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Cutin composition of selected northern berries and seeds
Food Chemistry 122 (2010) 137–144
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Cutin composition of selected northern berries and seeds Riikka Järvinen *, Mika Kaimainen, Heikki Kallio Department of Biochemistry and Food Chemistry, University of Turku, FI-20014 Turku, Finland
a r t i c l e
i n f o
Article history: Received 28 July 2009 Received in revised form 15 December 2009 Accepted 11 February 2010
Keywords: Berry Cutin Epoxy fatty acids Hydroxy fatty acids Mass spectrometry Seed
a b s t r a c t Monomeric compositions of the extractive-free cuticular polymer fractions of seven berries – rosehip (Rosa rugosa), black chokeberry (Aronia melanocarpa), strawberry (Fragaria x ananassa), raspberry (Rubus idaeus), cloudberry (Rubus chamaemorus), crowberry (Empetrum nigrum) and rowanberry (Sorbus aucuparia) – as well as the seeds from three berries – cloudberry, sea buckthorn (Hippophaë rhamnoides) and blackcurrant (Ribes nigrum) – were investigated. Depolymerisation of the cutin polymer was carried out using NaOMe-catalysed methanolysis and the composition of TMS-derivatised monomers was determined by GC using FID and MS detection. Two depolymerisation techniques were compared; 1.0 M NaOMe at ambient temperature and 1.3 M NaOMe under reflux conditions. The degree of depolymerisation of the cuticular membrane ranged from 2% (strawberry) to 84% (rosehip) for the CHCl3-soluble cutin monomers. Depolymerisation of seed samples was low (1–3%). The predominant monomers were C16 and C18 x-hydroxy acids with mid-chain functionalities, mainly epoxy and hydroxy groups, but suberin-like a,x-dicarboxylic acids, with mid-chain hydroxyl groups and C15 chain length monomers, were also found. Seed cutin monomers differed from the corresponding berry skin monomers. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Cuticular polymer cutin is the main lipid polymer in the skin of fruits and berries, as well as in the outer layers of the leaves and stems of vascular plants. Ester-linked monomers of cutin consist of a complex mixture of long-chain x-hydroxy acids, with typical chain lengths of 16 and 18 carbons, together with fatty acids, fatty alcohols and phenolic compounds, such as cinnamic acid derivatives and glycerol. Suberin-like a,x-diacids and their hydroxylated derivatives have also been detected. Cutin, together with nondepolymerisable cutan and cuticular waxes, make up the cuticle of vascular plants, which acts as a barrier and gateway between the plant and its environment (Graça, Schreiber, Rodrigues, & Pereira, 2002; Kolattukudy, 2001). The cuticle is attached to the epidermal cells of plants by the pectic layer (Kolattukudy, 2001). Cell walls of berry skin, and the surface of other plants cells, consist of a network of cellulose and hemicelluloses together with pectins. The composition of polysaccharides differs between berries and in the different parts of berries, for example between the skin, pulp and seeds (Hilz, Bakx, Schols, & Voragen, 2005). One major current use of berries is in industrial juice production. Together with seeds, polysaccharides and the polyphenolic compounds, the cuticular polymers account for the bulk of the pressed residue in juice production. This byproduct could be utilised as a valuable source of dietary fibre or * Corresponding author. Tel.: +358 2 333 6874; fax: +358 2 6860. E-mail address: riikka.jarvinen@utu.fi (R. Järvinen). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.02.030
specialty chemicals, but the lack of detailed compositional information hinders its usage. Berry is a common term for fleshy, juicy fruits with one or typically more seeds, including grape, gooseberry, blackcurrant, cranberry, sea buckthorn, tomato, strawberry and raspberry (Bender, 2005). Some are actually accessory fruits, even though they are generally considered berries. For example, a strawberry is not a berry but a pseudocarp, the edible red pulp of which is a swollen receptacle of the flower. Stone fruits, raspberries and cloudberries are also aggregate fruits with multiple drupes. The peel of a berry has a major influence on how berries are preserved in nature. Cuticular polymers together with associated waxes are thought to be the major contributors to morphology and preservation (Kolattukudy, 2001). To our knowledge, there is no published information about the cuticular components of berry seeds. Previous investigations have concentrated on other issues, including seed weight (Johansson, Laakso, & Kallio, 1997), sugar composition (Hilz et al., 2005), oil content and nutritional value (Tahvonen, Schwab, Yli-Jokipii, Mykkänen, & Kallio, 2005), fatty acid composition of oils (Yang & Kallio, 2001) and the effect of growth conditions on composition (Johansson, Kuusisto et al., 1997). Helbig, Böhm, Wagner, Scubert, and Jahreis (2008) recently studied the composition of berry seed press residue, but not the cuticular polymers of the seeds. Soybean seeds (Shao, Meyer, Ma, Peterson, & Bernards, 2007) and some angiosperm seeds (Arabidopsis thaliana, Brassica napus) (Molina, Bonaventure, Ohlrogge, & Pollard, 2006; Molina, Ohlrogge, & Pollard, 2008) have been examined for their composition, including
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cutin monomers. Cutin composition of the inner seed coat of the apple (Velcheva, Espelie, & Ivanov, 1981) and grapefruit (Espelie, Davis, & Kolattukudy, 1980) have also been investigated. Results have indicated that seeds possess two thin cuticular layers; one in the outer seed coat and another associated with the inner seed coat. Furthermore, lamellar suberin structures have been found throughout the cell walls of grapefruit seeds (Espelie et al., 1980) and suberin-type monomers have been released from some angiosperm seeds (A. thaliana, B. napus) (Molina et al., 2008). We have previously described the cutin monomer composition of edible northern berries, including sea buckthorn (Hippophaë rhamnoides), blackcurrant (Ribes nigrum), cranberry (Vaccinium oxycoccos), lingonberry (Vaccinium vitis-idaea) and bilberry (Vaccinium myrtillus), which are all in common use, especially in Scandinavia (Kallio, Nieminen, Tuomasjukka, & Hakala, 2006). In this paper, we describe for the first time the amount of extractive-free cuticular polymers and cutin monomer composition of several northern berries: rosehip (Rosa rugosa), black chokeberry (Aronia melanocarpa), strawberry (Fragaria x ananassa), raspberry (Rubus idaeus), cloudberry (Rubus chamaemorus), crowberry (Empetrum nigrum) and rowanberry (Sorbus aucuparia). The seeds of three berries – cloudberry, sea buckthorn and blackcurrant – were also investigated. With this second report of the composition of cuticular hydroxyl acids in northern berries, our aim is to acquire knowledge on the cuticular material of northern berries, as a part of the exploitation of Nordic natural resources, regarding chemical composition, nutritional value and sensory properties.
2. Materials and methods 2.1. Berry peel and seed samples Rosehips and black chokeberries were obtained from MTT Agrifood Research Finland (Jokioinen, Finland) and peels separated from frozen whole berries by manual peeling, washing with water and drying. Strawberries, raspberries and cloudberries were bought from a local market (Turku, Finland) and pressed with a hydraulic juice extractor to yield juice press residues from whole berries. Crowberry and rowanberry samples were frozen strained by-products obtained from Kiantama Oy (Suomussalmi, Finland). The seeds were manually separated from all berry samples after drying in an oven at 60 °C. Berries were collected at optimal ripeness for industrial berry processing. Cloudberries, crowberries and rowanberries were wild berries, whilst others were industrially obtained mixed cultivars. Sea buckthorn and blackcurrant seeds were obtained from Aromtech Ltd. (Tornio, Finland). Cloudberry seeds were obtained from the above-mentioned strained by-product by manual separation from the press residue. Seeds were crushed in a mortar with the aid of liquid nitrogen and dried in an oven at 60 °C.
2.2. Isolation of cuticular membranes The cuticular membranes of berry peels were isolated using the method previously described (Kallio et al., 2006). Briefly, the procedure was a combination of enzymatic treatments with cellulase (5.0 g/l Econase CE, AB Enzymes, Darmstadt, Germany) and pectinase (1.0 g/l Pectinex Ultra SP-L, Novozymes, Bagswaerd, Denmark) in acetate buffer. The reaction mixture was washed with water and the residue dried and weighed. The dried residue was exhaustively extracted using a Soxhlet apparatus with CHCl3 and MeOH (14– 20 h each), yielding extractive-free cuticular membranes. After repeating the isolation process, the product was washed with water and dried in an oven at 60 °C before depolymerisation. The
crushed seeds were treated once with the same combined enzymatic and extraction method. 2.3. Depolymerisation of cuticular membranes Depolymerisation of the isolated cuticular membranes was carried out using a method previously described by Holloway and Deas (1973), and further modified by our research group (Kallio et al., 2006). Dried samples of the extractive-free cuticular membrane (50– 100 mg) were refluxed for 3 h in 25 ml of a 1.3 M solution of NaOMe in MeOH. The reagent was freshly prepared by dissolving metallic sodium in dry methanol. Precautions were taken to eliminate moisture from the reaction. The reaction mixtures were filtered and the filtrates were acidified by adding 2 M H2SO4 in MeOH and dried using a rotary evaporator. The residue was suspended in 50 mL of H2O and extracted with CHCl3 (2 50 ml). The CHCl3-soluble monomers were dried with anhydrous Na2SO4 and after filtration, solvent was evaporated. Cutin monomers were determined gravimetrically and stored in CHCl3 at 18 °C for further analysis. In addition to the traditional reflux method, a small-scale closed-bottle method (test tube method) was developed. Dried samples of extractive-free cuticular membrane (20 mg) were treated with 3 ml of freshly prepared 1.0 M NaOMe in dry MeOH. Methanolysis was carried out overnight in a shaker (at approx. 500 rpm) at ambient temperature. The reaction mixture was acidified with 2 M H2SO4 in MeOH and the supernatant was separated by centrifugation (1315g, 10 min). Then, 10 ml H2O were added and the cutin monomers extracted with CHCl3 (2 10 ml). The mixture of monomers in CHCl3 was dried with anhydrous Na2SO4, filtered and solvent evaporated. Cutin monomers were determined gravimetrically and stored in CHCl3 at 18 °C for further analysis. 2.4. Analysis of monomeric compounds Cutin monomer methyl esters were further derivatised before the chromatographic analysis. CHCl3 was evaporated, and the remaining monomers were dried under a stream of nitrogen and kept in a desiccator overnight before trimethylsilylation with TriSil reagent (HMDS and TMCS in pyridine) (Pierce Chemicals, Rockford, IL). After adding the reagent, the sample was vigorously shaken at room temperature for 5 min and kept at 60 °C for 15 min. The composition of monomers was determined by gas chromatography electron impact mass spectrometry (GC–EI-MS) with a Shimadzu GC–MS-QP5000 (Shimadzu, Kyoto, Japan) under the following chromatographic conditions: 30 m 0.25 mm 0.25 lm DB-1MS column (Agilent, Folsom, CA); split injection (14:1); carrier gas He (linear velocity 42 cm/s); injector and detector temperatures 300 °C. The oven temperature was programmed from 125 °C at 10 °C/min to 220 °C, from 220 °C at 3 °C/min to 290 °C, and kept at 290 °C for 2 min. Mass range m/z 45–550 was acquired. In addition to GC–MS, all samples were analysed in parallel with gas chromatography-flame ionisation detection (GC-FID) with a Shimadzu GC 17A with the chromatographic conditions mentioned above, to compare the different detection methods. Compounds were identified by comparing the EI-MS spectra of the derivatives (as methyl ester TMS ether or TMS ester TMS ether) with published spectra (Deas, Baker, & Holloway, 1974; Eglinton & Hunneman, 1968; Eglinton, Hunneman, & McCormick, 1968; Holloway & Deas, 1971; Holloway & Deas, 1973; Järvinen et al., 2009; Kallio et al., 2006; Rontani & Aubert, 2004), spectra of reference compounds and interpreting the structures by common fragmentation patterns and retention times. Positions of the double bonds were not confirmed by chemical methods.
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2.5. Statistical analysis Each depolymerisation reaction with test tube method was carried out in triplicate and the reflux method in duplicate. Statistical analyses were carried out using the statistical software SPSS for Windows (Version 14.0; SPSS Inc., Chicago, IL). All results were expressed as means and standard deviations (SD). The t-test was used to separately compare the depolymerisation and detection methods. Differences reaching a minimal confidence level of 95% were considered statistically significant. 3. Results and discussion 3.1. Isolation of cuticular membranes
idues from an industrial process (no enzymes used) and closely resembled the material of the abovementioned juice pressing. Rosehip and black chokeberry samples were the peel from manually peeled whole berries, resulting in clean skin fractions without fruit flesh. The high amount of cuticular material (Table 1) obtained from seeds evidently contained not only cuticular components, but also other components remaining after the isolation process. The procedure of isolation was probably insufficient, as unlike the berry skins it was not repeated. The structure and composition of seeds deviating substantially from the skins may have also reduced the efficacy of enzymes and solvents. Seeds are complex organs with polyesters in many distinct cell layers (Molina et al., 2006). Their locations were not studied in this investigation, which mainly concentrated on the monomer types released from the seed material.
The first enzymatic treatment with cellulase and pectinase removed approx. 59% of raspberry, 72% of cloudberry, 35% of black chokeberry, 60% of rowanberry, 54% of rosehip, 66% of strawberry and 36% of crowberry raw materials. The first extraction (MeOH and CHCl3, 14–20 h each) of the washed residue removed a further 0% of raspberry, 8% of cloudberry, 13% of black chokeberry and rowanberry, 15% of rosehip and strawberry, and 31% of crowberry raw materials, calculated from the original seedless berry peel material. The second enzymatic treatment additionally reduced another 2–12% of the total mass, calculated from the raw material before the enzymatic treatments, with the exceptions of a 35% and 39% reduction in cloudberry and raspberry, respectively. The results clearly showed that more material was removed from press residue of the soft-skin berries (strawberry, raspberry and cloudberry) in the enzymatic treatments, compared with rosehip and black chokeberry peels and the crowberry straining residue (Table 1). The extractable compounds (including waxes, polyphenolics) were removed in the first extraction with MeOH and CHCl3 according to gravimetric measurement. Interestingly, raspberry press residue contained no solvent extractable material. Raw materials of the cuticular membranes were different because of the morphological and structural differences of the berries and the varying pre-treatments of the raw materials. This led to difficulties when comparing the amounts of extractive-free cuticular polymers between the berries. However, the results obtained show the high variation in extractive-free cuticular material in the different berries (Table 1). The peel of the soft-skinned strawberry (accessory fruit), as well as raspberry and cloudberry (aggregate fruits), was obtained from pressing the berries with a hydraulic juice extractor. Thus, the material was a residue, which also contained some non-water-soluble polysaccharides of the fruit flesh. Crowberry and rowanberry materials were strained res-
The traditional and laborious reflux method for depolymerisation of the cutin polymer by transmethylation with sodium methoxide in methanol is greatly affected by moisture in the reaction. It also requires large amounts of solvents. Separation of the residue and the CHCl3-soluble monomers by filtration and evaporation further exposures the reaction to moisture and may result in the loss of monomers. For these reasons, a small-scale closed-bottle test tube procedure was developed. Centrifugation was used to isolate the monomers from the acidified reaction mixture of methanolysis. The new method was carried out parallel to the reflux method, to determine its applicability for cutin depolymerisation. The two methods have previously been compared with potato suberin with analogous results (Järvinen et al., 2009). The two tested methods provided slightly different results depending on the cuticular materials (Table 2). For rowanberry, raspberry and strawberry, the methods generated identical results but for rosehip, black chokeberry and cloudberry the results were smaller in the test tube reaction. A statistically significant difference was obtained for cloudberry and rosehip, although its importance is questionable as the samples were small aliquots of natural material. The differences in results may be due to the varying composition and ratios of the cuticular components in different samples. The fact that samples were not homogenised extremely effectively may provide another explanation for the deviation between the two methods. The test tube method may be more susceptible to gravimetric errors because of its smaller scale than that of the reflux method (20 mg vs. 100 mg of sample, respectively), although extreme care was taken with all steps. However, the test tube method was found to be fast and reliable for
Table 1 Proportion of the extractive-free cuticular material of the raw materials.
Table 2 Comparison of depolymerisable cutin in selected berries and seeds (average percentage and standard deviation of CHCl3-soluble monomers of raw cutin).
a b c
Berry
Extractive-free cuticular material of raw materials (%)
Rosehip Black chokeberry Crowberry Rowanberry Strawberry Raspberry Cloudberry
38a 51a 42b 35b 29c 29c 18c
Seed Cloudberry Blackcurrant Sea buckthorn
58 71 74
Peel. Commercial straining by-product. Juice press residue.
3.2. Depolymerisation of cuticular polymer
a
Berry
Test tube N
AV (%)
SD
N
AV (%)
SD
Rosehipa Crowberry 2004 Crowberry 2005 Black chokeberry Strawberry Rowanberry Raspberry Cloudberrya
3 3 3 3 3 3 3 3
72.3 51.2 60.3 15.4 2.3 26.0 5.9 37.7
2.0 3.0 4.2 1.4 0.2 1.0 0.1 3.1
2 2 2 2 2 2 2 2
83.7 56.2 66.2 21.8 2.4 29.7 6.7 47.7
1.8 2.8 1.3 3.9 0.1 4.2 0.4 3.0
Seed Blackcurrant Sea buckthorn Cloudberry
3 3 3
1.5 1.3 3.2
0.3 0.0 0.6
2 2 2
2.0 1.2 3.0
0.0 0.7 0.5
p < 0.05.
Reflux
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providing a qualitative description of the cutin monomer composition and was preferred over the reflux method. The cuticle and monomer composition have been found to change during maturation (Kolattukudy, 2001; Molina et al., 2008). The aim was to harvest each of the berries at the stage of optimal ripeness according to common commercial practices and the personal but non-scientific evaluation of a berry specialist. The industrial berries were purchased, as usual, from various sources, which decreased the homogeneity of the raw materials. The amount of depolymerised, CHCl3-soluble cutin monomers (Table 2) again revealed the differences between cuticular polymers in different berries. The proportion of depolymerised material varied from 2% in strawberry to 70–80% in rosehip. In earlier investigations, the cutin monomer yield was determined for sea buckthorn (46%), blackcurrant (8%), cranberry (27%), lingonberry (30%) and bilberry (6%) (Kallio et al., 2006). The abundant residue remaining non-depolymerised in methanolysis of the isolated berry cuticular membrane deserves further compositional and structural investigation. Special attention should be paid to cutan-type polymer and polysaccharide complexes. According to the results obtained, there is no evident correlation between the amounts of cuticular material and ester-bound cutin in berry cuticles. As cutin is known to be a protective barrier against water loss together with cuticular waxes, in addition to genetic background, the growth conditions and climate may also be major explanations of the differences between berries. The amount of the cuticular material, cutin:cutan ratio and also the monomeric units together with the cuticular waxes all have a crucial influence on the preservation of different berries in nature and when collected for food. The composition and structure of the surface tissues ultimately define the characteristic-preserving properties of berries against drying, chemical attacks, mechanical injuries and microbial infections. For example, crowberries and lingonberries maintain their basic morphology and composition for almost a year in nature, whereas strawberries, raspberries and cloudberries are typically destroyed by drying or infections in early autumn. Earlier studies have also shown the amount of cutin in seed coats to be very small, whilst seed cutin has been determined separately for whole seeds (outer coat) and the inner coat (Molina et al., 2006). The small amount of cutin in seeds may be because of the location of seeds inside the berry as there is no evident need for an effective barrier against desiccation or pathogens, for example. Although the cuticular polymer values obtained from berry seeds may not fully explain the amount of cutin polymer, we were able to obtain enough monomers for profile assessment. As a curiosity, crowberries from consecutive years (2004 and 2005) were compared. The berries collected in 2004 contained about 10% less ester-bound cutin monomers than those collected in 2005. Since we compared only two years and growth locations were not specified, these results are inadequate for further conclusion. However, if the effects of weather conditions and growth places ever want to be investigated, careful analyses of berries from several years and from specified locations would have to be carried out. Cuticular polymers should evidently be included in non-soluble dietary fibre, although this has not been stated in common definitions. Dietary fibre is defined as the edible parts of plants resistant to digestion and absorption in the human small intestine, with complete or partial fermentation in the large intestine (AACC, 2001). This definition includes polysaccharides, oligosaccharides, lignins and associated plant substances, to which cuticular polyesters together with suberin and waxes can be included. Thus, the knowledge of these plant substances is evidently important. Plaami, Kumpulainen, and Tahvonen (1992) have previously examined the total dietary fibre of strawberries, blackcurrants, redcurrants, bilberries and lingonberries, finding that these berries
contain on average 18–19, 58, 47–53, 33 and 26 g/kg of fibre in each fresh berry, respectively. Bilberries and lingonberries were also studied for insoluble (27 and 22 g/kg, respectively) and soluble fibre (7 and 4 g/kg, respectively). These values also contain the weight of seeds, so conclusions about the correlation between cuticular material and fibre cannot be drawn based on these values. Dietary fibre powders of chokeberry, bilberry and blackcurrant (Wawer, Wolniak, & Paradowska, 2006) have been analysed with 13 C CP/MAS NMR and the fibre content was also determined using enzymatic hydrolysis of the berry powders. The total dietary fibre was 66–72% and insoluble fibre was 60–66% of the powder. Only 5–7% was found to be soluble fibre. In NMR spectra, cellulose and other polysaccharides, in addition to lignin and cutin, were recognised but the cutin–cutan relationship was not studied. In our investigation, some berry cuticles were found to be highly resistant to ester-breaking reactions, which may be because of the high amount of cutan-type compounds with C–C and ether bonds present. This assumption requires further confirmation with spectroscopic methods suitable for non-soluble material research. FT-IR and solid state NMR spectroscopy have been used to outline non-depolymerisable material from the cuticular fractions of lime (Pacchiano et al., 1993) and tomato, pepper and apple fruits (Johnson, Dorot, Liu, Chefetz, & Xing, 2007), but not applied to the isolated cuticular polymers of berries. 3.3. Cutin monomers of berries CHCl3-soluble cutin monomers of the berries and seeds were determined by gas chromatography using both MS and FID detection techniques (Table 3). We obtained better resolution and separation of peaks with MS, but traditionally FID detection has been considered more reliable for quantitative analysis. Our goal was to display chromatographic monomer profiles and, as the detection methods did not differ statistically (p > 0.05) when the major monomer groups were compared, MS detection was chosen as the preferred method. The relative proportions of different monomers in Table 3 are mean values from the MS analysis also previously used for berry cutin analysis (Kallio et al., 2006). Mean ± SD values for monomers were calculated from at least triplicate chromatographic analyses. Fig. 1 shows examples of the GC–MS total ion chromatograms of cutin analyses of some berries and seeds. Identification of the cutin monomers was performed with GC– MS after TMS derivatisation of the monomers released. GC separation of the complex mixture of cutin monomers, followed by MS detection, is necessary for analysis and identification, as reference compounds are only commercially available for some monomers. TMS ester and ether derivatives of cutin monomers provide diagnostic spectra that make interpretation relatively unambiguous. The location of a mid-chain hydroxyl group is easy to detect, as a-cleavage at either side of the carbon bearing the TMS-derivatised functional group, or cleavages between carbons bearing the functional groups in the case of adjacent groups, are most preferred in the ionisation (Eglinton & Hunneman, 1968; Eglinton et al., 1968; Hunneman & Eglinton, 1972). Long-chain x-hydroxy acids and a,x-diacids form diagnostic rearrangement ions when analysed as TMS derivatives (Rontani & Aubert, 2004). Tables gathering the information of the diagnostic ions for cutin and suberin monomers have been previously published by our research team (Järvinen et al., 2009; Kallio et al., 2006). Although precautions were taken to eliminate moisture from the reflux reaction, there remained varying amounts of saponification products in all the end products obtained with the reflux method. Methyl acetate as a competitor for saponification was tried but resulted in the formation of several acetylated derivatives of cutin monomers, which in turn made identification difficult. This reaction has been used by Molina et al. (2006), but with complete
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R. Järvinen et al. / Food Chemistry 122 (2010) 137–144 Table 3 Composition (mean ± SD) of cutin monomers released from berries by methanolysis (test tube reaction) as a percentage of total peak areas in GC–MS chromatogram. Peaka 1 2 3 4 5 7
8 9 10 11 12
Cutin monomerb
Black chokeberry
Rosehip
Cloudberry
Raspberry
Strawberry
Rowanberry
Crowberry
x-Hydroxy acids and hydroxy a,x-diacids
79.4 ± 0.7 0.8 ± 0.1 0.7 ± 0.0 16.8 ± 0.6 0.7 ± 0.2 1.6 ± 0.2 2.8 ± 0.2
94.4 ± 0.7 5.9 ± 0.3 4.5 ± 0.2 78.7 ± 1.2
91.9 ± 1.0 3.1 ± 0.1 4.6 ± 0.3 73.9 ± 2.0 0.3 ± 0.0 0.1 ± 0.0 1.7 ± 0.1 1.5 ± 0.2
83.3 ± 0.5 9.0 ± 0.2 3.6 ± 0.2 48.7 ± 1.4 3.7 ± 0.1 4.3 ± 0.1 8.7 ± 0.2 1.2 ± 0.4
61.4 ± 1.2 2.8 ± 0.0 1.8 ± 0.4 37.8 ± 1.3 0.6 ± 0.1 0.3 ± 0.0 4.6 ± 0.4 0.9 ± 0.1 2.1 ± 0.1 3.3 ± 0.1 2.5 ± 0.7 0.4 ± 0.4 1.7 ± 0.1 1.3 ± 0.1 0.2 ± 0.2 0.9 ± 0.1
83.8 ± 2.9 1.8 ± 0.2 4.4 ± 0.2 52.2 ± 5.0
93.3 ± 0.4 4.7 ± 0.2 1.0 ± 0.1 28.6 ± 0.2 0.6 ± 0.1 3.3 ± 0.1 19.1 ± 0.3
16-OH-16:0 Mono-OH-1,16-dioic 16:0d (major 7/8) di OH-16:0 (major 9,16/10,16) 18-OH-18:3c,g 18-OH-18:2c 18-OH-18:1c 18-OH-18:0e 18-OH-9,10-epoxy 18:2c,f,g 18-OH-9,10-epoxy 18:1c,f 18-OH-9,10-epoxy 18:0f 9,10,18-triOH-18:2c,g 9,10,18-triOH-18:1c 9,10,18-triOH-18:0 20-OH-20:0 22-OH-22:0 24-OH-24:0 6/7-OH-1.15-dioic-15:0 9,15-DiOH-15:0 9/10-OH-15:0
Other compound groups ar Aromatic compounds da Diacids fa Fatty acids ol Alcohols un Unidentified monomers TOTAL
0.0 ± 0.0 3.7 ± 0.8
2.9 ± 0.4 23.5 ± 0.2 17.7 ± 0.5
tr 0.5 ± 0.1 0.7 ± 0.1 4.5 ± 0.6
4.8 ± 0.6 6.6 ± 0.9 tr 0.3 ± 0.1 1.0 ± 0.2 0.5 ± 0.1
0.8 ± 0.1
0.2 ± 0.0 0.3 ± 0.0 0.9 ± 0.1 1.0 ± 0.0 0.2 ± 0.0 0.6 ± 0.0 0.3 ± 0.0 0.5 ± 0.1
tr
100.0
1.1 ± 0.2 1.8 ± 0.3 2.8 ± 0.3 100.0
6.9 ± 0.4 2.3 ± 0.2 0.9 ± 0.2 8.2 ± 0.6 4.3 ± 0.3 0.2 ± 0.0 0.2 ± 0.0
11.5 ± 0.4 22.2 ± 0.7 0.7 ± 0.2 1.4 ± 0.6
0.5 ± 0.1 0.2 ± 0.0
0.1 ± 0.0 0.5 ± 0.0 1.2 ± 0.2 2.4 ± 0.4 0.1 ± 0.0 16.5 ± 0.0
0.2 ± 0.0 1.1 ± 0.1 0.4 ± 0.3
0.6 ± 0.1 2.9 ± 0.3 4.6 ± 0.6 100.0
0.6 ± 0.0 3.5 ± 0.2 6.1 ± 0.3 0.3 ± 0.0 6.2 ± 0.9 100.0
0.1 ± 0.0 6.0 ± 0.6 3.6 ± 0.3 22.8 ± 2.2 0.7 ± 0.3 5.5 ± 0.6
0.9 ± 0.1 2.2 ± 0.2 1.7 ± 0.4 0.2 ± 0.0 11.2 ± 2.3
100.0
100.0
0.6 ± 0.0 0.3 ± 0.1 2.1 ± 0.1 3.6 ± 0.4 100.0
tr < 0.1%. a Peak number in Fig. 1. b Compounds determined as methyl ester TMS ether and/or TMS ether TMS ester. c Position of double bond not confirmed by chemical methods. d Value also contains 16-OH 10-oxo 16:0 in rosehip and rowanberry. e Not separated from DiOH-16:0 in black chokeberry and rowanberry. f Determined as corresponding methoxyhydrin compounds. g Tentative identification.
acetylation afterwards as derivatisation for GC analysis. The reflux and test tube methods resulted in identical monomers but slightly different relative proportions, which varied between the berries (data not shown). In addition to the nature of the samples the differences may also be because of the complexity of monomer mixtures obtained with the reflux method, which hampered calculations due to overlapping peaks. The formation of several different methyl and/or TMS derivatives, especially in the case of hydroxy–dioic acids, resulted in major overlapping of peaks. Depolymerisation in a test tube with dried reagents resulted in sole methyl ester derivatives of cutin monomers at best and at worst both ester derivatives, but only from the very major peaks, which ensured calculations were straightforward. Thus, the monomer profile proportions in Table 3 have been calculated from test tube reactions. Monomer profiles for crowberries were compared from berries collected in two different years. Hydroxy acids and diacids comprised 93.3 ± 0.4% and 94.2 ± 1.0% for berries collected in 2005 and 2004, respectively. The monomer profiles were fairly equal and in the range of standard deviations; thereby the values shown in Table 3 were derived from berries collected in 2005. The cutin monomers identified from berry cutins comprised mainly long-chain x-hydroxy acids (Table 3), with mid-chain functionalities known to commonly exist in cutin polymers of different plant materials, including berries (Kallio et al., 2006). Compounds with chain lengths C16 and C18 dominated all berry cutins studied, but also minor amounts of compounds with chain lengths C15 and C20 to C24 were found. C15 monomers have not been found earlier from berry cutins, but are known to exist in the cutin of several gymnosperms (Hunneman & Eglinton, 1972). Trace amounts of
short-chain hydroxy acids (
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Fig. 1. Total ion chromatograms with major peaks numbered (Table 3) of raspberry (A), cloudberry peel (B), sea buckthorn seed (C) and cloudberry seed (D) cutins.
previously found in blackcurrant cutin (10–14%) and citrus fruit (Deas et al., 1974; Kallio et al., 2006). Three monomeric compounds were tentatively identified to be 18-hydroxy-octadecatrienoic acid, 9,10,18-trihydroxyoctadecadienoic acid and 18-hydroxy-9,10-epoxyoctadecadienoic acid. Identifications were based on their retention times and similar mass spectral patterns as the corresponding mono or diunsaturated compounds. To our knowledge, the mass spectra have not been published and reference compounds were not available, thereby identification is considered tentative only and requires further work to become definite. Aromatic compounds were found in minor quantities (<1%) in black chokeberries, raspberries, rowanberries and crowberries. In strawberries, aromatic compounds comprised 6% of total monomers, being mainly coumaric acid (3.1%) and hydroxy-benzoic acid (1.8%). Other aromatic compounds were ferulic/isoferulic acid, hydroxybenzaldehyde, hydroxymethoxybenzoic acid and dihy-
droxybenzoic acid (all less than 1%). Rosehip and cloudberry cutin contained no aromatic compounds. Unidentified compounds comprised 2.8–16.5% of the total monomers. Single compounds were minor (<0.5%), except for some unknowns in black chokeberry, which accounted for 5.7% (UN1) and 4.3% (UN2) of the total monomers. Peak UN1 had a retention time of 23.8 min and its mass spectrum had diagnostic ions of a long-chain methyl ester TMS ether of x-hydroxy acids (e.g., rearrangement ions at m/z 146 and 159) (Rontani & Aubert, 2004), a major cleavage ion (possible a-cleavage) at m/z 285 and other important ions at m/z 271, 335, 425 (possible M-47) and 443 (possible M-15). Peak UN2 had a retention time of 24.7 min and its mass spectrum also showed diagnostic ions for a long-chain methyl ester TMS ether of x-hydroxy acids. According to MS, it was also a mixture of two compounds eluting practically at the same time. The first compound had fragment ions at m/z 271, 285, 315 and 329, indicating a mixture of positional isomers. The
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latter compound had significant ions at m/z 253, 290, 412 and also m/z 147, which indicates the presence of several TMS groups (Rontani & Aubert, 2004). These compounds were also present in many other berries but in lower proportions. Other unidentified monomers comprised similar diagnostic ion patterns as the three compounds above, indicating a series of related monomers. About 11% of the rowanberry cutin monomers remained unidentified, amongst which we found indications of branched chain hydroxy acids in trace quantities. Raspberries and rowanberries had just above the traceable limit (0.1%) of hydroxypentadecanoic acid, in which the hydroxy group was not in the x-position but in the 9/10 position. Other non-xhydroxy compounds (e.g., 10-OH-16:0) and epoxydiacids (e.g., 9,10-epoxy-1,18-dioic acid) were also detected, but did not exist above trace levels in any sample. Fatty acids were found in analyte mixtures both as methyl esters and TMS esters. According to NMR analysis, it is possible that free acids are present amongst the esterified monomers (Deshmukh, Simpson, & Hatcher, 2003). Fatty acids usually existed in small proportions (<3%), but in strawberries and raspberries they accounted for 22.8% and 6.1% of the total monomers, respectively. Alkanols were found in very minor amounts only and glycerol was not determined in this study because of the method used to recover monomers after depolymerisation. However, it is known
to be a relevant monomer (1–14% of total monomers) of some cutins (Graça et al., 2002).
3.4. Cutin monomers of seeds Cutin monomer analysis of cloudberry, blackcurrant and sea buckthorn seeds was carried out similarly to the peel cutin analysis. Hydroxy fatty acids and hydroxyl diacids were the major monomer groups, comprising 74.9%, 82.0% and 31.1% of the abovementioned berry seeds, respectively (Fig. 2A). Sea buckthorn seed monomers contained 48.1% of fatty acids, which has to be because of the insufficient isolation process; these seeds contain 7–11% oil vs. seed weight depending on variety (Yang & Kallio, 2001). Cloudberry seed cutin monomer profiles most resembled the corresponding berry cutin (Fig. 1), whereas that of blackcurrant and sea buckthorn differed markedly from the respective berry skin compounds (Kallio et al., 2006). Major monomers of blackcurrant seed cutin were epoxy-substituted compounds (Fig. 2B), comprising 47.2% of total monomers, compared with 11% of total berry cutin monomers (Kallio et al., 2006). Blackcurrant seeds also contained epoxy and dihydroxysubstituted octadecanedioic acid, which has not been found from the corresponding berry skin. Dihydroxyhexadecanoic acid (mainly
A
B
100 %
100 %
90 %
90 %
80 %
80 %
70 %
70 % others Unknown
60 %
Hydrocarbons
hydroxy-C18 diacid
60 %
trihydroxy C18
Diacids 50 %
Fatty acids
hydroxy C18 50 %
epoxy-hydroxy C18 b hydroxy-C16 diacid
Aromatics 40 %
Hydroxy acids and diacids
dihydroxy-C16
40 %
hydroxy C16 a
30 %
30 %
20 %
20 %
10 %
10 %
0%
0% Cloudberry Black currant Buckthorn
Sea
Cloudberry
Black currant Buckthorn
Sea
Fig. 2. Seed cutin monomer profiles by methanolysis (test tube reaction) as a percentage of total peak areas in GC–MS chromatogram (A) and with hydroxy acids and diacids separated in compound classes by chain length and functional groups (B).
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9/10–16-isomers), a major monomer in blackcurrant berry cutin (30%) (Kallio et al., 2006), was present in seed cutin at only 7.3%. The most prominent sea buckthorn seed cutin monomer was dihydroxyhexadecanoic acid (mainly 9/10–16-isomers: 14.2%) instead of epoxy-substituted C18 compounds (0.9%), which dominated the corresponding berry cutin (71%) (Kallio et al., 2006). Sea buckthorn seeds were also rich in monohydroxyhexadecanedioic acid (9.3%), which was not found in berry skin in our earlier studies. Shao et al. (2007) have determined that cutin of soybean seeds differ from the other parts of the plant (the leaves or pods) and contain unusual 2-hydroxy fatty acids as their main compounds, in addition to x-hydroxy fatty acids. The soybeans contained no mid-chain hydroxylated monomers at all. Molina et al. (2006) have suggested that most of the C16–C20 fatty acids found in seed polyesters of B. napus and A. thaliana could originate from ‘‘physically trapped” triacylglycerols, which is probably the reason for such a high content of fatty acids in sea buckthorn seed cutin as well. Molina et al. (2006) also suspected that 2-hydroxy acids are derived from sphingolipid acyl groups and not from cutin polyesters. In our studies of berry seed cutin, we have not found 2-hydroxysubstituted monomers, but confirmed that the monomer profile is different in different parts of berries. The composition profiles of cutins in different berries are crucial for evaluating their physiological properties for the plant and nutritional qualities when consumed as food. Polymers mainly composed of x-hydroxy acids and a,x-diacids without mid-chain functional groups or mid-chain substituted monomers with epoxy or oxo-functionalities are essentially linear as they do not have mid-chain groups that could be part of ester linkages forming cross-linkages. Polymers mainly with di- and/or trihydroxy-substituted monomers make up a totally different polymer via crosslinking. Glycerol has been described to be a common link between different monomers (Graça et al., 2002), but ester bonds without glycerol have also been recovered when analysing oligomers with MS and NMR techniques (Ray, Chen, & Stark, 1998). Differences in the extent of ester-linked cutin vs. resistant cutan, variable cutin monomers affecting the cross-linking and structure of cutin polymers and the composition of cutan in berries all have a strong influence on the various functions of cuticles in berries, and also as a dietary fibre. Further studies on structures of cuticular polymers and on their effects on human nutrition are required for a more comprehensive understanding of the evident significance of this polymer. Acknowledgements This research has been supported financially by the ABS – Graduate School (Applied Bioscience Bioengineering, Food and Nutrition, Environment) and the Finnish Funding agency for Technology and Innovation, TEKES in a project LIPFUN. The author would like to thank Jenni Kumpula for assistance in technical work. References AACC. (2001). Report of the dietary fiber definition committee to the board of directors of the American association of cereal chemists. The definition of dietary fiber. St. Paul, MN: American Association of Cereal Chemists. Bender, D. A. (2005). A dictionary of food and nutrition. Oxford: Oxford University Press. Deas, A. H. B., Baker, E. A., & Holloway, P. J. (1974). Identification of 16hydroxyoxohexadecanoic acid monomers in plant cutins. Phytochemistry, 13, 1901–1905.
Deshmukh, A. P., Simpson, A. J., & Hatcher, P. G. (2003). Evidence for cross-linking in tomato fruit cutin using HR-MAS NMR spectroscopy. Phytochemistry, 64, 1163–1170. Eglinton, G., & Hunneman, D. H. (1968). Gas chromatographic–mass spectrometric studies of long-chain hydroxyacids I. Phytochemistry, 7, 313–322. Eglinton, G., Hunneman, D. H., & McCormick, A. (1968). Gas chromatographic–mass spectrometric studies of long-chain hydroxyacids III. Organic Mass Spectrometry, 1, 593–611. Espelie, K. E., Davis, R. W., & Kolattukudy, P. E. (1980). Composition, ultrastructure and function of the cutin- and suberin-containing layers in the leaf, fruit peel, juice-sac and inner seed coat of grapefruit (citrus paradise macfed.). Planta, 149, 498–511. Graça, J., Schreiber, L., Rodrigues, J., & Pereira, H. (2002). Glycerol and glyceryl esters of x-hydroxyacids in cutins. Phytochemistry, 61, 205–215. Helbig, D., Böhm, V., Wagner, A., Scubert, R., & Jahreis, G. (2008). Berry seed press residues and their valuable ingredients with special regard to black currant press residues. Food Chemistry, 111, 1043–1049. Hilz, H., Bakx, E. J., Schols, H. A., & Voragen, A. G. J. (2005). Cell wall polysaccharides in blackcurrants and bilberries – Characterization in berries, juice and press cake. Carbohydrate Polymers, 59, 477–488. Holloway, P. J., & Deas, A. H. B. (1971). Occurrence of positional isomers of dihydroxyhexadecanoic acid in plant cutins and suberins. Phytochemistry, 10, 2781–2785. Holloway, P. J., & Deas, A. H. B. (1973). Epoxyoctadecanoic acids in plant cutins and suberins. Phytochemistry, 12, 1721–1735. Hunneman, D. H., & Eglinton, G. (1972). The constituent acids of gymnosperm cutins. Phytochemistry, 11, 1989–2001. Järvinen, R., Silvestre, A. J. D., Holopainen, U., Kaimainen, M., Nyyssölä, A., Gil, A. M., et al. (2009). Suberin of potato (Solanum tuberosum var Nikola) – Comparison of the effect of cutinase CcCut1 hydrolysis with chemical depolymerization. Journal of Agricultural and Food Chemistry, 57, 9016–9027. Johansson, A., Kuusisto, P. H., Laakso, P. H., Derome, K. K., Sepponen, P. J., Katajisto, J. K., et al. (1997). Geographical variations in seed oils from Rubus chaemaemorus and Empetrum nigrum. Phytochemistry, 44, 1421–1427. Johansson, A., Laakso, P., & Kallio, H. (1997). Characterization of seed oils of wild, edible Finnish berries. Zeitschrift für Lebensmittel-Untersuchung und -Forschung, 204, 300–307. Johnson, E. L., Dorot, O., Liu, J., Chefetz, B., & Xing, B. (2007). Spectroscopic evaluation of aliphatic moieties in four plant cuticles. Communications in Soil Science and Plant Analysis, 38, 2461–2478. Kallio, H., Nieminen, R., Tuomasjukka, S., & Hakala, M. (2006). Cutin composition of five northern berries. Journal of Agricultural and Food Chemistry, 54, 457– 462. Kolattukudy, P. E. (2001). Polyesters in higher plants. In W. Babel & A. Steinbüchel (Eds.), Advances in biochemical engineering/biotechnology (pp. 1–49). Berlin, Heidelberg, Germany: Springer-Verlag. Molina, I., Bonaventure, G., Ohlrogge, J., & Pollard, M. (2006). The lipid polyester composition of Arabidophis thaliana and Brassica napus seeds. Phytochemistry, 67, 2597–2610. Molina, I., Ohlrogge, J. B., & Pollard, M. (2008). Deposition and localization of lipid polyester in developing seeds of Brassica napus and Arabidophis thaliana. The Plant Journal, 53, 437–449. Pacchiano, R. A., Jr., Sohn, W., Chlanda, V. L., Garbow, J. R., & Stark, R. E. (1993). Isolation and spectral characterization of plant-cuticle polyesters. Journal of Agricultural and Food Chemistry, 41, 78–83. Plaami, S. P., Kumpulainen, J. T., & Tahvonen, R. L. (1992). Total dietary fiber contents of vegetables, fruits and berries consumed in Finland. Journal of the Science of Food and Agriculture, 59, 545–549. Ray, A. K., Chen, Z.-J., & Stark, R. E. (1998). Chemical depolymerization studies of the molecular architecture of lime fruit cuticle. Phytochemistry, 49, 65–70. Rontani, P.-F., & Aubert, C. (2004). Trimethylsilyl transfer during electron ionization spectral fragmentation of some x-hydroxycarboxylic and x-dicarboxylic acid trimethylsilyl derivatives and the effect of chain length. Rapid Communications in Mass Spectrometry, 18, 1889–1895. Shao, S., Meyer, C. J., Ma, F., Peterson, C. A., & Bernards, M. A. (2007). The outermost cuticle of soybean seeds: Chemical composition and function during imbibitions. Journal of Experimental Botany, 58, 1071–1082. Tahvonen, R. L., Schwab, U. S., Yli-Jokipii, K. M., Mykkänen, H. M., & Kallio, H. P. (2005). Blackcurrant seed oil and fish oil supplements differ in their effects on fatty acid profiles of plasma lipids, and concentrations of serum total and lipoprotein lipids, plasma glucose and insulin. Journal of Nutritional Biochemistry, 16(6), 353–359. Velcheva, M. P., Espelie, K. E., & Ivanov, C. P. (1981). Aliphatic composition of cutin from inner seed coat of apple. Phytochemistry, 20, 2225–2227. Wawer, I., Wolniak, M., & Paradowska, K. (2006). Solid state NMR study of dietary fiber powders from aronia, bilberry, blackcurrant and apple. Solid State Nuclear Magnetic Resonance, 30, 106–113. Yang, B., & Kallio, H. P. (2001). Fatty acid composition of lipids in sea buckthorn (Hippophaë rhamnoides L.) berries of different origins. Journal of Agricultural and Food Chemistry, 49, 1939–1947.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Cutin composition of selected northern berries and seeds Riikka Järvinen *, Mika Kaimainen, Heikki Kallio Department of Biochemistry and Food Chemistry, University of Turku, FI-20014 Turku, Finland
a r t i c l e
i n f o
Article history: Received 28 July 2009 Received in revised form 15 December 2009 Accepted 11 February 2010
Keywords: Berry Cutin Epoxy fatty acids Hydroxy fatty acids Mass spectrometry Seed
a b s t r a c t Monomeric compositions of the extractive-free cuticular polymer fractions of seven berries – rosehip (Rosa rugosa), black chokeberry (Aronia melanocarpa), strawberry (Fragaria x ananassa), raspberry (Rubus idaeus), cloudberry (Rubus chamaemorus), crowberry (Empetrum nigrum) and rowanberry (Sorbus aucuparia) – as well as the seeds from three berries – cloudberry, sea buckthorn (Hippophaë rhamnoides) and blackcurrant (Ribes nigrum) – were investigated. Depolymerisation of the cutin polymer was carried out using NaOMe-catalysed methanolysis and the composition of TMS-derivatised monomers was determined by GC using FID and MS detection. Two depolymerisation techniques were compared; 1.0 M NaOMe at ambient temperature and 1.3 M NaOMe under reflux conditions. The degree of depolymerisation of the cuticular membrane ranged from 2% (strawberry) to 84% (rosehip) for the CHCl3-soluble cutin monomers. Depolymerisation of seed samples was low (1–3%). The predominant monomers were C16 and C18 x-hydroxy acids with mid-chain functionalities, mainly epoxy and hydroxy groups, but suberin-like a,x-dicarboxylic acids, with mid-chain hydroxyl groups and C15 chain length monomers, were also found. Seed cutin monomers differed from the corresponding berry skin monomers. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Cuticular polymer cutin is the main lipid polymer in the skin of fruits and berries, as well as in the outer layers of the leaves and stems of vascular plants. Ester-linked monomers of cutin consist of a complex mixture of long-chain x-hydroxy acids, with typical chain lengths of 16 and 18 carbons, together with fatty acids, fatty alcohols and phenolic compounds, such as cinnamic acid derivatives and glycerol. Suberin-like a,x-diacids and their hydroxylated derivatives have also been detected. Cutin, together with nondepolymerisable cutan and cuticular waxes, make up the cuticle of vascular plants, which acts as a barrier and gateway between the plant and its environment (Graça, Schreiber, Rodrigues, & Pereira, 2002; Kolattukudy, 2001). The cuticle is attached to the epidermal cells of plants by the pectic layer (Kolattukudy, 2001). Cell walls of berry skin, and the surface of other plants cells, consist of a network of cellulose and hemicelluloses together with pectins. The composition of polysaccharides differs between berries and in the different parts of berries, for example between the skin, pulp and seeds (Hilz, Bakx, Schols, & Voragen, 2005). One major current use of berries is in industrial juice production. Together with seeds, polysaccharides and the polyphenolic compounds, the cuticular polymers account for the bulk of the pressed residue in juice production. This byproduct could be utilised as a valuable source of dietary fibre or * Corresponding author. Tel.: +358 2 333 6874; fax: +358 2 6860. E-mail address: riikka.jarvinen@utu.fi (R. Järvinen). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.02.030
specialty chemicals, but the lack of detailed compositional information hinders its usage. Berry is a common term for fleshy, juicy fruits with one or typically more seeds, including grape, gooseberry, blackcurrant, cranberry, sea buckthorn, tomato, strawberry and raspberry (Bender, 2005). Some are actually accessory fruits, even though they are generally considered berries. For example, a strawberry is not a berry but a pseudocarp, the edible red pulp of which is a swollen receptacle of the flower. Stone fruits, raspberries and cloudberries are also aggregate fruits with multiple drupes. The peel of a berry has a major influence on how berries are preserved in nature. Cuticular polymers together with associated waxes are thought to be the major contributors to morphology and preservation (Kolattukudy, 2001). To our knowledge, there is no published information about the cuticular components of berry seeds. Previous investigations have concentrated on other issues, including seed weight (Johansson, Laakso, & Kallio, 1997), sugar composition (Hilz et al., 2005), oil content and nutritional value (Tahvonen, Schwab, Yli-Jokipii, Mykkänen, & Kallio, 2005), fatty acid composition of oils (Yang & Kallio, 2001) and the effect of growth conditions on composition (Johansson, Kuusisto et al., 1997). Helbig, Böhm, Wagner, Scubert, and Jahreis (2008) recently studied the composition of berry seed press residue, but not the cuticular polymers of the seeds. Soybean seeds (Shao, Meyer, Ma, Peterson, & Bernards, 2007) and some angiosperm seeds (Arabidopsis thaliana, Brassica napus) (Molina, Bonaventure, Ohlrogge, & Pollard, 2006; Molina, Ohlrogge, & Pollard, 2008) have been examined for their composition, including
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cutin monomers. Cutin composition of the inner seed coat of the apple (Velcheva, Espelie, & Ivanov, 1981) and grapefruit (Espelie, Davis, & Kolattukudy, 1980) have also been investigated. Results have indicated that seeds possess two thin cuticular layers; one in the outer seed coat and another associated with the inner seed coat. Furthermore, lamellar suberin structures have been found throughout the cell walls of grapefruit seeds (Espelie et al., 1980) and suberin-type monomers have been released from some angiosperm seeds (A. thaliana, B. napus) (Molina et al., 2008). We have previously described the cutin monomer composition of edible northern berries, including sea buckthorn (Hippophaë rhamnoides), blackcurrant (Ribes nigrum), cranberry (Vaccinium oxycoccos), lingonberry (Vaccinium vitis-idaea) and bilberry (Vaccinium myrtillus), which are all in common use, especially in Scandinavia (Kallio, Nieminen, Tuomasjukka, & Hakala, 2006). In this paper, we describe for the first time the amount of extractive-free cuticular polymers and cutin monomer composition of several northern berries: rosehip (Rosa rugosa), black chokeberry (Aronia melanocarpa), strawberry (Fragaria x ananassa), raspberry (Rubus idaeus), cloudberry (Rubus chamaemorus), crowberry (Empetrum nigrum) and rowanberry (Sorbus aucuparia). The seeds of three berries – cloudberry, sea buckthorn and blackcurrant – were also investigated. With this second report of the composition of cuticular hydroxyl acids in northern berries, our aim is to acquire knowledge on the cuticular material of northern berries, as a part of the exploitation of Nordic natural resources, regarding chemical composition, nutritional value and sensory properties.
2. Materials and methods 2.1. Berry peel and seed samples Rosehips and black chokeberries were obtained from MTT Agrifood Research Finland (Jokioinen, Finland) and peels separated from frozen whole berries by manual peeling, washing with water and drying. Strawberries, raspberries and cloudberries were bought from a local market (Turku, Finland) and pressed with a hydraulic juice extractor to yield juice press residues from whole berries. Crowberry and rowanberry samples were frozen strained by-products obtained from Kiantama Oy (Suomussalmi, Finland). The seeds were manually separated from all berry samples after drying in an oven at 60 °C. Berries were collected at optimal ripeness for industrial berry processing. Cloudberries, crowberries and rowanberries were wild berries, whilst others were industrially obtained mixed cultivars. Sea buckthorn and blackcurrant seeds were obtained from Aromtech Ltd. (Tornio, Finland). Cloudberry seeds were obtained from the above-mentioned strained by-product by manual separation from the press residue. Seeds were crushed in a mortar with the aid of liquid nitrogen and dried in an oven at 60 °C.
2.2. Isolation of cuticular membranes The cuticular membranes of berry peels were isolated using the method previously described (Kallio et al., 2006). Briefly, the procedure was a combination of enzymatic treatments with cellulase (5.0 g/l Econase CE, AB Enzymes, Darmstadt, Germany) and pectinase (1.0 g/l Pectinex Ultra SP-L, Novozymes, Bagswaerd, Denmark) in acetate buffer. The reaction mixture was washed with water and the residue dried and weighed. The dried residue was exhaustively extracted using a Soxhlet apparatus with CHCl3 and MeOH (14– 20 h each), yielding extractive-free cuticular membranes. After repeating the isolation process, the product was washed with water and dried in an oven at 60 °C before depolymerisation. The
crushed seeds were treated once with the same combined enzymatic and extraction method. 2.3. Depolymerisation of cuticular membranes Depolymerisation of the isolated cuticular membranes was carried out using a method previously described by Holloway and Deas (1973), and further modified by our research group (Kallio et al., 2006). Dried samples of the extractive-free cuticular membrane (50– 100 mg) were refluxed for 3 h in 25 ml of a 1.3 M solution of NaOMe in MeOH. The reagent was freshly prepared by dissolving metallic sodium in dry methanol. Precautions were taken to eliminate moisture from the reaction. The reaction mixtures were filtered and the filtrates were acidified by adding 2 M H2SO4 in MeOH and dried using a rotary evaporator. The residue was suspended in 50 mL of H2O and extracted with CHCl3 (2 50 ml). The CHCl3-soluble monomers were dried with anhydrous Na2SO4 and after filtration, solvent was evaporated. Cutin monomers were determined gravimetrically and stored in CHCl3 at 18 °C for further analysis. In addition to the traditional reflux method, a small-scale closed-bottle method (test tube method) was developed. Dried samples of extractive-free cuticular membrane (20 mg) were treated with 3 ml of freshly prepared 1.0 M NaOMe in dry MeOH. Methanolysis was carried out overnight in a shaker (at approx. 500 rpm) at ambient temperature. The reaction mixture was acidified with 2 M H2SO4 in MeOH and the supernatant was separated by centrifugation (1315g, 10 min). Then, 10 ml H2O were added and the cutin monomers extracted with CHCl3 (2 10 ml). The mixture of monomers in CHCl3 was dried with anhydrous Na2SO4, filtered and solvent evaporated. Cutin monomers were determined gravimetrically and stored in CHCl3 at 18 °C for further analysis. 2.4. Analysis of monomeric compounds Cutin monomer methyl esters were further derivatised before the chromatographic analysis. CHCl3 was evaporated, and the remaining monomers were dried under a stream of nitrogen and kept in a desiccator overnight before trimethylsilylation with TriSil reagent (HMDS and TMCS in pyridine) (Pierce Chemicals, Rockford, IL). After adding the reagent, the sample was vigorously shaken at room temperature for 5 min and kept at 60 °C for 15 min. The composition of monomers was determined by gas chromatography electron impact mass spectrometry (GC–EI-MS) with a Shimadzu GC–MS-QP5000 (Shimadzu, Kyoto, Japan) under the following chromatographic conditions: 30 m 0.25 mm 0.25 lm DB-1MS column (Agilent, Folsom, CA); split injection (14:1); carrier gas He (linear velocity 42 cm/s); injector and detector temperatures 300 °C. The oven temperature was programmed from 125 °C at 10 °C/min to 220 °C, from 220 °C at 3 °C/min to 290 °C, and kept at 290 °C for 2 min. Mass range m/z 45–550 was acquired. In addition to GC–MS, all samples were analysed in parallel with gas chromatography-flame ionisation detection (GC-FID) with a Shimadzu GC 17A with the chromatographic conditions mentioned above, to compare the different detection methods. Compounds were identified by comparing the EI-MS spectra of the derivatives (as methyl ester TMS ether or TMS ester TMS ether) with published spectra (Deas, Baker, & Holloway, 1974; Eglinton & Hunneman, 1968; Eglinton, Hunneman, & McCormick, 1968; Holloway & Deas, 1971; Holloway & Deas, 1973; Järvinen et al., 2009; Kallio et al., 2006; Rontani & Aubert, 2004), spectra of reference compounds and interpreting the structures by common fragmentation patterns and retention times. Positions of the double bonds were not confirmed by chemical methods.
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2.5. Statistical analysis Each depolymerisation reaction with test tube method was carried out in triplicate and the reflux method in duplicate. Statistical analyses were carried out using the statistical software SPSS for Windows (Version 14.0; SPSS Inc., Chicago, IL). All results were expressed as means and standard deviations (SD). The t-test was used to separately compare the depolymerisation and detection methods. Differences reaching a minimal confidence level of 95% were considered statistically significant. 3. Results and discussion 3.1. Isolation of cuticular membranes
idues from an industrial process (no enzymes used) and closely resembled the material of the abovementioned juice pressing. Rosehip and black chokeberry samples were the peel from manually peeled whole berries, resulting in clean skin fractions without fruit flesh. The high amount of cuticular material (Table 1) obtained from seeds evidently contained not only cuticular components, but also other components remaining after the isolation process. The procedure of isolation was probably insufficient, as unlike the berry skins it was not repeated. The structure and composition of seeds deviating substantially from the skins may have also reduced the efficacy of enzymes and solvents. Seeds are complex organs with polyesters in many distinct cell layers (Molina et al., 2006). Their locations were not studied in this investigation, which mainly concentrated on the monomer types released from the seed material.
The first enzymatic treatment with cellulase and pectinase removed approx. 59% of raspberry, 72% of cloudberry, 35% of black chokeberry, 60% of rowanberry, 54% of rosehip, 66% of strawberry and 36% of crowberry raw materials. The first extraction (MeOH and CHCl3, 14–20 h each) of the washed residue removed a further 0% of raspberry, 8% of cloudberry, 13% of black chokeberry and rowanberry, 15% of rosehip and strawberry, and 31% of crowberry raw materials, calculated from the original seedless berry peel material. The second enzymatic treatment additionally reduced another 2–12% of the total mass, calculated from the raw material before the enzymatic treatments, with the exceptions of a 35% and 39% reduction in cloudberry and raspberry, respectively. The results clearly showed that more material was removed from press residue of the soft-skin berries (strawberry, raspberry and cloudberry) in the enzymatic treatments, compared with rosehip and black chokeberry peels and the crowberry straining residue (Table 1). The extractable compounds (including waxes, polyphenolics) were removed in the first extraction with MeOH and CHCl3 according to gravimetric measurement. Interestingly, raspberry press residue contained no solvent extractable material. Raw materials of the cuticular membranes were different because of the morphological and structural differences of the berries and the varying pre-treatments of the raw materials. This led to difficulties when comparing the amounts of extractive-free cuticular polymers between the berries. However, the results obtained show the high variation in extractive-free cuticular material in the different berries (Table 1). The peel of the soft-skinned strawberry (accessory fruit), as well as raspberry and cloudberry (aggregate fruits), was obtained from pressing the berries with a hydraulic juice extractor. Thus, the material was a residue, which also contained some non-water-soluble polysaccharides of the fruit flesh. Crowberry and rowanberry materials were strained res-
The traditional and laborious reflux method for depolymerisation of the cutin polymer by transmethylation with sodium methoxide in methanol is greatly affected by moisture in the reaction. It also requires large amounts of solvents. Separation of the residue and the CHCl3-soluble monomers by filtration and evaporation further exposures the reaction to moisture and may result in the loss of monomers. For these reasons, a small-scale closed-bottle test tube procedure was developed. Centrifugation was used to isolate the monomers from the acidified reaction mixture of methanolysis. The new method was carried out parallel to the reflux method, to determine its applicability for cutin depolymerisation. The two methods have previously been compared with potato suberin with analogous results (Järvinen et al., 2009). The two tested methods provided slightly different results depending on the cuticular materials (Table 2). For rowanberry, raspberry and strawberry, the methods generated identical results but for rosehip, black chokeberry and cloudberry the results were smaller in the test tube reaction. A statistically significant difference was obtained for cloudberry and rosehip, although its importance is questionable as the samples were small aliquots of natural material. The differences in results may be due to the varying composition and ratios of the cuticular components in different samples. The fact that samples were not homogenised extremely effectively may provide another explanation for the deviation between the two methods. The test tube method may be more susceptible to gravimetric errors because of its smaller scale than that of the reflux method (20 mg vs. 100 mg of sample, respectively), although extreme care was taken with all steps. However, the test tube method was found to be fast and reliable for
Table 1 Proportion of the extractive-free cuticular material of the raw materials.
Table 2 Comparison of depolymerisable cutin in selected berries and seeds (average percentage and standard deviation of CHCl3-soluble monomers of raw cutin).
a b c
Berry
Extractive-free cuticular material of raw materials (%)
Rosehip Black chokeberry Crowberry Rowanberry Strawberry Raspberry Cloudberry
38a 51a 42b 35b 29c 29c 18c
Seed Cloudberry Blackcurrant Sea buckthorn
58 71 74
Peel. Commercial straining by-product. Juice press residue.
3.2. Depolymerisation of cuticular polymer
a
Berry
Test tube N
AV (%)
SD
N
AV (%)
SD
Rosehipa Crowberry 2004 Crowberry 2005 Black chokeberry Strawberry Rowanberry Raspberry Cloudberrya
3 3 3 3 3 3 3 3
72.3 51.2 60.3 15.4 2.3 26.0 5.9 37.7
2.0 3.0 4.2 1.4 0.2 1.0 0.1 3.1
2 2 2 2 2 2 2 2
83.7 56.2 66.2 21.8 2.4 29.7 6.7 47.7
1.8 2.8 1.3 3.9 0.1 4.2 0.4 3.0
Seed Blackcurrant Sea buckthorn Cloudberry
3 3 3
1.5 1.3 3.2
0.3 0.0 0.6
2 2 2
2.0 1.2 3.0
0.0 0.7 0.5
p < 0.05.
Reflux
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R. Järvinen et al. / Food Chemistry 122 (2010) 137–144
providing a qualitative description of the cutin monomer composition and was preferred over the reflux method. The cuticle and monomer composition have been found to change during maturation (Kolattukudy, 2001; Molina et al., 2008). The aim was to harvest each of the berries at the stage of optimal ripeness according to common commercial practices and the personal but non-scientific evaluation of a berry specialist. The industrial berries were purchased, as usual, from various sources, which decreased the homogeneity of the raw materials. The amount of depolymerised, CHCl3-soluble cutin monomers (Table 2) again revealed the differences between cuticular polymers in different berries. The proportion of depolymerised material varied from 2% in strawberry to 70–80% in rosehip. In earlier investigations, the cutin monomer yield was determined for sea buckthorn (46%), blackcurrant (8%), cranberry (27%), lingonberry (30%) and bilberry (6%) (Kallio et al., 2006). The abundant residue remaining non-depolymerised in methanolysis of the isolated berry cuticular membrane deserves further compositional and structural investigation. Special attention should be paid to cutan-type polymer and polysaccharide complexes. According to the results obtained, there is no evident correlation between the amounts of cuticular material and ester-bound cutin in berry cuticles. As cutin is known to be a protective barrier against water loss together with cuticular waxes, in addition to genetic background, the growth conditions and climate may also be major explanations of the differences between berries. The amount of the cuticular material, cutin:cutan ratio and also the monomeric units together with the cuticular waxes all have a crucial influence on the preservation of different berries in nature and when collected for food. The composition and structure of the surface tissues ultimately define the characteristic-preserving properties of berries against drying, chemical attacks, mechanical injuries and microbial infections. For example, crowberries and lingonberries maintain their basic morphology and composition for almost a year in nature, whereas strawberries, raspberries and cloudberries are typically destroyed by drying or infections in early autumn. Earlier studies have also shown the amount of cutin in seed coats to be very small, whilst seed cutin has been determined separately for whole seeds (outer coat) and the inner coat (Molina et al., 2006). The small amount of cutin in seeds may be because of the location of seeds inside the berry as there is no evident need for an effective barrier against desiccation or pathogens, for example. Although the cuticular polymer values obtained from berry seeds may not fully explain the amount of cutin polymer, we were able to obtain enough monomers for profile assessment. As a curiosity, crowberries from consecutive years (2004 and 2005) were compared. The berries collected in 2004 contained about 10% less ester-bound cutin monomers than those collected in 2005. Since we compared only two years and growth locations were not specified, these results are inadequate for further conclusion. However, if the effects of weather conditions and growth places ever want to be investigated, careful analyses of berries from several years and from specified locations would have to be carried out. Cuticular polymers should evidently be included in non-soluble dietary fibre, although this has not been stated in common definitions. Dietary fibre is defined as the edible parts of plants resistant to digestion and absorption in the human small intestine, with complete or partial fermentation in the large intestine (AACC, 2001). This definition includes polysaccharides, oligosaccharides, lignins and associated plant substances, to which cuticular polyesters together with suberin and waxes can be included. Thus, the knowledge of these plant substances is evidently important. Plaami, Kumpulainen, and Tahvonen (1992) have previously examined the total dietary fibre of strawberries, blackcurrants, redcurrants, bilberries and lingonberries, finding that these berries
contain on average 18–19, 58, 47–53, 33 and 26 g/kg of fibre in each fresh berry, respectively. Bilberries and lingonberries were also studied for insoluble (27 and 22 g/kg, respectively) and soluble fibre (7 and 4 g/kg, respectively). These values also contain the weight of seeds, so conclusions about the correlation between cuticular material and fibre cannot be drawn based on these values. Dietary fibre powders of chokeberry, bilberry and blackcurrant (Wawer, Wolniak, & Paradowska, 2006) have been analysed with 13 C CP/MAS NMR and the fibre content was also determined using enzymatic hydrolysis of the berry powders. The total dietary fibre was 66–72% and insoluble fibre was 60–66% of the powder. Only 5–7% was found to be soluble fibre. In NMR spectra, cellulose and other polysaccharides, in addition to lignin and cutin, were recognised but the cutin–cutan relationship was not studied. In our investigation, some berry cuticles were found to be highly resistant to ester-breaking reactions, which may be because of the high amount of cutan-type compounds with C–C and ether bonds present. This assumption requires further confirmation with spectroscopic methods suitable for non-soluble material research. FT-IR and solid state NMR spectroscopy have been used to outline non-depolymerisable material from the cuticular fractions of lime (Pacchiano et al., 1993) and tomato, pepper and apple fruits (Johnson, Dorot, Liu, Chefetz, & Xing, 2007), but not applied to the isolated cuticular polymers of berries. 3.3. Cutin monomers of berries CHCl3-soluble cutin monomers of the berries and seeds were determined by gas chromatography using both MS and FID detection techniques (Table 3). We obtained better resolution and separation of peaks with MS, but traditionally FID detection has been considered more reliable for quantitative analysis. Our goal was to display chromatographic monomer profiles and, as the detection methods did not differ statistically (p > 0.05) when the major monomer groups were compared, MS detection was chosen as the preferred method. The relative proportions of different monomers in Table 3 are mean values from the MS analysis also previously used for berry cutin analysis (Kallio et al., 2006). Mean ± SD values for monomers were calculated from at least triplicate chromatographic analyses. Fig. 1 shows examples of the GC–MS total ion chromatograms of cutin analyses of some berries and seeds. Identification of the cutin monomers was performed with GC– MS after TMS derivatisation of the monomers released. GC separation of the complex mixture of cutin monomers, followed by MS detection, is necessary for analysis and identification, as reference compounds are only commercially available for some monomers. TMS ester and ether derivatives of cutin monomers provide diagnostic spectra that make interpretation relatively unambiguous. The location of a mid-chain hydroxyl group is easy to detect, as a-cleavage at either side of the carbon bearing the TMS-derivatised functional group, or cleavages between carbons bearing the functional groups in the case of adjacent groups, are most preferred in the ionisation (Eglinton & Hunneman, 1968; Eglinton et al., 1968; Hunneman & Eglinton, 1972). Long-chain x-hydroxy acids and a,x-diacids form diagnostic rearrangement ions when analysed as TMS derivatives (Rontani & Aubert, 2004). Tables gathering the information of the diagnostic ions for cutin and suberin monomers have been previously published by our research team (Järvinen et al., 2009; Kallio et al., 2006). Although precautions were taken to eliminate moisture from the reflux reaction, there remained varying amounts of saponification products in all the end products obtained with the reflux method. Methyl acetate as a competitor for saponification was tried but resulted in the formation of several acetylated derivatives of cutin monomers, which in turn made identification difficult. This reaction has been used by Molina et al. (2006), but with complete
141
R. Järvinen et al. / Food Chemistry 122 (2010) 137–144 Table 3 Composition (mean ± SD) of cutin monomers released from berries by methanolysis (test tube reaction) as a percentage of total peak areas in GC–MS chromatogram. Peaka 1 2 3 4 5 7
8 9 10 11 12
Cutin monomerb
Black chokeberry
Rosehip
Cloudberry
Raspberry
Strawberry
Rowanberry
Crowberry
x-Hydroxy acids and hydroxy a,x-diacids
79.4 ± 0.7 0.8 ± 0.1 0.7 ± 0.0 16.8 ± 0.6 0.7 ± 0.2 1.6 ± 0.2 2.8 ± 0.2
94.4 ± 0.7 5.9 ± 0.3 4.5 ± 0.2 78.7 ± 1.2
91.9 ± 1.0 3.1 ± 0.1 4.6 ± 0.3 73.9 ± 2.0 0.3 ± 0.0 0.1 ± 0.0 1.7 ± 0.1 1.5 ± 0.2
83.3 ± 0.5 9.0 ± 0.2 3.6 ± 0.2 48.7 ± 1.4 3.7 ± 0.1 4.3 ± 0.1 8.7 ± 0.2 1.2 ± 0.4
61.4 ± 1.2 2.8 ± 0.0 1.8 ± 0.4 37.8 ± 1.3 0.6 ± 0.1 0.3 ± 0.0 4.6 ± 0.4 0.9 ± 0.1 2.1 ± 0.1 3.3 ± 0.1 2.5 ± 0.7 0.4 ± 0.4 1.7 ± 0.1 1.3 ± 0.1 0.2 ± 0.2 0.9 ± 0.1
83.8 ± 2.9 1.8 ± 0.2 4.4 ± 0.2 52.2 ± 5.0
93.3 ± 0.4 4.7 ± 0.2 1.0 ± 0.1 28.6 ± 0.2 0.6 ± 0.1 3.3 ± 0.1 19.1 ± 0.3
16-OH-16:0 Mono-OH-1,16-dioic 16:0d (major 7/8) di OH-16:0 (major 9,16/10,16) 18-OH-18:3c,g 18-OH-18:2c 18-OH-18:1c 18-OH-18:0e 18-OH-9,10-epoxy 18:2c,f,g 18-OH-9,10-epoxy 18:1c,f 18-OH-9,10-epoxy 18:0f 9,10,18-triOH-18:2c,g 9,10,18-triOH-18:1c 9,10,18-triOH-18:0 20-OH-20:0 22-OH-22:0 24-OH-24:0 6/7-OH-1.15-dioic-15:0 9,15-DiOH-15:0 9/10-OH-15:0
Other compound groups ar Aromatic compounds da Diacids fa Fatty acids ol Alcohols un Unidentified monomers TOTAL
0.0 ± 0.0 3.7 ± 0.8
2.9 ± 0.4 23.5 ± 0.2 17.7 ± 0.5
tr 0.5 ± 0.1 0.7 ± 0.1 4.5 ± 0.6
4.8 ± 0.6 6.6 ± 0.9 tr 0.3 ± 0.1 1.0 ± 0.2 0.5 ± 0.1
0.8 ± 0.1
0.2 ± 0.0 0.3 ± 0.0 0.9 ± 0.1 1.0 ± 0.0 0.2 ± 0.0 0.6 ± 0.0 0.3 ± 0.0 0.5 ± 0.1
tr
100.0
1.1 ± 0.2 1.8 ± 0.3 2.8 ± 0.3 100.0
6.9 ± 0.4 2.3 ± 0.2 0.9 ± 0.2 8.2 ± 0.6 4.3 ± 0.3 0.2 ± 0.0 0.2 ± 0.0
11.5 ± 0.4 22.2 ± 0.7 0.7 ± 0.2 1.4 ± 0.6
0.5 ± 0.1 0.2 ± 0.0
0.1 ± 0.0 0.5 ± 0.0 1.2 ± 0.2 2.4 ± 0.4 0.1 ± 0.0 16.5 ± 0.0
0.2 ± 0.0 1.1 ± 0.1 0.4 ± 0.3
0.6 ± 0.1 2.9 ± 0.3 4.6 ± 0.6 100.0
0.6 ± 0.0 3.5 ± 0.2 6.1 ± 0.3 0.3 ± 0.0 6.2 ± 0.9 100.0
0.1 ± 0.0 6.0 ± 0.6 3.6 ± 0.3 22.8 ± 2.2 0.7 ± 0.3 5.5 ± 0.6
0.9 ± 0.1 2.2 ± 0.2 1.7 ± 0.4 0.2 ± 0.0 11.2 ± 2.3
100.0
100.0
0.6 ± 0.0 0.3 ± 0.1 2.1 ± 0.1 3.6 ± 0.4 100.0
tr < 0.1%. a Peak number in Fig. 1. b Compounds determined as methyl ester TMS ether and/or TMS ether TMS ester. c Position of double bond not confirmed by chemical methods. d Value also contains 16-OH 10-oxo 16:0 in rosehip and rowanberry. e Not separated from DiOH-16:0 in black chokeberry and rowanberry. f Determined as corresponding methoxyhydrin compounds. g Tentative identification.
acetylation afterwards as derivatisation for GC analysis. The reflux and test tube methods resulted in identical monomers but slightly different relative proportions, which varied between the berries (data not shown). In addition to the nature of the samples the differences may also be because of the complexity of monomer mixtures obtained with the reflux method, which hampered calculations due to overlapping peaks. The formation of several different methyl and/or TMS derivatives, especially in the case of hydroxy–dioic acids, resulted in major overlapping of peaks. Depolymerisation in a test tube with dried reagents resulted in sole methyl ester derivatives of cutin monomers at best and at worst both ester derivatives, but only from the very major peaks, which ensured calculations were straightforward. Thus, the monomer profile proportions in Table 3 have been calculated from test tube reactions. Monomer profiles for crowberries were compared from berries collected in two different years. Hydroxy acids and diacids comprised 93.3 ± 0.4% and 94.2 ± 1.0% for berries collected in 2005 and 2004, respectively. The monomer profiles were fairly equal and in the range of standard deviations; thereby the values shown in Table 3 were derived from berries collected in 2005. The cutin monomers identified from berry cutins comprised mainly long-chain x-hydroxy acids (Table 3), with mid-chain functionalities known to commonly exist in cutin polymers of different plant materials, including berries (Kallio et al., 2006). Compounds with chain lengths C16 and C18 dominated all berry cutins studied, but also minor amounts of compounds with chain lengths C15 and C20 to C24 were found. C15 monomers have not been found earlier from berry cutins, but are known to exist in the cutin of several gymnosperms (Hunneman & Eglinton, 1972). Trace amounts of
short-chain hydroxy acids (
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R. Järvinen et al. / Food Chemistry 122 (2010) 137–144
Fig. 1. Total ion chromatograms with major peaks numbered (Table 3) of raspberry (A), cloudberry peel (B), sea buckthorn seed (C) and cloudberry seed (D) cutins.
previously found in blackcurrant cutin (10–14%) and citrus fruit (Deas et al., 1974; Kallio et al., 2006). Three monomeric compounds were tentatively identified to be 18-hydroxy-octadecatrienoic acid, 9,10,18-trihydroxyoctadecadienoic acid and 18-hydroxy-9,10-epoxyoctadecadienoic acid. Identifications were based on their retention times and similar mass spectral patterns as the corresponding mono or diunsaturated compounds. To our knowledge, the mass spectra have not been published and reference compounds were not available, thereby identification is considered tentative only and requires further work to become definite. Aromatic compounds were found in minor quantities (<1%) in black chokeberries, raspberries, rowanberries and crowberries. In strawberries, aromatic compounds comprised 6% of total monomers, being mainly coumaric acid (3.1%) and hydroxy-benzoic acid (1.8%). Other aromatic compounds were ferulic/isoferulic acid, hydroxybenzaldehyde, hydroxymethoxybenzoic acid and dihy-
droxybenzoic acid (all less than 1%). Rosehip and cloudberry cutin contained no aromatic compounds. Unidentified compounds comprised 2.8–16.5% of the total monomers. Single compounds were minor (<0.5%), except for some unknowns in black chokeberry, which accounted for 5.7% (UN1) and 4.3% (UN2) of the total monomers. Peak UN1 had a retention time of 23.8 min and its mass spectrum had diagnostic ions of a long-chain methyl ester TMS ether of x-hydroxy acids (e.g., rearrangement ions at m/z 146 and 159) (Rontani & Aubert, 2004), a major cleavage ion (possible a-cleavage) at m/z 285 and other important ions at m/z 271, 335, 425 (possible M-47) and 443 (possible M-15). Peak UN2 had a retention time of 24.7 min and its mass spectrum also showed diagnostic ions for a long-chain methyl ester TMS ether of x-hydroxy acids. According to MS, it was also a mixture of two compounds eluting practically at the same time. The first compound had fragment ions at m/z 271, 285, 315 and 329, indicating a mixture of positional isomers. The
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latter compound had significant ions at m/z 253, 290, 412 and also m/z 147, which indicates the presence of several TMS groups (Rontani & Aubert, 2004). These compounds were also present in many other berries but in lower proportions. Other unidentified monomers comprised similar diagnostic ion patterns as the three compounds above, indicating a series of related monomers. About 11% of the rowanberry cutin monomers remained unidentified, amongst which we found indications of branched chain hydroxy acids in trace quantities. Raspberries and rowanberries had just above the traceable limit (0.1%) of hydroxypentadecanoic acid, in which the hydroxy group was not in the x-position but in the 9/10 position. Other non-xhydroxy compounds (e.g., 10-OH-16:0) and epoxydiacids (e.g., 9,10-epoxy-1,18-dioic acid) were also detected, but did not exist above trace levels in any sample. Fatty acids were found in analyte mixtures both as methyl esters and TMS esters. According to NMR analysis, it is possible that free acids are present amongst the esterified monomers (Deshmukh, Simpson, & Hatcher, 2003). Fatty acids usually existed in small proportions (<3%), but in strawberries and raspberries they accounted for 22.8% and 6.1% of the total monomers, respectively. Alkanols were found in very minor amounts only and glycerol was not determined in this study because of the method used to recover monomers after depolymerisation. However, it is known
to be a relevant monomer (1–14% of total monomers) of some cutins (Graça et al., 2002).
3.4. Cutin monomers of seeds Cutin monomer analysis of cloudberry, blackcurrant and sea buckthorn seeds was carried out similarly to the peel cutin analysis. Hydroxy fatty acids and hydroxyl diacids were the major monomer groups, comprising 74.9%, 82.0% and 31.1% of the abovementioned berry seeds, respectively (Fig. 2A). Sea buckthorn seed monomers contained 48.1% of fatty acids, which has to be because of the insufficient isolation process; these seeds contain 7–11% oil vs. seed weight depending on variety (Yang & Kallio, 2001). Cloudberry seed cutin monomer profiles most resembled the corresponding berry cutin (Fig. 1), whereas that of blackcurrant and sea buckthorn differed markedly from the respective berry skin compounds (Kallio et al., 2006). Major monomers of blackcurrant seed cutin were epoxy-substituted compounds (Fig. 2B), comprising 47.2% of total monomers, compared with 11% of total berry cutin monomers (Kallio et al., 2006). Blackcurrant seeds also contained epoxy and dihydroxysubstituted octadecanedioic acid, which has not been found from the corresponding berry skin. Dihydroxyhexadecanoic acid (mainly
A
B
100 %
100 %
90 %
90 %
80 %
80 %
70 %
70 % others Unknown
60 %
Hydrocarbons
hydroxy-C18 diacid
60 %
trihydroxy C18
Diacids 50 %
Fatty acids
hydroxy C18 50 %
epoxy-hydroxy C18 b hydroxy-C16 diacid
Aromatics 40 %
Hydroxy acids and diacids
dihydroxy-C16
40 %
hydroxy C16 a
30 %
30 %
20 %
20 %
10 %
10 %
0%
0% Cloudberry Black currant Buckthorn
Sea
Cloudberry
Black currant Buckthorn
Sea
Fig. 2. Seed cutin monomer profiles by methanolysis (test tube reaction) as a percentage of total peak areas in GC–MS chromatogram (A) and with hydroxy acids and diacids separated in compound classes by chain length and functional groups (B).
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9/10–16-isomers), a major monomer in blackcurrant berry cutin (30%) (Kallio et al., 2006), was present in seed cutin at only 7.3%. The most prominent sea buckthorn seed cutin monomer was dihydroxyhexadecanoic acid (mainly 9/10–16-isomers: 14.2%) instead of epoxy-substituted C18 compounds (0.9%), which dominated the corresponding berry cutin (71%) (Kallio et al., 2006). Sea buckthorn seeds were also rich in monohydroxyhexadecanedioic acid (9.3%), which was not found in berry skin in our earlier studies. Shao et al. (2007) have determined that cutin of soybean seeds differ from the other parts of the plant (the leaves or pods) and contain unusual 2-hydroxy fatty acids as their main compounds, in addition to x-hydroxy fatty acids. The soybeans contained no mid-chain hydroxylated monomers at all. Molina et al. (2006) have suggested that most of the C16–C20 fatty acids found in seed polyesters of B. napus and A. thaliana could originate from ‘‘physically trapped” triacylglycerols, which is probably the reason for such a high content of fatty acids in sea buckthorn seed cutin as well. Molina et al. (2006) also suspected that 2-hydroxy acids are derived from sphingolipid acyl groups and not from cutin polyesters. In our studies of berry seed cutin, we have not found 2-hydroxysubstituted monomers, but confirmed that the monomer profile is different in different parts of berries. The composition profiles of cutins in different berries are crucial for evaluating their physiological properties for the plant and nutritional qualities when consumed as food. Polymers mainly composed of x-hydroxy acids and a,x-diacids without mid-chain functional groups or mid-chain substituted monomers with epoxy or oxo-functionalities are essentially linear as they do not have mid-chain groups that could be part of ester linkages forming cross-linkages. Polymers mainly with di- and/or trihydroxy-substituted monomers make up a totally different polymer via crosslinking. Glycerol has been described to be a common link between different monomers (Graça et al., 2002), but ester bonds without glycerol have also been recovered when analysing oligomers with MS and NMR techniques (Ray, Chen, & Stark, 1998). Differences in the extent of ester-linked cutin vs. resistant cutan, variable cutin monomers affecting the cross-linking and structure of cutin polymers and the composition of cutan in berries all have a strong influence on the various functions of cuticles in berries, and also as a dietary fibre. Further studies on structures of cuticular polymers and on their effects on human nutrition are required for a more comprehensive understanding of the evident significance of this polymer. Acknowledgements This research has been supported financially by the ABS – Graduate School (Applied Bioscience Bioengineering, Food and Nutrition, Environment) and the Finnish Funding agency for Technology and Innovation, TEKES in a project LIPFUN. The author would like to thank Jenni Kumpula for assistance in technical work. References AACC. (2001). Report of the dietary fiber definition committee to the board of directors of the American association of cereal chemists. The definition of dietary fiber. St. Paul, MN: American Association of Cereal Chemists. Bender, D. A. (2005). A dictionary of food and nutrition. Oxford: Oxford University Press. Deas, A. H. B., Baker, E. A., & Holloway, P. J. (1974). Identification of 16hydroxyoxohexadecanoic acid monomers in plant cutins. Phytochemistry, 13, 1901–1905.
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