural

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Research and Higher Education Center, CATIE (Turrialba, Costa Rica). For light and electron

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transmission microscopy, fresh fruits were used as described below. For bioaccessibility

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assays and HPLC-DAD-MSn analyses of the carotenoid profile, the fruits were transported by

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plane to the University of Hohenheim (Stuttgart, Germany) and stored wrapped in newspapers

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at 23 ºC until full maturity in agreement with Costa Rican consumer habits. After manual

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peeling, the obtained pulp from different fruits was pooled to ensure sample homogeneity, cut

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into small cubes of approximately 1.5 cm3, frozen with liquid nitrogen, packed into airtight

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aluminum pouches, and stored at -80 ºC until analyses. Red bell pepper fruits (Capsicum

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annum L.) and salmon (Oncorhynchus nerka) (Sockeye Wildlachs, Suempol, Germany) were

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obtained from a local market in Stuttgart, and stored at 7 ºC until further analyses.

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Reagents. Ammonium oxalate, calcium carbonate (p.a.), calcium chloride dihydrate (p.a.),

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ethyl acetate (p.a.), ethanol, methyl tert-butyl ether (MTBE) (HPLC gradient grade),

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potassium chloride (p.a.), potassium dihydrogen phosphate (p.a.), NaOH (Titrisol, 1 M),

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sodium hydrogen carbonate (p.a.) and aqueous hydrochloric acid (37%) were purchased from

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Merck (Darmstadt, Germany). 2,6-di-tert-butyl-p-cresol (BHT) was obtained from Fluka

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Chemie GmbH (Buchs, Switzerland). Sodium chloride, magnesium chloride hexahydrate (Ph.

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Eur.), methanol (HPLC grade), and petroleum spirit (b. p. 40−60 °C, GPR rectapur) were

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from VWR International (Leuven, Belgium). Porcine pancreatic α-amylase (46.6 U/mg),

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porcine bile extract, cholesterol esterase from porcine pancreas (42.9 U/mg), pepsin from



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porcine gastric mucosa, and pancreatin from porcine pancreas were purchased from Sigma-

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Aldrich Chemie (Steinheim, Germany). Sodium phosphate dibasic anhydrous and sodium

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phosphate monobasic monohydrate were obtained from Fisher Scientific (Hampton, NH).

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Carotenoid standards for β-carotene and astaxanthin were obtained from Sigma Aldrich

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Chemie GmbH (Taufkirchen, Germany). Glutaraldehyde (70% in water), paraformaldehyde,

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Spurr’s low viscosity resin, crystalline osmium tetroxide, lead citrate and uranyl acetate were

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purchased from Electron Microscopy Sciences (Hatfield, PA). Ultrapure water was prepared

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by an arium 611 Ultrapure Water System (Sartorius, Göttingen, Germany) and used

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throughout the study, except for light microscopy where tap water was used.

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In vitro digestion model. The employed in vitro digestion model involved oral, gastric and

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intestinal phases according to methods of Garrett et al.21, Bengtsson et al.22 and Schweiggert

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et al.9 Test food preparation was carried out as follows. After cutting into small cubes, an

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aliquot of each above mentioned test food (sapote, red pepper, salmon) was used for the

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bioaccessibility assay of raw samples, while another fraction of each test food was

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supplemented with 1% (w/w) of soybean oil (Sojola, Herford, Germany) and then cooked for

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30 min at 90 °C in a beaker placed in a water bath. Heat-treated samples were stored at -80 ºC

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prior to subjecting to the digestion model. Aliquots of each raw and cooked test food were

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stored at -80 ºC for carotenoid analyses using HPLC-DAD-MSn.

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The amount of test food subjected to the digestion model was 10 g for red pepper and salmon,

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and 20 g for mamey sapote, mainly to ensure sufficient HPLC-DAD signal in the micellized

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fraction after digestion. The protocol followed for digesting 10 g of red pepper and salmon is

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described below. All digestive fluids were doubled when digesting mamey sapote. First,

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samples were manually squashed in a mortar for 45 s to mimic the chewing process.

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Subsequently, an aliquot of 10 g (20 g for mamey sapote) was transferred to an amber glass

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bottle. After adding 10 mL of artificial saliva solution (50 mM NaCl, 10 mM KH2PO4, 2 mM 6 ACS Paragon Plus Environment

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CaCl2•6H2O, 40 mM NaHCO3), the pH was adjusted to 6.9 with aqueous 1 M NaOH or 1 M

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HCl and 100 µL of an α-amylase solution (25 U) were added. Each flask was gently shaken

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for 30 s. For the subsequent gastric phase, 3 mL of gastric solution (51 mM NaCl, 14.7 mM

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KCl, 10 mM CaCl2•2H2O, 3.7 mM KH2PO4, 3.4 MgCl2•6H2O) were added. The pH was

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adjusted to 4 with 1 M HCl. Afterwards, 2 mL of a porcine pepsin solution (40 mg/mL in 0.1

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M HCl) were added, and the pH was re-adjusted to pH 2 with 1 M HCl. After flushing the

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headspace of the flask with nitrogen, samples were kept in a shaking bath at 37 ºC for 1 h at

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95 rpm. Subsequently, the intestinal phase was initiated by adjusting to pH 5.3 with 0.9 M

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NaHCO3, and 9 mL of bile extract/pancreatin solution (12 mg/mL porcine bile extract and 2

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mg/mL pancreatin in aqueous 0.1 M NaHCO3 solution) were added. Additionally, 174 µL of a

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cholesterol esterase solution (5 U in 0.1 M Na2HPO4 buffer, pH 7) was included, and pH was

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re-adjusted to 7.5 with 1 M NaOH. Headspace of the flask was flushed again with nitrogen

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and samples were kept in a shaking bath at 37 ºC for 2 h at 95 rpm. Subsequently, samples

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were made up with deionized water to 50 mL (100 mL for mamey sapote), and centrifuged for

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60 min at 10 ºC in a Avanti J-26 XP/XPI centrifuge (Beckman Coulter, Krefeld, Germany)

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with a JA-25.50 rotor at 75,000 x g for 60 min. Thereby, the precipitate and aqueous

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supernatant containing the carotenoids liberated from the test foods into the simulated

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duodenal fluid were separated. Half of the supernatant was stored at -80 ºC, while the other

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half was filtered through a 0.2 µm syringe filter cellulose acetate-based (Klaus Ziemer,

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Mannheim, Germany) to separate the micellar fraction. The micellar fraction was stored at -80

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ºC until carotenoid analyses.

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Carotenoid extraction. Carotenoids from test foods were extracted according to a method of

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Murillo et al.15 with slight modifications. After briefly homogenizing in a porcelain mortar, an

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aliquot of 1.0 ± 0.1 g of the macerate was placed in a tube containing 0.1 g of NaHCO3.

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Carotenoids were extracted using acetone enriched with 0.1% (w/v) BHT and a sonopuls HD

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3100 probe-sonicator (Sonopuls, Germany) equipped with a MS 72 probe. After brief 7 ACS Paragon Plus Environment


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centrifugation (3000 x g, 3 min), the liquid supernatant was collected, and the solid

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remainders were re-extracted at least 4 times until colorless. A final extraction cycle was done

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with n-hexane. The supernatants were combined with 4 mL diethyl ether/n-hexane (1:1, v/v,

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0.1% BHT) and 2 mL of deionized water, vortexed, and centrifuged for phase separation. The

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upper phase containing the extracted carotenoids was collected and washed with water once.

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The washed extract was evaporated under nitrogen atmosphere, and stored at -80 °C until

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carotenoid analysis. Prior to HPLC analyses, the dried extracts were dissolved in

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MTBE/methanol (1:1, v/v), and filtered through a 0.45 µm PTFE membrane into amber

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HPLC vials prior to HPLC analyses.

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Carotenoid extraction of the supernatant and the micellar fraction of the digestion model was

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performed as follows. An aliquot of 12 mL of the respective fraction was extracted two times

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by simultaneously adding both 3 mL acetone (0.1% BHT) and 6 mL of a ternary mixture

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(1:1:1, v/v/v) of methanol, ethyl acetate and petroleum spirit. The aforementioned probe-

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sonicator was used to enhance the extraction. The organic phases were collected, combined,

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evaporated under nitrogen atmosphere, and stored at -80 °C. For HPLC analyses, samples

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were dissolved in MTBE/methanol (1:1, v/v), and filtered through a 0.45 µm pore PTFE

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membrane into amber HPLC vials prior to HPLC analyses.

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HPLC-DAD-MSn analyses. Carotenoid separation was achieved using a 1100 series HPLC

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(Agilent, Waldbronn, Germany) equipped with a G1379A degasser, a G1312A binary

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gradient pump, a G1313A autosampler, a G1316A column oven, and a G1315B diode array

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detector. The column used was a 150 x 3.0 mm i.d., 3 µm, reverse phase C30 with a 10 × 3.0

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mm i.d. guard column of the same material (YMC Europe, Dinslaken, Germany), and was

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operated at 40 °C. The mobile phase consisted of methanol and water (90:10, v/v, eluent A) as

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well as methanol, MTBE, and water (20:78:2, v/v/v, eluent B). Both eluents A and B

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contained 1.5 g ammonium acetate/L. For mamey sapote samples the gradient was 8 ACS Paragon Plus Environment

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programmed as follows: isocratic 100% A for 5 min, from 100-25% A in 73 min, from 25%-

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0% A in 4 min and from 0%-100% A in 8 min. Total run time was 90 min at a flow rate of 0.8

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mL/min. For red bell pepper and salmon, the same eluents were used and the gradient

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programmed as follows: isocratic 100% A for 5 min, from 100-0% A in 47 min, isocratic 0%

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A for 3 min and from 0-100% a in 5 min. Total run time was 60 min at a flow rate of 0.5

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mL/min. Carotenoids were monitored at 450 nm and additional UV/Vis spectra were recorded

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in the range of 200−600 nm.

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Carotenoid identification was performed by coupling the above-described HPLC system to a

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3000+ ion trap mass spectrometer (Bruker, Bremen, Germany), equipped with an APCI

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source operating in positive mode. Parameters for analysis were set as described by

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Schweiggert et al.23 Identification of carotenoids was accomplished by comparison of

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retention times, UV/Vis absorption, and mass spectrometric behavior with those of authentic

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standards. When standards were unavailable, pigments were tentatively identified by

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comparing their UV/Vis absorption spectra and mass spectral behavior with previously

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published data.23-25

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Quantitation of carotenoids was achieved by HPLC-DAD and external calibration curves of

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β-carotene and astaxanthin. When standards were unavailable, quantitation was performed

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using the β-carotene calibration curve. Molecular weight correction factors (MWCF) were

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used when necessary, representing the ratio of the molecular weight of the compound to be

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quantitated and that of β-carotene. Similarly, unknown compounds were quantitated using the

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β-carotene calibration curve for estimating the total carotenoid content.

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Light microscopy. Fresh, free-hand sections of mamey sapote, red bell pepper and salmon

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were cut with razor blades and mounted on glass slides without staining. Slides were observed



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in an inverted Olympus IX-51 microscope (Tokyo, Japan). Photoshop CS6 (Adobe Systems,

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San Jose, CA) was used to adjust contrast and brightness if necessary.

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Transmission electron microscopy. Sections of mamey sapote mesocarp of approximately 1

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mm3 were obtained with a razor blade and immediately fixed in a modified Karnovsky

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solution (2.5% w/v glutaraldehyde, 2% w/v paraformaldehyde) in 0.1 M sodium phosphate

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buffer (pH 7.4) for at least 4 h at 4 ºC. Samples were then washed three times for 15 min with

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0.1 M sodium phosphate buffer. After post-fixation with a 2% (w/v) osmium tetroxide

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solution for 2 h at room temperature, samples were washed three times for 15 min with water,

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and subsequently, dehydrated with an aqueous acetone series (30%, 50%, 70%, 90% and 3 x

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100%). Samples were then embedded in Spur’s medium, and polymerized at 60 °C for 48 h.

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Ultra-thin sections were obtained with a diamond knife using a Power Tome PC

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ultramicrotome (RMC Products, Tucson, AZ) and collected on copper grids. Samples were

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then stained with uranyl acetate and lead citrate prior to observation in a Hitachi H-7100

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transmission electron microscope (Tokyo, Japan) at 100 kV. Photoshop CS6 (Adobe Systems,

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San Jose, CA) was used to adjust contrast and brightness if necessary.

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Statistical analysis. A one-way analysis of variance (ANOVA) was conducted to determine

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significant differences of means. Shapiro-Wilk’s test was used to test normality of the data,

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and homogeneity of variances was assessed by Levene’s test. For non-parametric samples, a

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Kruskall-Wallis test was conducted. All analyses were performed with the program SAS JMP

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8 (SAS Institute, Cary, NC). All in vitro digestions were performed in triplicate, and all

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carotenoid extractions in duplicate. The in vitro liberation and bioaccessibility of carotenoids

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was calculated as the relative amount of carotenoids that were transferred from the test food to

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the supernatant phase recovered after ultracentrifugation (liberated carotenoids) and to the

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micellar fraction obtained after microfiltration (bioaccessible carotenoids), respectively, as

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described previously by Bengtsson et al.22 10 ACS Paragon Plus Environment

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RESULTS AND DISCUSSION

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Identification and quantitation of carotenoids in test foods

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Only those carotenoids that were found in the duodenal fluids after simulated digestion of the

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mamey sapote (test food) were considered in this section. Particularly, numerous carotenoid

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epoxides contained in the fruits were degraded during the simulated digestion, being in

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agreement with previous reports.7

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The major nutritionally relevant pigments in mamey sapote were the potentially provitamin

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A-active carotenoids sapotexanthin (234-250 µg/100 g FW), cryptocapsin laurate (158-190

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µg/100 g FW), and cryptocapsin myristate (133-221 µg/100 g FW), being shown to be present

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in the intestinal fluids after simulated digestion (Figure 2A). They were identified by

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comparing our HPLC-DAD-MSn data with those reported by Murillo et al.24 Total carotenoid

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levels of mamey sapote amounted to 1,719 µg/100 g of FW, being only slightly degraded

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upon thermal treatment (90 °C, 30 min) as shown in Table 1. To the best of our knowledge,

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previous quantitative data on mamey sapote carotenoids are unavailable.

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In agreement with Schweiggert et al.23 , the main carotenoids of red bell pepper were β-

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carotene (4,816-4,840 µg/100 g), capsanthin (833-1,482 µg/100 g FW), and various

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capsanthin esters (6,896-13,108 µg/100 g FW), as illustrated in Figure 2B. Substantially

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exceeding those of mamey sapote, total carotenoid levels were approximately 50 mg/100 g

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FW bell pepper, in agreement with previous reports.26 Thermal treatment diminished the

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carotenoid levels to 32 mg/100 g FW (Table 1). In salmon, astaxanthin (approximately 4.4

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mg/100 g of FW) was the sole carotenoid, present mainly in the (all-E)-form (87%) and its

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two (Z)-isomers (3 and 9%) (Table 1, Figure 2C) as identified according to a previous

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report.27 Total astaxanthin content appear not to be affected after heat treatment. Astaxanthin



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levels in salmon were similar to those of previous reports, although they may vary

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substantially depending on the feed of the fish.11

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Noteworthy, excessive thermal treatments might cause the EZ (or trans-cis) isomerization of

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carotenoids,27,28 particularly in the presence of added lipids.28 However, in our study, the (all-

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E)-forms of all reported carotenoids were predominant in both the raw and heated test foods

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and quantifiable amounts of (Z)-isomers were not detected, except for those of astaxanthin. In

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agreement, substantial carotenoid isomerization was previously observed at heat exposures

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and fat contents being significantly higher (e.g., 127°C, 40 min, 10% oil)28 than those of our

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study (90 °C, 30 min, 1% fat content).

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Carotenoid deposition in mamey sapote, red pepper, and salmon

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Due to its previously suggested impact on carotenoid bioaccessibility,9 the deposition forms

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of carotenoids in mamey sapote, red bell pepper, and salmon were examined based on our

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own and previously published light and transmission electron micrographs. Light micrographs

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of mamey sapote mesocarp cells showed small round-shaped yellow and orange colored

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structures corresponding to chromoplasts (Figure 3). Large crystalloid structures as previously

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found in carrot and tomato were absent.9 Starch granules were also observed (Figure 3).

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Transmission electron micrographs of mamey mesocarp revealed chromoplasts containing

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tubular and numerous globular elements (Figures 4A and B), therefore, being unambiguously

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categorized as globular-tubular chromoplasts. This type of chromoplast has been previously

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described in mango cv. ‘Tommy Atkinsʼ,28 papaya,29 and other fruits as summarized

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previously by Schweiggert and Carle.5 Besides globular and tubular elements, some of the

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observed chromoplasts contained apparent stroma thylakoid remnants (Figure 4C). In

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addition, starch granules were found next to tubular elements, revealing the presence of so-

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called amylo-chromoplasts as shown in Figure 4D. Amylo-chromoplasts have recently been

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reported in yellow peach palm mesocarp and tissue near the peel.30 Furthermore, such plastids 12 ACS Paragon Plus Environment

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were observed in mango fruits,28 and in nectaries of tobacco flowers, where they were

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described to be an intermediate stage in the conversion of amyloplasts into chromoplasts.31

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In brief, globular and tubular elements were the sole carotenoid-bearing structures observed in

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mamey sapote chromoplasts. In the lipid globules, carotenoids are stored in a lipid-dissolved

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physical state, while in tubular elements, carotenoids are believed to be deposited in a liquid

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crystalline core surrounded by a mono-layer of polar lipids and proteins (Figure 5A).32

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Carotenoid biosynthesis has been reported to widely occur at the membrane of the globules,

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accumulating carotenoids in the lipid core of the globule.33 When surpassing saturation

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concentrations, carotenoid aggregation, driven by structure-related self-assembly,32 will

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inevitably lead to the formation of either crystalloid or tubular elements. For instance,

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lycopene-rich fruits and vegetables, such as tomato and watermelon, were previously shown

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to contain chromoplasts with large crystalloid structures.34,35 Lycopene aggregates were

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reported to represent tight assemblies with small intermolecular distances, forming rigid solid

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crystals.35 In contrast, chromoplasts of fruits rich in carotenoid esters were most frequently

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observed to contain tubular elements with a core of presumably liquid-crystalline carotenoids.

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In agreement, aggregates of carotenoid esters have been previously described to represent

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comparably

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aggregation.35,36 Since mamey sapote contained high amounts of carotenoid esters, with

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cryptocapsin esters being the most abundant, we suggested that these might be mainly

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deposited in the tubular elements. This in accordance with the description and evaluation of

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tubule self-assembly,19,32 showing carotenoid esters to be more efficiently arranged into

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tubules than free carotenoids. If the slightly more polar sapotexanthin and several carotenoid

286

epoxides found in mamey sapote fruits might be preferentially stored in the abundantly

287

observed globules or also within tubules, however, remains unknown. Therefore, further

288

studies are needed to confirm the putative structure-related discrimination in chromoplastidal

loose

associations,

naturally

forming

nematic

liquid

crystals

upon



Page 14 of 36

289

deposition. Both discussed storage forms of mamey sapote are schematically presented in

290

Figure 5.

291

Similarly to those of mamey sapote, the chromoplasts of red bell pepper have been previously

292

reported to be of the globular-tubular type.5 Their high content in carotenoid esters23 might be

293

related with the occurrence of tubular chromoplastidal elements. Figure 5B shows light

294

micrographs of the red bell pepper used in this study. Besides the keto-carotenoid-rich mamey

295

sapote and red bell pepper, we included sockeye salmon into our deposition and

296

bioaccessibility studies due to its high content of the keto-carotenoid astaxanthin, and its

297

substantially different deposition form in the muscle tissue. Astaxanthin was shown to be

298

deposited in the muscle fibers. Earlier, it was believed that astaxanthin might be bound in the

299

actin and myosin protein complex by weak unspecific hydrophobic bonds.37 However,

300

Matthews et al.20 found this carotenoid to be bound to the hydrophobic core of a specific

301

protein within the actin-myosin complex, the so-called α-actinin (Figure 5C). Astaxanthin

302

presents two hydroxyl and one keto functions allowing a stronger binding than other

303

carotenoids present in salmon.37

304

Carotenoid liberation and bioaccessibility

305

Irrespective of being lipid-dissolved, liquid-crystalline, solid-crystalline, or protein-bound, all

306

biological aggregates of hydrophobic carotenoids from fruits and vegetables are surrounded

307

by an aqueous environment, and, in order to be absorbed by humans, they need to be liberated

308

from the food matrix and incorporated into micelles in the intestine.38 In agreement with

309

previous reports,7,9 the following sections will consider the carotenoids present in the

310

supernatant fraction obtained after ultracentrifugation as “liberated carotenoids”, while

311

carotenoids present in the fraction recovered after microfiltration (0.2 µm pore size) of the

312

above mentioned supernatant were considered as micellized, thus being bioaccessible. Only


Page 15 of 36


313

bioaccessible carotenoids are sufficiently stable and efficiently micellized during digestion to

314

be potentially bioavailable in humans.

315

Mamey sapote

316

Carotenoids liberated from the mamey sapote fruit matrix into the simulated duodenal fluid

317

were mainly sapotexanthin and cryptocapsin esters, although free cryptocapsin, and several

318

unidentified compounds were also detected in trace amounts. After simulated digestion,

319

sapotexanthin recovered from the liberated fraction amounted to 6.7 µg/100 g of FW of the

320

digested test food, while 2.2 µg/100 g of FW of digested test food were obtained from the

321

micellar fractions (Table 2). Thus, only 2.7 and 0.9% of the sapotexanthin contained in the

322

test meal were liberated and bioaccessible when digesting raw sapote, respectively (Figures

323

6A and B). However, heat treatment (90 °C, 30 min) and oil addition (1%) boosted both

324

sapotexanthin liberation and bioaccessibility by 1.8-2.8-fold to 5% and 2.7% of the fed

325

sapotexanthin dose, respectively, equaling 11.8 and 6.3 µg of liberated and micellized

326

sapotexanthin per 100 g of FW of digested test food, respectively (Figure 6 and Table 2).

327

Compared to sapotexanthin, a lower bioaccessibility of cryptocapsin laurate and myristate

328

(0.2%) was observed. Absolute levels are shown in Table 2. By analogy to sapotexanthin,

329

liberation and bioaccessibility of both esters was enhanced at least 2-fold after heat treatment

330

and 1% oil addition to 3.3-5 and 1.4-2.1%, respectively (Figures 6A and B). It should be

331

noted that carotenoid esters have not yet been reported in human blood, as they were assumed

332

to be efficiently cleaved during absorption in the intestine.10,39,40 However, in our study,

333

cryptocapsin esters were found in both liberated and micellized fractions after simulated

334

digestion, while free cryptocapsin was observed only in traces. Possibly, our model was

335

unable to cleave cryptocapsin esters, since Breithaupt et al.41 previously reported that ester

336

cleavage rates can be different depending on the carotenoid, being particularly low when 15 ACS Paragon Plus Environment


Page 16 of 36

337

cleaving esterified keto-carotenoids, e.g. capsanthin diesters. Nevertheless, since both

338

sapotexanthin and cryptoxanthin esters remained stable during digestion and were detected in

339

significant amounts within the bioaccessible micellar fractions, they are hypothetically

340

available for absorption. Since a study proving the qualitative bioavailability of sapotexanthin

341

and cryptocapsin in humans is currently unavailable, it remains to be elucidated whether the

342

reported bioaccessibility of both potentially provitamin A-active keto-carotenoids may

343

translate into their actual absorption to the human blood stream.

344

Red bell pepper

345

After mimicking the digestion of raw red bell pepper, capsanthin (194 µg/100 g FW of

346

digested test food) and capsanthin esters (2,301 µg/100 g FW of digested test food) (Table 2)

347

were the main keto-carotenoids liberated from the food matrix into the simulated duodenal

348

fluids. Their high absolute levels in the liberated fraction may simply correspond to their

349

strikingly high amount in the bell pepper (15,774 µg total capsanthin/100 g FW of test food).

350

However, only free capsanthin was found to be micellized and bioaccessible (279 µg/100 g

351

FW of digested test food), while capsanthin esters were undetectable after digesting raw bell

352

pepper (Table 2). In addition, the enhanced hydrophilicity of free capsanthin as compared to

353

the esterified capsanthin might have fostered its incorporation into the mixed micelles as

354

suggested previously by Tyssandier et al.42. However, the apparently high bioaccessibility of

355

free capsanthin (18.8% of the fed dose of free capsanthin) appears to be deceiving, since

356

capsanthin esters may have been hydrolyzed to yield additional free capsanthin during the

357

simulated digestion phase. In agreement, Breithaupt43 has previously shown lipase-catalyzed

358

cleavage of capsanthin esters from red pepper pods, although occurring at a comparably slow

359

rate. When relating to the total capsanthin levels of the test foods, only 1.8% of the capsanthin

360

was observed to be bioaccessible (Table 2). By analogy to the keto-carotenoids from sapote,

361

the bioaccessibility of total capsanthin was increased to 4.4% after heat treatment and lipid 16 ACS Paragon Plus Environment

Page 17 of 36


362

addition. In addition, upon the latter treatment, micellization of esterified capsanthin (2.7% of

363

the initial dose) was observed, presumably by providing a more lipophilic environment that

364

facilitated the incorporation of the hydrophobic esters into the micelles (Table 2). The second

365

most abundant carotenoid in red bell pepper was β-carotene, which will serve as comparison.

366

A total of 19% of the β-carotene from the bell pepper subjected to the digestion model was

367

liberated into the duodenal fluids, while only 0.4% was micellized, thus being considered as

368

bioaccessible. Hence, β-carotene was approximately 5.4-fold less bioaccessible from raw bell

369

pepper than capsanthin. However, upon heat treatment and lipid addition, bioaccessibility of

370

β-carotene increased to approximately 5%, surpassing that of capsanthin from processed bell

371

pepper (4.4%). Although not dealing with capsanthin and its derivatives, O’Sullivan

372

previously reported the bioaccessibility of β-carotene from red bell peppers, observing values

373

(6.2% ± 0.5%) similar to those of our study.44

374

Salmon

375

In contrast to sapote and bell pepper, differences in the bioaccessibility of total astaxanthin

376

from heat-treated salmon (1.2%) and that from raw salmon (1.9%) (Figure 6A) were

377

insignificant. For salmon test meals, thermal treatment and adding lipids might have been

378

ineffective, because of its high genuine fat content (approximately 3.4%), in contrast to sapote

379

and bell pepper. Possibly, adding lipids beyond a certain threshold might be ineffective to

380

enhance bioaccessibility. In addition, the supposed enhancing effect of added lipids might

381

have been antagonized by the thermal treatment, which may result in denaturation of

382

myofibrillar proteins, including water loss and tissue shrinkage.45 Consequently, astaxanthin

383

might have been entrapped within the matrix, possibly diminishing any potential benefit from

384

the added lipids. A previous study by Sy et al.46 revealed the bioaccessibility from Norwegian

385

smoked salmon (6.4%) to be substantially lower than that from a formulation containing lipid-



Page 18 of 36

386

dissolved astaxanthin (49.7%), thus indicating that complex matrix played an essential role in

387

astaxanthin release and incorporation into mixed micelles.43

388

Comparison of keto-carotenoid bioaccessibility from different food matrices

389

A comparison of carotenoid bioaccessibility across the different test foods might be highly

390

intricate due to the complexity of influence factors as outlined below. Nevertheless, the

391

bioaccessibility of the investigated keto-carotenoids from processed mamey sapote, bell

392

pepper, and salmon may be ranked as follows: total capsanthin (4.4%, from pepper) >

393

sapotexanthin (2.7%, from sapote) > cryptocapsin laurate (2.1%, from sapote) > cryptocapsin

394

myristate (1.4%, from sapote) > astaxanthin (1.2%, from salmon). The highest relative

395

bioaccessibility as well as the highest absolute micellar levels were observed for total

396

capsanthin after digestion of bell pepper, being characterized by a variable proportion of the

397

polar free capsanthin (45-100% of the total micellized capsanthin) (Table 2). Polar

398

carotenoids such as lutein and β-cryptoxanthin were previously reported to be more

399

bioaccessible than apolar carotenoids like β-carotene and lycopene, presumably since the

400

more polar xanthophylls are preferably incorporated into mixed micelles during digestion.9,47

401

Therefore, the lower bioaccessibility of sapotexanthin and cryptoxanthin esters from sapote as

402

compared to capsanthin from bell pepper might be attributed to their different chemical

403

structure. However, a number of other factors should be considered. For instance, the

404

chromoplastidal deposition form has been previously assumed to be decisive for the release

405

efficiency of carotenoids from plant foods.5 Nevertheless, as shown in this study, mamey

406

sapote contained a globular-tubular type of chromoplasts (Figure 4), and previous reports on

407

bell pepper also showed a globular-tubular type of chromoplasts.48 Furthermore, total dietary

408

fiber contents of mamey sapote fruits (7.95%) were considerably higher than that of red bell

409

peppers (2.2%),49 and might have attributed to its lower bioaccessibility, since dietary fibers

410

have been previously reported to reduce carotenoid bioavailability.50,51 18 ACS Paragon Plus Environment

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411

In conclusion, the two potentially provitamin A-active keto-carotenoids sapotexanthin and

412

cryptocapsin occur in globular-tubular chromoplasts of mamey sapote fruits, being deposited

413

in a lipid-dissolved presumably liquid-crystalline physical state. Irrespective of mimicking the

414

digestion of raw or processed test foods, the bioaccessibility of sapotexanthin and

415

cryptocapsin from mamey sapote exhibited intermediate values (0.2 - 3%) as compared to

416

other keto-carotenoids like astaxanthin from salmon (approximately 1.9%), and total

417

capsanthin from red bell pepper (1.8-4.4%). However, unlike astaxanthin and capsanthin, both

418

the qualitative and quantitative bioavailability of sapotexanthin and cryptoxanthin in humans,

419

and, in particular, their metabolic conversion to vitamin A remain to be proven.

420 421

ABBREVIATIONS USED

422

BHT: 2,6-di-tert-butyl-p-cresol; FW: Fresh Weight; MTBE: Methyl tert-butyl ether; MWCF:

423

Molecular weight correction factors; PTFE: Polytetrafluoroethylene; TAG: Triacylglyceride.

424

ACKNOWLEDGEMENTS

425

The authors thank the Tropical Agricultural Research and Higher Education Center (CATIE)

426

in Costa Rica for providing mamey sapote fruits. The Alexander von Humboldt Foundation

427

(Bonn, Germany) is acknowledged for partially funding this study in the framework of the

428

Research Group Linkage Program. Further partial funding was provided by the University of

429

Costa Rica (project VI-735-B2-A16). TCO acknowledges the scholarship provided by the

430

Baden-Württemberg Stiftung, and the financial support of the Post-Graduate Studies System

431

of the University of Costa Rica (SEP), as well as the Research Center on Microscopic

432

Structures (CIEMic) and the Research Vice-Rectory of the University of Costa Rica for the

433

scholarship provided to conduct the electron microscopy studies (project VI-810-B3-183).



Page 20 of 36

434

TCO, PE and VMJ thank the German Academic Exchange Service (DAAD) for financial

435

support.

436

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437

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569



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FIGURE CAPTIONS Figure 1. Structure of keto-carotenoids of mamey sapote, red bell pepper and salmon. Dashed lines mark potential cleavage sites to obtain vitamin A. Figure 2. Chromatograms of liberated fractions from raw test foods. Peak assignment: 1: sapotexanthin, 2: cryptocapsin laurate, 3: cryptocapsin myristate, 4: capsanthin, 5: capsanthin laurate, 6: β-carotene, 7: capsanthin myristate, 8-12: capsanthin diesters, 13, 15: astaxanthin (Z)-isomers, 14: astaxanthin. Figure 3. Mamey sapote fruit and light micrograph of fruit mesocarp. Arrows: chromoplasts, S: starch grains. Figure 4. A. Electron micrographs of globular-tubular chromoplast in fully ripe red-orange fleshed mamey sapote fruits. B. Detailed view of tubular elements. C. Globular chromoplast containing some apparent stroma thylakoid remnants. D. Amylo-chromoplast presenting starch granules as well as tubular elements. Arrows: tubular elements, Arrowheads: thylakoid remnant, G: lipid globule, M: mitochondrion, CM: chromoplast membrane, CW: cell wall, S: starch granule,*: osmophilic structures. Figure 5. Deposition forms of keto-carotenoids in mamey sapote, red bell pepper, and salmon as shown by light microscopy images and detailed schematic diagrams of their carotenoid storage structures. A. Mamey sapote fruits in globular-tubular chromoplasts, detailed view of lipid globule containing lipid-dissolved carotenoids. B. Red bell pepper in tubular chromoplasts, detailed view of tubule containing a presumably liquid-crystalline carotenoid phase. C. Salmon with astaxanthin associated to α-actinin in the actin-myosin complex inside muscle cells. The illustrations were based of content reported previously by Sitte32 and Matthews et al.20


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Figure 6. A. Liberation, and B. bioaccessibility of keto-carotenoids from raw as well as from thermally treated (90 °C, 30 min) and lipid-enriched (+ 1% oil) test foods. Liberated carotenoids correspond to those recovered from the supernatant after centrifugation. Bioaccessible carotenoids and micellar fraction correspond to those obtained after microfiltration (0.2 µm) of the supernatant. Different letters indicate significant differences (p < 0.05) of the means between raw and cooked samples of the corresponding compound.



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Table 1. Carotenoid Content in Test Foods for the In vitro Digestion Assays. carotenoid content* test food

carotenoids

raw

cooked (90 °C, 30 min) with 1% oil

sapotexanthin

249.6 ± 6.4

234.2 ± 33.1

mamey

cryptocapsin laurate

188.6 ± 4.0

157.7 ± 46.2

sapote

cryptocapsin myristate

133.2 ± 8.7

221.0 ± 51.0

1,719.3 ± 29.2

1,690.7 ± 405.0

β-carotene

4,816.0 ± 1,944.3

4,839.8 ± 495.8

capsanthin

1,482.2 ± 530.2

833.6 ± 203.8

14,2991.9 ± 4,877.3

6,896.2 ± 1,862.7

15,774.1 ± 5,407.6

7,729.8 ± 2,066.5

50,025.1 ± 12,425.8

32,555.7 ± 14,970.0

3,882.8 ± 108.4

3,845.3 ± 303.0

(13Z)-astaxanthin

404.4 ± 383.1

440.1 ± 206.4

astaxanthin (Z)-isomer

149.4 ± 15.3

151.9 ± 36.5

total carotenoid content

4,436.6 ± 506.8

4,438.1 ± 133.1

total carotenoid content+

red bell pepper

capsanthin esters total capsanthin+ total carotenoid content all-trans-astaxanthin

salmon

+

* Mean ± standard deviation in µg/100 g of FW, n = 2. + Total capsanthin content includes those of the free and esterified forms. Total carotenoid content includes further non-keto-carotenoids present in the samples.


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Table 2. Carotenoid Content in Liberated and Micellar Fractions (raw and cooked with 1% soybean oil) from the In vitro Digestion Assays. test food

carotenoids sapotexanthin

mamey cryptocapsin laurate sapote cryptocapsin myristate β-carotene capsanthin red bell capsanthin esters pepper total capsanthin+ salmon

total astaxanthin

in liberated fraction in micellar fraction cooked cooked raw (90 °C, 30 min) raw (90 °C, 30 min) with 1% oil with 1% oil 6.7 ± 0.9 11.8 ± 3.5 2.2 ± 0.3 6.3 ± 1.7 (2.7%) (5.0%) (0.9%) (2.7%) 3.6 ± 0.5 7.9 ± 2.8 0.5 ± 0.2 3.2 ± 0.8 (1.9%) (5.0%) (0.2%) (2.1%) 4.2 ± 0.6 7.4 ± 2.7 0.3 ± 0.1 3.1 ± 0.8 (3.2%) (3.3%) (0.2%) (1.4%) 914.3 ± 551.7 409.9 ± 25.1 18.0 ± 8.8 271.7 ± 29.1 (19.0%) (3.4%) (0.4%) (5.6%) 194.9 ± 73.7 158.6 ± 4.7 278.6 ± 10.7 153.4 ± 6.2 (13.1%) (19.03%) (18.8%) (18.4%) 2,301.7 ± 1,198.6 967.7 ± 108.1 186.2 ± 30.0 (16.1%) (14.0%) (2.7%) 2,496.6 ± 1,272.1 1,166.5 ± 40.3 278.6 ± 10.7 339.5 ± 34.4 (15.8%) (14.6%) (1.8%) (4.4%) 116.3 ± 48.1 21.0 ± 8.1 84.3 ± 37.8 54.1 ± 19.7 (2.6%) (0.5%) (1.9%) (1.2%)

* Mean ± standard deviation in µg/100 g of FW (% liberated or bioaccessible, related to the carotenoid content in the test food), n = 3. + Total capsanthin content includes those of the free and esterified forms.



Figure 1. Structure of keto-carotenoids of mamey sapote, red bell pepper and salmon. Dashed lines mark potential cleavage sites to obtain vitamin A. 84x66mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Figure 2. Chromatograms of liberated fractions from raw test foods. Peak assignment: 1: sapotexanthin, 2: cryptocapsin laurate, 3: cryptocapsin myristate, 4: capsanthin, 5: capsanthin laurate, 6: β-carotene, 7: capsanthin myristate, 8-12: capsanthin diesters, 13, 15: astaxanthin (Z)-isomers, 14: astaxanthin. 177x156mm (300 x 300 DPI)



Figure 3. Mamey sapote fruit and light micrograph of fruit mesocarp. Arrows: chromoplasts, S: starch grains. 84x48mm (300 x 300 DPI)


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Figure 4. A. Electron micrographs of globular-tubular chromoplast in fully ripe red-orange fleshed mamey sapote fruits. B. Detailed view of tubular elements. C. Globular chromoplast containing some apparent stroma thylakoid remnants. D. Amylo-chromoplast presenting starch granules as well as tubular elements. Arrows: tubular elements, Arrowheads: thylakoid remnant, G: lipid globule, M: mitochondrion, CM: chromoplast membrane, CW: cell wall, S: starch granule, *: osmophilic structures. 155x136mm (300 x 300 DPI)



Figure 5. Deposition forms of keto-carotenoids in mamey sapote, red bell pepper, and salmon as shown by light microscopy images and detailed schematic diagrams of their carotenoid storage structures. A. Mamey sapote fruits in globular-tubular chromoplasts, detailed view of lipid globule containing lipid-dissolved carotenoids. B. Red bell pepper in tubular chromoplasts, detailed view of tubule containing a presumably liquid-crystalline carotenoid phase. C. Salmon with astaxanthin associated to α-actinin in the actin-myosin complex inside muscle cells. The illustrations were based of content reported previously by Sitte32 and Matthews et al.20 177x124mm (300 x 300 DPI)


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Figure 6. A. Liberation, and B. bioaccessibility of keto-carotenoids from raw as well as from thermally treated (90 °C, 30 min) and lipid-enriched (+ 1% oil) test foods. Liberated carotenoids correspond to those recovered from the supernatant after centrifugation. Bioaccessible carotenoids and micellar fraction correspond to those obtained after microfiltration (0.2 µm) of the supernatant. Different letters indicate significant differences (p < 0.05) of the means between raw and cooked samples of the corresponding compound. 85x121mm (300 x 300 DPI)



TOC Graphic 82x44mm (300 x 300 DPI)


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Deposition Form and Bioaccessibility of Keto-carotenoids from Mamey

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