Carotenoid stability and lipid oxidation during storage of low-fat carrot and tomato based systems

LWT - Food Science and Technology 80 (2017) 470e478

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Carotenoid stability and lipid oxidation during storage of low-fat carrot and tomato based systems Leonard Mutsokoti, Agnese Panozzo, Jeritah Tongonya, Biniam T. Kebede, Ann Van Loey, Marc Hendrickx* a Laboratory of Food Technology and Leuven Food Science and Nutrition Research Centre (LFoRCe), Department of Microbial and Molecular Systems (M2S), KU Leuven, Kasteelpark Arenberg 22, 3001 Heverlee, Belgium

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 July 2016 Received in revised form 3 February 2017 Accepted 11 March 2017 Available online 16 March 2017

Thermally processed (F0 ¼ 5min, process temperature 117  C) tomato and carrot purees containing 5% olive oil were stored in the dark at 20, 30 and 40  C for 6 months and investigated for carotenoids and lipid stability. Lipid oxidation (peroxide value and hexanal) and carotenoids (lycopene, a- and b-carotene) were analyzed and monitored during storage. Carotenoid bioaccessibility of the samples during storage was also studied. Under the storage conditions studied, the samples did not undergo significant lipid oxidation. Moreover, carotenoid bioaccessibility remained (P > 0.05) unaffected by storage. Regardless of storage temperature, carotenoids were stable with a retention of 98% and color (L*, a*, b* and DE) changes were imperceptible after 6 months. The results suggests that through formulation and careful selection of processing and storage conditions, carotenoid stability in lipid-containing fruit- and vegetable-based foods can potentially be guaranteed. This can be important to define optimal control measures to favor carotenoid stability and acceptable organoleptic properties during the storage of similar foods. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Carotenoids Thermal processing Storage Lipid oxidation Bioaccessibility

1. Introduction Carotenoids are fat soluble phytochemicals largely responsible for the red, orange and yellow color of fruits and vegetables. Interest in these compounds arises from their purported health benefits. The increased awareness of the health benefits associated with carotenoids has brought a surge of interest in identifying specific food formulations and processing conditions so as to maximize the potential of carotenoid-rich foods to confer the health benefits (Liu, 2003; Van Duyn & Pivonka, 2000; Astorg, ndez & Mínguez-Mosquera 2007). To this re1997; Hornero-Me gard, the inclusion of lipids into food formulations during processing (high pressure homogenization and/or thermal processing) can be a strategy to potentially enhance the nutritional quality of carrot and tomato based products (Colle et al., 2013; Mutsokoti, Panozzo, Musabe, Van Loey, & Hendrickx, 2015). In fact, processing can be used to favor the mass transfer of lycopene, b-carotene

* Corresponding author. KU Leuven, Department of Microbial and Molecular Systems, Laboratory of Food Technology, Kasteelpark Arenberg 22 postbox 2457, B3001 Leuven, Belgium. E-mail address: [email protected] (M. Hendrickx). http://dx.doi.org/10.1016/j.lwt.2017.03.021 0023-6438/© 2017 Elsevier Ltd. All rights reserved.

and a-carotene from the matrix to the oil phase, thus obtaining a carotenoid-rich lipid phase prior to digestion. This allows circumventing matrix-related factors and the low acidity gastric conditions that hinders the transfer of carotenoids into the oil phase during digestion (Rich, Fillery-Travis, & Parker, 1998). The transfer of carotenoids to oil is crucial for their bioaccessibility, i.e. the fraction of the nutrient that is released from the food matrix and subsequently incorporated into micelles during digestion before n, Diaz, & Svanberg, being absorbed by the enterocytes (Hedre 2002). To consider the effectiveness of the aforementioned strategy for food applications, it is necessary for the carotenoid enriched lipid phase in the food system to maintain its stability and functionality during storage. Therefore, investigation of the effects of storage conditions on the quality of lipid-containing fruit and vegetable based systems is needed. Lipids can undergo peroxidation, during processing and storage (Bonnie & Choo, 1999). Lipid oxidation is a complex process where unsaturated fatty acids react with molecular oxygen via a free radical mechanism or in a photosensitized or enzyme-catalyzed oxidation process (Christensen, Edelenbos, & Kreutzmann, 2007). Consequently, nonvolatile hydroperoxides are formed (primary oxidation) that further decompose to volatile compounds

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(secondary oxidation) which are responsible for quality changes in  , Majcher, & Dziadas, foods (Decker & McClements, 2000; Jelen 2012). Important to note is that the radical species that can be produced during the peroxidation process not only degrade fatty acids, but also other components of the lipid fraction, such as carotenoids, resulting in reduced nutritional quality (Hornerorez-G Mendez, Pe alvez, & Mínguez-Mosquera, 2001). In fresh and processed lipid-containing foods, lipid oxidation is one of the main causes of deterioration and reduced stability and quality and is often a determining factor in their shelf-life (van Ruth, Roozen, Posthumus, & Jansen, 1999). Therefore, the control of lipid oxidation is a major issue. The type of lipid can influence not only carotenoid bioaccessibility (Colle, Van Buggenhout, Lemmens, Loey, & Hendrickx, 2012; Huo, Ferruzzi, Schwartz, & Failla, 2007) but also carotenoid stability during storage. Specifically, the oxidative stability of lipids depends on a number of factors such as the degree of unsaturation of fatty acids and presence of other naturally occurring compounds that may inhibit lipid peroxidation during storage (Parker, Adams, Zhou, Harris, & Yu, 2003). Therefore, careful selection of the lipid substrate, as an ingredient, and food formulation design can be a strategy to control lipid oxidation, and as a consequence maintain carotenoid stability in carotenoid-rich food systems. In the Mediterranean diet, extra virgin olive oil (EVOO) represents the major edible vegetable oil and is becoming increasingly popular in other parts of the world liveau, & due to its unique health benefits (Lamy, Ouanouki, Be Desrosiers, 2014; Saleh & Saleh, 2011; Tuck & Hayball, 2002). In the food industry, EVOO is not only used as a filling medium of canned products, but also represents the lipid phase of a number of food formulations such as salad dressings, sauces and chilled and frozen ready-to-eat products (Calligaris, Sovrano, Manzocco, & Nicoli, 2006). To date, carotenoid stability studies during storage have been conducted in both model and real food systems. It is known that carotenoid degradation reactions during storage are accelerated at high temperature, oxygen and light exposure, and very low moisture content (Xianquan, Shi, Kakuda, & Yueming, 2005). In the case of fruit and vegetable based food systems (e.g. purees, juices, dehydrated carrots and tomatoes pieces), the general conclusion from shelf-life studies is that significant carotenoid degradation can occur during storage (Giovanelli & Paradiso, 2002) implying that storage conditions play a crucial role in impacting the final nutritional quality. Therefore, the aim of this study was to evaluate carotenoid stability during the storage of thermally processed lipidcontaining fruit and vegetable based matrices. To this purpose, lycopene and b-carotene in tomato and a- and b-carotene in carrot purees containing 5% (w/w) olive oil were considered. Aiming at shelf-stable food systems, industrially relevant thermal processing 10  C conditions were selected (F121:1 ¼ 5min, holding  c (F0)  temperature ¼ 117 C). These processing conditions will transfer a large portion of carotenoids to the oil fraction (Mutsokoti, Panozzo, Van Loey, & Hendrickx, 2016). The thermally processed purees were stored at 20, 30 and 40  C for 6 months and the stability of the samples monitored by measuring carotenoid content (all-trans and the cis isomers) and lipid oxidation (both primary and secondary) products. The effect of storage on the bioaccessibility of carotenoids was also investigated. This work is important for the design of effective control measures to promote carotenoid stability and acceptable organoleptic properties during the storage of shelfstable lipid-containing fruit and vegetable based formulations. Ultimately, this research has broad implications on the development and elaboration of carotenoid-containing functional foods.

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2. Materials and methods 2.1. Materials All chemicals and reagents used were of analytical or HPLCgrade. L-a-phosphatidylcholine and carotenoid standards (alltrans lycopene, all-trans b-carotene and all-trans a-carotene) were purchased from Sigma-Aldrich (Borne, Belgium). 5-cis lycopene, 9cis, 13-cis and 15-cis b-carotene were purchased from CaroteNature (Lupsingen, Switzerland). Olive oil (extra virgin) was kindly donated by Vandemoortele (Ghent, Belgium). Red ripe tomatoes (Lycopersicon esculentum cv Prunus) and orange carrots (Daucus carota cv Nerac) were obtained from a local shop in Belgium and stored at 4  C for 1 day prior to use. 2.2. Sample pre-treatments Tomatoes and carrots were sorted and washed under running deionized water. Carrots were peeled, cut into cylinders, while tomatoes were cut into thirds. The pieces were vacuum-packed in low density polythene bags. To prevent enzymatic reactions during processing and storage, tomatoes were blanched at 95  C for 8 min (Kebede et al., 2014). Carrots were pre-treated at 95  C for 20 min in a water bath (Haak W15 DC-10, Germany) in order to facilitate the softening of the cell wall as a result of b-eliminative depolymerisation of pectin (Sila, Smout, Elliot, Loey, & Hendrickx, 2006). The blanched plastic bags were immediately cooled in ice water, frozen in liquid nitrogen and stored for 3 days at 40  C until puree preparation. 2.3. Low fat puree preparation The pre-treated tomato and carrot samples were thawed overnight at 4  C. The pre-treated tomato pieces were blended (Waring Commercial, Torrington, CT, USA) for 1 min and then sieved (pore size 1.0 mm) to remove the seeds and the excess skin while the pretreated carrot pieces were mixed with deionized water in a 1:1 ratio and then blended using the same conditions previously described. On the one hand, to the carrot puree, extra virgin olive oil (EVOO) (5%, w/w) was added and the mixtures blended (Waring Commercial, Torrington, CT, USA) further for 10 s. The carrot puree/ oil mixture was then high pressure homogenized (Panda 2K, Gea Niro Soavi, Parma, Italy) at 100 MPa over one cycle for matrix disruption to aid carotenoid release and stabilize the puree/oil mixture. On the other hand, the tomato puree was first high pressure homogenized at 100 MPa during one cycle followed by the addition of EVOO (5%, w/w). The tomato puree/oil mixture was blended for 10 s and then further high pressure homogenized using the same conditions previously described. 2.4. Thermal processing The thermal treatment was carried out in a static steriflow pilot retort (Barriquand, Paris, France). The thermal treatment was done simultaneously for both low fat tomato and carrot purees. Glass jars, (100 mL volume, 95 mm height, and 45 mm diameter) were filled with 90 ± 0.5 g of the homogenized puree/oil mixtures, from here on referred to as simply puree, and closed with metal lids. The glass jars were loaded into the retort and sterilized at a process temperature of 117  C to achieve an F0 value of 5 min, with a holding time of 29.9 min. Temperature profiles in the retort and at the coldest point within the product were recorded using type T thermocouples (Ellab, Hillerod, Denmark). Following the thermal treatment, glass jars were immediately cooled in an ice bath.

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2.5. Storage study The thermally processed samples were placed in incubators, in the dark, at three different isothermal temperatures: 20, 30, and 40  C for a period of 24 weeks. Samples to which no storage time was applied following the thermal treatment represented the reference (time 0) samples. Puree samples that were not stored were taken as the control samples at time 0. At fixed points in time, stored samples were randomly selected, (3 jars per temperature for each matrix) transferred in a cooling room into falcon tubes (50 mL). The headspace of the tubes was flushed with N2. Thereafter, the samples were frozen in liquid nitrogen, and stored at 80  C until further analysis. All samples were thawed overnight at 4  C before the analyses. All samples were analyzed at once at the end of the 24 weeks storage period. 2.6. Static in vitro digestion The samples of tomato and carrot purees stored (0e24 weeks) at 20  C were subjected to a static in vitro digestion procedure according to Minekus et al. (2014) with some modifications, in which the gastric and intestinal phases were simulated. To simulate the gastric phase, 10 mL of emulsion and 7.5 mL of simulated gastric fluid (SGF, pH 3.04) (0.064% KCl, 0.015% KH2PO4, 0.263% NaHCO3, 0.345% NaCl, 0.003% MgCl2$(H2O)6 and 0.006% (NH4)2CO3) were added to the samples. This was followed by the addition of 1.6 mL of porcine pepsin solution (25 000 U/mL, made up in SGF) and 5 mL of CaCl2 (0.3 M). The pH was then adjusted to 3 ± 0.05 by addition of 10 mL of HCl (2 M). Thereafter 885 mL deionized water was added to bring the final volume of the gastric chyme to 20 mL. Subsequently, the headspace of the samples was flushed with N2 for 10s. The samples were incubated in the dark at 37  C while shaking end-over-end for 2 h. Once the gastric digestion was concluded, simulation of the intestinal phase was initiated by adding 11 mL of simulated intestinal fluid (SIF, pH 7.03) (0.063% KCl, 0.014% KH2PO4, 0.893% NaHCO3, 0.281% NaCl and 0.008% MgCl2$(H2O)6) to the gastric chyme (20 mL). Five milliliters of pancreatin solution [pancreatin, (800 U/mL, based on trypsin activity), lipase, 1600 U/mL, atocopherol 1.4% (w/w) and pyrogallol 0.6% (w/w) made up in SIF] was then added. Forty microliters of CaCl2 (0.3 M), 1.46 mL of deionized water and 2.5 mL of bile solution (160 mM, made up in SIF) were then added followed by flushing the headspace of the samples with N2 for 10 s. Thereafter, the samples were incubated for 120 min at the same conditions as mentioned previously. After simulation of the intestinal phase was concluded, part of the digest was ultra-centrifuged (Beckman Optima XPN-100 Ultracentrifuge, Brea, CA, USA) at 65 000 g for 1 h and 8 min at 4  C in order to separate the micellar fraction. The micellar fraction (supernatant) and the whole digest were then analyzed for carotenoid concentration. The static in vitro digestion procedure was performed in three replicates (each replicate analyzed once) and the experiment repeated twice. Carotenoid bioaccessibility (% BAC) was expressed as carotenoid concentration in the micellar phase (Cm) relative to its concentration in the digest (Cd):

by Palmero, Lemmens, Hendrickx, and Van Loey (2014) and Palmero, Panozzo, Simatupang, Hendrickx, and Van Loey (2014). The procedure was performed by mixing 1 g puree or 5 g digest with 25 mL of the extraction solution [hexane/acetone/ethanol (50:25:25 v/v/v) containing 0.1% of butylated hydroxytoluene (BHT)] and 0.5 g of NaCl. The mixture was stirred for 20 min at 4  C. Reagent grade water (7.5 mL, 18.2 MU cm) was then added and stirring continued for 10 min at 4  C. The mixtures were then placed in separation funnels to collect the organic phase. The isolated organic phase was filtered (Chromafil PET filters, 0.2 mm pore sizee25 mm diameter) and transferred into a dark vial for HPLC analysis. The identification and quantification of carotenoids were performed using a HPLC system equipped with a C30-column (3 mm  150 mm  4.6 mm, YMC Europe, Dinslaken, Belgium) and a diode array detector (Agilent Technologies 1200 Series, Dinslaken, Belgium). The temperature of the column was kept constant at 25  C during the analyses. A mobile phase of reagent grade water (18.2 MU$cm) (A), methanol (B) and methyl-t-butyl-ether (C) was used. The gradient elution for the analysis of carotenoids from the carrot purees was as follows: 0 min: 4% (A), 81% (B); 15% (C); 12e17min: 4% (A), 41% (B), 55% (C); 17e24 min: 4% (A), 81% (B), 15% (C). For the analysis of carotenoids from tomato purees, the gradient elution was as follows: 0 min: 4% (A), 81% (B), 15% (C); 3e9 min: 4% (A), 36% (B), 60% (C); 9e24 min: 4% (A), 28% (B), 68% (C); 24e38 min: 4% (A), 16% (B), 80% (C); 38e44 min: 4% (A), 81% (B), 15% (C). The flow rate was set at 1 mL/min and the gradient was built up in 44 min for all-trans and cis-lycopene and 24 min for alltrans a-carotene, all-trans b-carotene and cis b-carotene analyses. Carotenoid identification was performed at 472 nm for all-trans and cis-lycopene and at 450 nm for all-trans a-carotene, all-trans bcarotene and cis b-carotene on the basis of retention times and spectral characteristics of pure standards as described by Colle, Lemmens, Van Buggenhout, Van Loey, and Hendrickx (2010) and Lemmens, Van Buggenhout, Oey, Van Loey, and Hendrickx (2009). Carotenoids were quantified with the use of the corresponding calibration curves. 2.8. Peroxide value Lipid hydroperoxides were measured in duplicate using the ndez et al. (2001) with some method described by Hornero-Me modifications. Oil samples (0.05 g) were weighed into a 10 mL screw-caped test tube and dissolved in a mixture of chloroform and acetic acid (1 mL; 2:3 v/v) with the addition of 100 ml of Fe (II), vortexed for 15 s and incubated in the dark for 10 min. To remove the carotenoids, hexane (4 mL; containing 7 ppm BHT w/v) and reagent grade water (2 mL; 18.2 MU cm) were added, and the mixture vortexed for 15 s. The organic phase was discarded and the remainder of hexane removed under nitrogen gas current for 10 s. Thereafter, 1 mL of the aqueous phase was transferred to an Eppendorf tube, 100 mL of saturated ammonium thiocyanate solution added and the mixture incubated in the dark for 10 min. The absorbance was measured at 470 nm by an Ultrospec 2100 Pro UV Vis Spectrophotometer (Amersham Biosciences, Sweden). The concentration of hydroperoxides was calculated from the calibration curve of Fe (III).

Carotenoid bioaccessibilityð% BACÞ ¼

Carotenoid concentration in micelles ðC m Þ x100 Carotenoid concentration in digest ðCd Þ

2.7. Carotenoid concentration Carotenoids were quantified according to the method described

2.9. Hexanal Headspace hexanal was measured with a gas chromatograph system (6890N, Keysight Technologies, Diegem, Belgium) coupled to a mass selective detector (MSD) (5973N, Keysight Technologies, Diegem, Belgium), and equipped with a CompiPAL autosampler (CTC Analytics, Zwingen, Switzerland) based on the method described by Kebede et al. (2015). Concentrations were determined

L. Mutsokoti et al. / LWT - Food Science and Technology 80 (2017) 470e478

by use of the standard addition technique. Each sample was divided into four portions of 2.5 g each, and transferred into 10 mL amber glass vials (VWR International, Radnor, PA, USA). To each vial, 2.5 mL saturated salt solution was added. The vials were tightly closed using screw-caps with silicon septum seal (Grace, Columbia, MD). Under the fume hood, different volumes (0, 100, 200, and 300 mL) of hexanal working solution (1 mg/mL), were added through the septum to each of the four vials using a chilled syringe. Different amounts of deionized water were then added to obtain a standard volume and the vials vortexed. The vials were transferred to a cooling tray of the autosampler, thermostated at 10  C. A divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/ PDMS) stable flex solid phase microextraction (SPME) fiber (Supelco, Bellefonte, PA) was inserted through the vial septum and exposed to the sample headspace for 20 min at 40  C under agitation at 500 rpm. The volatiles on the SPME fiber were desorbed at 230  C for 2 min in the GC detector at a split ratio of 1:5. The chromatographic separation of the volatile aldehydes was carried out on an HP-5MS capillary column (30 m  0.25 mm inner diameter (i.d), x 0.25 mm film thickness; coated with 5%-phenylmethylpolysiloxane) (Agilent Technologies, Santa Clara, CA) with helium as carrier gas at a constant flow of 1.3 mL/min. The GC oven temperature was programmed from a starting temperature of 40  C, which was maintained for 2 min, to 172  C at 4  C/min, then ramped to 300  C at 30  C/min and kept constant at 300  C for 2 min before cooling back to 40  C. Mass spectra were obtained in electron ionization (EI) mode at 70 eV, with a scanning range of m/z 35e400 and a scanning speed of 3.8 scans per second. The selected ions were m/z 44, 56, 72, and 82. MS ion source and quadrupole temperatures were 230  C and 150  C, respectively. The dwell time was 10 s. Scan and selected ion monitoring (SIM) was used as data acquisition modes. The concentration of hexanal in tomato and carrot samples was calculated as the intercept of the line obtained by fitting the peak area (ordinate) and the amount of standard added (abscissa). The determination coefficient R2 of the fitting lines was found to be > 95 and > 97 for tomato and carrot samples, respectively. 2.10. Color Color analysis was carried out using a Hunterlab ColorQuest colorimeter (Hunterlab, Reston, VA). The instrument was standardized against white and black tiles before measurements. Color measurements were carried out in triplicate with five readings for each sample. The color attributes of the samples were expressed as * ) values. The latter was calculated acL*, a*, b* and chroma (Cab cording to equation (2). * Cab ¼

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi a*2 þ b*2

(2)

The color change in the samples during storage was expressed as total color difference (DE) according to Eq (3).

DE ¼

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi DL*2 þ Da*2 þ Db  2

(3)

where DL*, Da* and Db* represent the differences between the L*, a* and b* values, respectively, of the samples at the different storage times and the reference sample (time 0). 2.11. Statistical analysis Data are reported as mean value ± standard deviation. The Tukey's Studentized Range test (SAS version 9.4, Cary, N$C., USA) was used to check for significant differences in the means. Means

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were considered significantly different at P < 0.05. 3. Results and discussion 3.1. Lipid oxidation in carrot and tomato purees during storage The fatty acid composition of the extra virgin olive oil (EVOO), as given by the supplier, used in the present research is reported in Table 1. The EVOO consisted of about 14%, 78% and 7% saturated, monounsaturated, and polyunsaturated fatty acids, respectively. In ndez et al. (2001), the method for the accordance with Hornero-Me determination of peroxide value in the present study was validated by the iodometric AOAC official method with a good correlation (R2 ¼ 0.939) between data obtained by both analytical methods (data not shown). The peroxide value (PV) for fresh EVOO was 3.03 ± 0.32 mequiv peroxide/kg sample which is in agreement with a peroxide value for fresh olive oil as reported by García et al. (1996). Primary lipid oxidation was measured in the oil fraction from the carrot and tomato purees after 4, 12, 20 and 24 weeks of storage in the dark at three different temperatures: 20, 30, and 40  C. The concentration of the hydroperoxides, in the oil fraction of carrot and tomato samples was below the detection limit of 0.044 ndez et al., 2001) for all mequiv peroxide/kg of sample (Hornero-Me the storage time/temperature conditions that were investigated. It is known that when a food system undergoes lipid oxidation, its PV increases for a certain time period, due to hydroperoxides formation. Thereafter, the PV decreases as a result of hydroperoxides degradation to secondary lipid oxidation products (García et al., 1996). Since the absence of hydroperoxides in a system does not necessarily imply the absence of primary lipid oxidation, we also considered secondary lipid oxidation in the stored puree samples. In fact, Barden, Vollmer, Johnson, and Decker (2015) pointed out that hydroperoxides may have shorter life, up to a few days, depending on the moisture content of the food, before the commencement of the decomposition into secondary products. Therefore, secondary lipid oxidation products in carrot and tomato purees after 4, 12, 20 and 24 weeks of storage in the dark at 20, 30, and 40  C were measured. Representative GC-MS total ion chromatograms of the volatiles identified from the headspace of untreated and treated samples (time 0) of carrot and tomato purees are presented in Fig. 1. Even though similar volatiles were detected in the headspace of the control samples of carrot and tomato purees, their abundance differed between the two matrices. The most abundant volatiles detected were pentane, hexane, hexanal, 3methyl butanal, and 2.4-dimethly hexane. Hexanal was found to be the major aldehyde generated from lipid oxidation of the samples in the present study. Despite the fact that oleic acid is the main fatty acid in olive oil (Table 1), formation of octanal and nonanal, which represents decomposition products of oleic acid based on the beta-scission reactions routes (Kochhar, 1996, pp. 168e225), was negligible compared to hexanal. Similarly to Elisia and Kitts

Table 1 Fatty acid composition of extra virgin olive oil used. Fatty acid

%

C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:0

10.73 0.79 2.75 77.15 6.03 0.78 0.38 0.27 0.11

Values are expressed as the percentage (weight %) of total fatty acid methyl esters.

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Fig. 1. Volatiles identified from (A) tomato puree and (B) Carrot purees by HS-SPME GC-MS. Compounds identified are 1. CO2; 2. Acetone; 3. Acetic acid methyl ester; 4. Ethyl ether; 5. Pentane; 6.2-methyl propanol; 7. Methacrolein; 8. 2-methyl furan; 9. 3-methyl furan; 10. 4-methyl-(Z)-2-pentene; 11. Hexane; 12. 3-methyl butanal; 13. Pentanal; 14. Heptane; 15. Hexanal; 16. 2, 4-dimethyl hexane. ( ) Untreated; ( ), week 0 of storage.

(2011), this finding can be ascribed to the relatively greater susceptibility of linoleic acid to lipid oxidation compared to oleic acid. While hexanal and pentanal are compounds derived from oxidation of n-6 fatty acids, the former is the most frequently used n, to indicate the level of lipid oxidation in foods (Barriuso, Astiasara & Ansorena, 2012). It is a main product of linoleic acid oxidation via the 13-hydroperoxide (Kochhar, 1996, pp. 168e225). Previous investigations have reported hexanal as the major aldehyde indicating lipid oxidation in lipid-containing or lipid-based food systems where oleic acid is present in significantly higher proportions compared to linoleic acid, as is the case in the present study. For example, hexanal was used to monitor lipid oxidation volatiles in meat and meat products (Shahidi & Pegg, 1994) infant , 2006) potato formulas (García-Llatas, Lagarda, Clemente, & Farre  s, Lo  pez-Herna ndez, & Paseirocrisps (Sanches-Silva, de Quiro Losada, 2004), wheat crackers (Barden et al., 2015), and olive oil (Kanavouras & Coutelieris, 2006). It is known that upon fruit and vegetable tissue disruption, enzyme catalyzed oxidation reactions result in the degradation of endogenous lipids to fatty acids, yielding hydroperoxides, being the source of hexanal (Galliard, Matthew, Wright, & Fishwick, 1977). In fact, hexanal concentrations of 0.06 mg/g and 0.17 mg/g in untreated carrot and tomato samples, respectively, were found. These results indicate that hexanal was formed during the pre-treatments prior to thermal processing. As can be seen (Fig. 1), the amount of hexanal increased after thermal processing (time zero) by 64.7% and 37.0% for carrot and tomato samples, respectively. Therefore, on the one hand, the presence of hexanal in the control samples could be expected since the first stage of enzyme catalyzed oxidation reaction may already be initiated when cutting the carrot and

tomatoes prior to blanching. On the other hand, the increase in hexanal can be attributed to, for example, to the oxidative breakdown of unsaturated fatty acids due to the high temperatures employed during thermal processing (Christensen et al., 2007). Recognizing that the onset of oxidation during the processing can continue during storage and can influence storage stability of the final product, hexanal concentration in the headspace of the samples was monitored during six months of storage (Fig. 2). On the one hand, at a storage temperature of 20  C, the hexanal concentration in carrot and tomato matrices remained fairly constant throughout the storage period of 6 months. On the other hand, for samples that were stored at 30 and 40  C, a slight increase in hexanal concentration followed by a decrease after week 20 was observed. Moreover, hexanal formation in samples stored at 40  C was much higher for tomato (Fig. 2B), compared to the carrot purees (Fig. 2A). Generally, it seemed that the release of hexanal into the headspace of the sample after its formation was influenced by storage temperature. The decrease in hexanal concentration can be ascribed to hexanal degradation to other compounds. Previous investigations have reported a similar trend to that obtained in the  , Obuchowska, Zawirska-Wojtasiak, and present study. Jelen Wasowicz (2000) reported a sharp increase in hexanal, among other volatile compounds, during storage (10 days at 60  C) of n, and Farre  rapeseed oil. García-Llatas, Lagarda, Romero, Abella (2007) observed that the hexanal concentration in lipid foods stored for 7 months was lower than in samples that were stored for 4 months. As described earlier, decomposition of hydroperoxides to volatile carbonyl compounds is favored at elevated temperatures. By comparing hexanal concentration at day 0 and week 24, the

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475

Fig. 2. Hexanal concentration (mg/g puree) of thermally processed (A) carrot and (B) tomato purees during storage. (C) 20  C; (,) 30  C; (B) 40  C.

increase was relatively small, 0.26e0.57 mg/g puree in tomato puree and 0.18e0.32 mg/g puree in carrot puree. The levels of hexanal concentrations that were found in the present study are rather low compared to the values that are reported in the literature for samples that undergo significant lipid oxidation during storage. In a study by Sanches-Silva et al. (2004), hexanal concentration of 0.5e0.6 mg/g sample was reported for fresh, non-oxidized potato crisps. Moreover, in oxidized low fat dehydrated foods, rancid flavors were detected at hexanal concentration levels above 5 ppm (Fritsch & Gale, 1977). 3.2. Carotenoid concentration changes during storage Lycopene and b-carotene exert antioxidant functions in lipid phases by quenching 1O2 or free radicals leading to losses through bleaching and degradation and consequently, altered nutritional function (Sies & Stahl, 1995). Therefore, carotenoid concentration changes in the stored samples was also monitored. The cis isomers contributed 18e23% of the total carotenoid content in the purees and no significant differences were also found in the concentrations of the individual cis isomers (9-cis, 13-cis, 15-cis b-carotene in carrot and 9-cis, 13-cis, 15-cis lycopene in tomato puree) during storage (data not shown). Therefore, the changes in the total concentration (all-trans þ cis) of lycopene and b-carotene from tomato and a- and b-carotene from carrot purees as function of storage temperature and time was considered (Fig. 3). Generally, at all temperatures, total carotenoid concentration did not change considerably with storage time. This observation was also reflected in a high carotenoid retention at the end of the 6 months storage. In particular, in lipid-containing (olive oil, 5% w/w) tomato puree lycopene and bcarotene showed 97.8 ± 3.2 and 97.7 ± 1.2, 98.9 ± 2.6 and 98.0 ± 2.3, 98.7 ± 1.6 and 96.9 ± 1.6 percentage retention after six months storage at 20, 30 and 40  C, respectively. Similarly, for a- and bcarotene in lipid-containing (olive oil, 5% w/w) carrot puree a percentage retention of 98.7 ± 4.2 and 101.2 ± 5.9, 100.7 ± 1.6 and 98.2 ± 4.5, 102.5 ± 3.7 and 99.1 ± 2.8 at 20, 30, 40  C, respectively, was found. Nevertheless, these results suggest that not only the food systems but also the combination of the storage conditions employed in the present study potentially promoted the stability of the lipid fraction and carotenoids therein. These results are in agreement with other findings showing that the relative concentration of lycopene and b-carotene in fruit and vegetable based food systems, with or without oil, did not vary significantly during storage. For example, lycopene from canned pulp, puree in glass bottles, and paste in aluminum tubes remained stable during 3 months of storage at 30, 40 and 50  C (Lavelli & Giovanelli, 2003). Similarly, all-trans b-carotene stability was observed in pumpkin puree during 180 day storage at 23  C

(Provesi, Dias, & Amante, 2011) and in mango puree during 168 squez-Caicedo, Schilling, Carle, & days of storage at 25  C (Va Neidhart, 2006). Provesi et al. (2011) linked the all-trans b-carotene stability to the exclusion of oxygen and protection from light. Moreover, the inactivation of spoilage microorganisms and enzymes during the pretreatments and subsequent thermal processing was also reported to favor carotenoid stability during squez-Caicedo et al. (2006) also concluded storage. Similarly, Va that oxygen exclusion and headspace minimization was crucial for carotenoid stability during storage. In the case where oil was added to the food system, Anese, Bot, Panozzo, Mirolo, and Lippe (2015) observed stability of all-trans lycopene in ultrasound treated tomato pulp containing 10% sunflower oil after storage (from 15 to 100 days at 5  C). In order to relate the carotenoid changes during storage to color, the color change in the samples during storage was considered. Therefore, the CIE L*(lightness, ranging from 0 (black) to 100 (white)), a* (greenness (negative) to redness (positive)) and, b*(blueness (negative) to yellowness (positive)) values of color * ) components of the samples were measured and the chroma (Cab was calculated. The average color values as well as the chroma over the 24 weeks period are reported in Table 2. On average, the total color change, DE, was 2.42 ± 0.86 and 3.77 ± 0.76 for tomato and carrot purees, respectively and no significant differences in this as * ) color parameters as a function well as in the other (i.e. L*, a*, b*, Cab of storage time and temperature were found. Overall, the hydroperoxides originally present in the samples (as inferred from the peroxide value) were possibly degraded during the thermal treatment (Madhavi, Deshpande, & Salunkhe, 1995; Velasco & Dobarganes, 2002). This, together with the fact that the consumption (by radicals formed during the thermal treatment) of residual oxygen in the headspace of the samples followed by the subsequent degradation of the hydroperoxides can explain why at time ¼ 0, no peroxides were detected in the samples. This implies that during the subsequent storage of the samples, very limited oxygen was left to participate in autoxidation reactions, hence the high carotenoid stability (Fig. 3 and Table 2) and limited lipid oxidation (0.044 mequiv peroxide/kg of sample) which could be confirmed by the color stability of the samples (Table 2) as well as by the limited increase in hexanal concentration (Fig. 2). 3.3. Bioaccessibility of carotenoids during storage of carrot and tomato purees In the present investigation, a significant proportion (80%) of the carotenoids was present in the lipid fraction of the carrot and tomato food systems as a result of the combined effect of the high pressure homogenization and the thermal treatment (Mutsokoti

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Fig. 3. Concentration, expresses as mg/g puree, of (A) b-carotene (all-trans þ cis) and (B) a-carotene in thermally treated carrot puree; (C) all-trans b-carotene and (D) lycopene (alltrans þ cis) in thermally treated tomato puree during storage. (C) 20  C; (,) 30  C; (B) 40  C.

Table 2 CIE color parameters of reference (week 0) and stored (week 24) carrot and puree samples. Matrix

t (weeks)

L*

Tomato

0 24 0 24

57.25 57.45 66.15 62.22

Carrot

a* ± ± ± ±

0.12a 1.37a 0.06a' 1.82b'

29.57 28.74 16.56 17.14

* Þ Chroma ðCab

b* ± ± ± ±

0.10b 1.22b 0.01c' 0.72c'

63.23 61.96 83.49 80.56

± ± ± ±

0.75c 2.16c 0.15d' 2.09d'

69.81 68.31 85.12 82.37

± ± ± ±

0.72d 2.45d 0.14e' 2.11e'

Data are the mean of 3 measurements ± standard deviation. Significant difference is indicated by different letters.

Fig. 4. Relative carotenoid bioaccessibility (%) from (A) carrot and (B) tomato purees during storage at 20  C. Significant differences in the relative bioaccessibility as a function of storage time are indicated for each type of carotenoid within each matrix by a different letter. A: - b-carotene, - a-carotene; B: ,b-carotene, ,lycopene.

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et al., 2015, 2016). Consequently, the food systems represented the possibility of introducing carotenoids already solubilized in a lipid phase prior to digestion. In order to investigate the effect of the storage conditions on the amount of carotenoids that can be released from the matrix during in vitro digestion, the in vitro bioaccessibility of lycopene, a- and b-carotene at time zero and during storage at 20  C was considered (Fig. 4). Carotenoid bioaccessibility ranged from 19 ± 4% to 41 ± 3%, with lycopene and bcarotene being the least and most bioaccessible in the product, respectively. These results (Fig. 4) are in line with other findings showing that the in vitro bioaccessibility of lycopene is generally lower compared to b-carotene (Svelander, Lopez-Sanchez, Pudney, Schumm, & Alminger, 2011; Tyssandier, Lyan, & Borel, 2001; van het Hof, West, Weststrate, & Hautvast, 2000), even when the two carotenoids are considered within the same matrix (Palmero et al., 2014). Higher lycopene in vitro bioaccessibility was found in the present study as compared to previously reported values. For example, Colle et al. (2013) reported all-trans lycopene bioaccessibility values of around 16% in high pressure homogenized and thermally treated tomato pulp, while Svelander et al. (2011) observed that only 1%e 3% of the total all-trans lycopene from tomato emulsions was present in the micellar phase. In both cases, as in the present study, the samples contained 5% olive oil. With regard to b-carotene, Knockaert, Lemmens, Van Buggenhout, Hendrickx, and Van Loey (2012) observed all-trans b-carotene in vitro bioaccessibility of up to 66%, expressed as bioaccessible b-carotene concentration mg/g dry matter. Considering both carrot and tomato matrices and also the carotenoids in each matrix, storage did not have a noticeable effect on the bioaccessibility of carotenoids, as no significant differences (P > 0.05) among the samples studied were observed (Fig. 4). Similarly, Benlloch-Tinoco et al. (2015) also observed no effect of storage (10  C for 63 days) on the relative bioaccessibility of b-carotene from kiwi puree. By contrast, Anese et al. (2015) observed a significant decrease in lycopene bioaccessibility during 60 days storage of ultrasound treated tomato pulp containing 10% sunflower oil. This decrease in lycopene bioaccessibility coincided with an increase in peroxide value which indicated oxidation of the lipid fraction. This in turn was linked to the losses of lycopene concentration (indicating degradation). Therefore, the results of the present study suggests that the susceptibility of the carotenoids to degradation is influenced differently depending on the proportion of unsaturated lipids of the lipid fraction. In fact, sunflower oil generally has a significantly higher proportion of unsaturated fats compared to olive oil (Monfreda, Gobbi, & Grippa, 2012). However the presence of other compounds in the food matrix that possess antioxidant capacity e.g. phenolic compounds (Hager & Howard, 2006; Tuck & Hayball, 2002) cannot be ruled out. 4. Conclusion The positive effect of the addition of oil combined with a thermal treatment on carotenoid bioaccessibility has been demonstrated in various studies. In the context of shelf-stable fruit and vegetable based formulations tailored to deliver a specific nutritional function, storage conditions play a crucial role in influencing various chemical and physical reactions responsible for altering nutritional and organoleptic quality of such formulations. This work was addressed to evaluate the stability of carotenoids and their bioaccessibility during the storage of thermally processed carrot and tomato purees containing 5% olive oil. The results clearly showed that lycopene and b-carotene in tomato and a-carotene and b-carotene in carrot purees were stable with retention of 97% after 6 months of storage in the dark at 20, 30 and 40  C. No significant differences in carotenoid

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concentrations during storage, also confirmed by the color measurements, were found. Moreover carotenoid bioaccessibility remained unchanged during storage. Overall, the results revealed that the food system and the combination of processing and storage conditions (limited headspace, absence of light) potentially promoted the protection of both the lipid fraction and carotenoids therein during 6 months of storage. These results in turn demonstrate that the processing, formulation and storage conditions applied in the present study represent a valuable strategy to obtain vegetable based products with very high quality and improved nutritional properties. Acknowledgements This research has been carried out with the financial support of the KU Leuven Research Council (METH/14/03) through its long term structural funding programdMethusalem funding by the Flemish Government. References Anese, M., Bot, F., Panozzo, A., Mirolo, G., & Lippe, G. (2015). Effect of ultrasound treatment, oil addition and storage time on lycopene stability and in vitro bioaccessibility of tomato pulp. Food Chemistry, 172, 685e691. Astorg, P. (1997). Food carotenoids and cancer prevention: An overview of current research. Trends in Food Science and Technology, 8(12), 406e413. Barden, L., Vollmer, D., Johnson, D., & Decker, E. (2015). Impact of iron, chelators, and free fatty acids on lipid oxidation in low-moisture crackers. Journal of Agricultural and Food Chemistry, 63(6), 1812e1818. Barriuso, B., Astiasar an, I., & Ansorena, D. (2012). A review of analytical methods measuring lipid oxidation status in foods: A challenging task. European Food Research and Technology, 236(1), 1e15. Benlloch-Tinoco, M., Kaulmann, A., Corte-Real, J., Rodrigo, D., MartínezNavarrete, N., & Bohn, T. (2015). Chlorophylls and carotenoids of kiwifruit puree are affected similarly or less by microwave than by conventional heat processing and storage. Food Chemistry, 187, 254e262. Bonnie, T. P., & Choo, Y. M. (1999). Oxidation and thermal degradation of carotenoids. Journal of Oil Palm Research, 2(1), 62e78. Calligaris, S., Sovrano, S., Manzocco, L., & Nicoli, M. C. (2006). Influence of crystallization on the oxidative stability of extra virgin olive oil. Journal of Agricultural and Food Chemistry, 54(2), 529e535. Christensen, L. P., Edelenbos, M., & Kreutzmann, S. (2007). Fruits and vegetables of moderate climate. In R. G. Berger (Ed.), Flavours and Fragrances (pp. 135e181). Berlin, Heidelberg: Springer Berlin Heidelberg. Colle, I. J. P., Lemmens, L., Van Buggenhout, S., Met, K., Van Loey, A. M., et al. (2013). Processing tomato pulp in the presence of lipids: The impact on lycopene bioaccessibility. Food Research International, 51(1), 32e38. Colle, I., Lemmens, L., Van Buggenhout, S., Van Loey, A., & Hendrickx, M. (2010). Effect of thermal processing on the degradation, isomerization, and bioaccessibility of lycopene in tomato pulp. Journal of Food Science, 75(9), C753eC759. Colle, I. J. P., Van Buggenhout, S., Lemmens, L., Van Loey, A. M., & Hendrickx, M. E. (2012). The type and quantity of lipids present during digestion influence the in vitro bioaccessibility of lycopene from raw tomato pulp. Food Research International, 45(1), 250e255. Decker, E. A., & McClements, D. J. (2000). Lipid oxidation in oil-in-water Emulsions: Impact of molecular environment on chemical. Journal of Food Science, 65(8), 1270e1282. Elisia, I., & Kitts, D. D. (2011). Quantification of hexanal as an index of lipid oxidation in human milk and association with antioxidant components. Journal of Clinical Biochemistry and Nutrition, 49(3), 147e152. Fritsch, C. W., & Gale, J. A. (1977). Hexanal as a measure of rancidity in low fat foods. Journal of the American Oil Chemists Society, 54(6), 225e228. Galliard, T., Matthew, J. A., Wright, A. J., & Fishwick, M. J. (1977). The enzymic breakdown of lipids to volatile and non-volatile carbonyl fragments in disrupted tomato fruits. Journal of the Science of Food and Agriculture, 28(9), 863e868. , R. (2006). Monitoring of García-Llatas, G., Lagarda, M. J., Clemente, G., & Farre headspace volatiles in milk-cereal-based liquid infant foods during storage. European Journal of Lipid Science and Technology, 108(12), 1028e1036. , R. (2007). García-Llatas, G., Lagarda, M. J., Romero, F., Abell an, P., & Farre A headspace solid-phase microextraction method of use in monitoring hexanal and pentane during storage: Application to liquid infant foods and powdered infant formulas. Food Chemistry, 101(3), 1078e1086. rrez, F., Castellano, J. M., Perdiguero, S., Morilla, A., & Albi, M. A. García, J. M., Gutie (1996). Influence of storage temperature on fruit ripening and olive oil quality. Journal of Agricultural and Food Chemistry, 44(1), 264e267. Giovanelli, G., & Paradiso, A. (2002). Stability of dried and intermediate moisture

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