Structural characterisation of polysaccharides from roasted hazelnut skins

Food Chemistry 286 (2019) 179–184

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Research Article

Structural characterisation of polysaccharides from roasted hazelnut skins ⁎

T

Zuzana Košťálová , Zdenka Hromádková Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Dúbravská cesta 9, SK-84538 Bratislava, Slovakia

A R T I C LE I N FO

A B S T R A C T

Keywords: Hazelnut skin Polysaccharides Pectin NMR

Two polysaccharide fractions sequentially extracted with water 1W and alkali 1A, were isolated from the hazelnut skins. The monosaccharide composition together with the FTIR and NMR analyses, indicated that both fractions are formed from a mixture of polysaccharides. The fraction 1W consists of methyl-esterified pectic polysaccharide with rhamnogalacturonan I blocks, branching with arabinose side chains, and with 1,5-, 1,3,5arabinan and galactan polysaccharides. The fraction 1A is a mixture of deesterified rhamnogalacturonan I and 1,5-, 1,3,5-arabinan and 4-O-Me-glucuronoxylan polysaccharides. The presence of unsaturated galacturonic acid and the heterogeneity of the molecular weights, which Mw ranged between 3.6 and 39 kg mol−1, indicated the pectin degradation during roasting.

1. Introduction The main hazelnut (Corylus avellana L.) production is concentrated on the Black Sea coast of Turkey. Accordance with the international nut and dried fruit statistic database, Turkey is the leading producer, accounting for up to 73% of world share. In 2017/2018 season, world hazelnut production amounted to over 490,000 tons (kernel basis) (International Nut and Dried Food Council, 2018). Hazelnut skin is the perisperm of the hazelnut kernel and forms approximately 2.5% of the total kernel weight but is often discarded during roasting (Zeppa et al., 2015). If not processed further, they become waste and their destruction can cause environmental problems. Therefore, processing industries are commercially interested in the valorisation of these by-products, i.e. by recovering potentially high value compounds. Almonds, hazelnuts, and possibly other nuts have a significant portion of polyphenols concentrated in their skin. Polyphenols can have covalent or strong non-covalent associations with plant proteins and polysaccharides (Le Bourvellec & Renard, 2012). Moreover, Montella, Coïsson, Travaglia, Locatelli, Bordiga, et al. (2013) and Zeppa et al. (2015) confirmed that nuts skins are a good source of dietary fibre, amounting to 58.3% of dried weight, of which about 7% is soluble fibre and about 93% insoluble fibre. Several complex oligosaccharides were isolated and characterized by gas chromatography (GC) and MALDI-FTICR MS from the hazelnut skin by Montella, Coïsson, Travaglia, Locatelli, Bordiga, et al. (2013). Galacturonic acid was a dominant component in the water extract of hazelnut (Montella, Coïsson, Travaglia, Locatelli, Bordiga, et al., 2013)

⁎

and also of almond skins (Mandalari et al., 2010). In the simulated digestion, the cell walls of almond skin may function as a useful source of fermentable fibre with beneficial implications for gut health (Mandalari et al., 2010). A similar effect was observed in hazelnut skin fibre with potential prebiotic activity towards L. crispatus P17631 and L. plantarum P17630, which helps to improve growth during in vitro fermentation (Montella, Coïsson, Travaglia, Locatelli, Malfa, et al., 2013). Therefore, the nuts skin can be potentially utilised as an additive in food production, for example, as an ingredient in bakery products (Anil, 2007; Cikrikci, Demirkesen, & Mert, 2016) or in fresh pasta products, to obtain a fortified food with high fibre content and antioxidant activity (Zeppa et al., 2015). Montella, Coïsson, Travaglia, Locatelli, Bordiga, et al. (2013), Zeppa et al. (2015) and Yilmaz and Tavman (2016) characterized the basic carbohydrate composition of the hazelnut skin. Even so, the polysaccharides of hazelnut skin are not well-described. Moreover there is also a question of whether the roasting process has an effect on the polysaccharides structure. The aim of this work is to move the research further into detailed structural descriptions of hazelnut polysaccharides. This information could lead to increased utilization of polysaccharides as a food ingredient or pharmaceutical additive. 2. Materials and methods 2.1. Material Roasted hazelnut skins from producers in the Black Sea Region of Turkey were kindly provided for this study by Prof. Sebnem Tavman (Ege University, Engineering Faculty, Food Engineering Department,

Corresponding author. E-mail address: [email protected] (Z. Košťálová).

https://doi.org/10.1016/j.foodchem.2019.01.203 Received 28 September 2018; Received in revised form 28 January 2019; Accepted 31 January 2019 Available online 07 February 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

Food Chemistry 286 (2019) 179–184

Z. Košťálová and Z. Hromádková

III HDX 600 MHz, equipped with a triple inverse TCI H-C/N-D-05-Z liquid He-cooled cryoprobe. One-dimensional 400 MHz or 600 MHz 1H spectra, as well as two-dimensional 1H-13C HSQC and HMBC, were used to the determination of 1H and 13C chemical shifts. The chemical shifts were referenced to the internal standard DSS (sodium 4,4-dimethyl-4silapentan-sulfonate from Sigma–Aldrich), with 1H and 13C at 0.00 ppm. The spectra were measured in D2O at 300 K. Size exclusion high performance liquid chromatography (HPSEC), with two HEMA-BIO 40 and 100 columns (Tesssek, 8 × 250 mm) connected in series, was used to estimate the weight-average molecular weights (Mw) and the number–average molecular weights (Mn). The analyses were performed on an Agilent LC 1260 GPC/SEC System equipped with DAD and RI detectors. The mobile phase was 0.1 M NaNO3 and the flow rate was 0.4 mL/min. A set of dextrans (American Polymer Standard Corporation, Mentor, OH, USA) with Mw 0.5, 5.2, 25.5, and 72.7 kg mol−1, was used for the calibration, and Cirrus GPC/ SEC 3.4.1 software for the calculation of the polydispersity (PDI). A standard calibration curve for the logarithm of the molecular weight versus the HPSEC retention time was obtained for all standards.

Izmir, Turkey). In this study, the hazelnut skins as a residue from the roasting process were used. Afterwards, they were pre-treated with ethanol and ether, then left to dry to obtain an alcohol insoluble residue (AIR). The grounded fine powder (particle size: 373 × 50 µm) was stored in the dark. For the detail characterisation of raw material see Yilmaz and Tavman (2016). Monosaccharide standards were obtained from Fluka (Schnelldorf, Germany). All the chemicals used were of analytical grade and were used without further purification. 2.2. Polysaccharide isolation The pre-treated skin powder (10 g) was extracted using 100 mL of 60 °C water for 1 h in a water bath with constant stirring. The mixture was filtered through a nylon filter 10 µm followed by S4 glass filtration. The residue was washed with 50 mL of pure water and left in the open to dry. The supernatant was evaporated to half volume and precipitated using 4 times this volume of ethanol. After staying overnight in the fridge, the sediment was separated by centrifugation (4000 rpm) 10 min, dialyzed in 3500 MWCO dialysis tubes and freeze dried, yielding the water extracted sample 1W. In the second step, the residue after water extraction was treated with 100 mL of 3% NaOH at 40 °C for 1 h in a water bath with constant stirring. The mixture was filtered through a nylon filter 10 µm following with S4 glass filtration. The residue was washed with 50 mL of pure water and left in the open to dry. The supernatant was neutralized to pH 7 with dilute HCl than evaporated, precipitated, centrifuged and dialyzed as a sample before yielding the alkali extracted sample 1A. Both samples, 1W and 1A were fractionated into water soluble (1Wws, 1Aws) and water insoluble fractions (1Wwis, 1Awis). In the separation, 0.1 g of each sample was dissolved in 50 mL of deionised water, stirred for 1 h, and then separated by centrifugation (9000 rpm) for 10 min, and finally freeze dried.

2.4. Statistical analysis Mean differences in chemical composition of the hazelnut polysaccharides were evaluated by one-way analyses of variance (ANOVA), and Fisher’s LSD post-hoc test at a confidence level of 0.95 using STATISTICA v 9.1 software (StatSoft Inc, Tulsa, OK, USA). 3. Results and discussion 3.1. Yields and composition analyses The hazelnut skins, as waste after the roasting process, were pretreated with ethanol and an ether soxhlet extraction to eliminate the alcohol soluble phenolic and lipidic components. The alcohol insoluble residue of the hazelnut skins, denominated as AIR, was treated with water and 3% NaOH to yield the water-extracted (1W) and alkali-extracted (1A) polysaccharide fractions. The polysaccharide yields were expressed as a percentage of the dry matter weight of AIR. The polysaccharide yield isolated with water was 2.4% of dry AIR (Table 1), with an alkali extraction 26.4% of dry AIR, and the residue was 41.5% of dry AIR, respectively. The rest of the material was formed by the parts non-precipitated in ethanol 1W/EtOH and 1A/EtOH (Table 1), and the probable losses during dialyses. All the extracted polysaccharides had a dark brown colour and were analysed by a suite of chemical and spectral tools to estimate the main polysaccharide components. The polysaccharides rich in galacturonic acid (59%) were extracted using water, whereas the glucose, galactose and arabinose dominated the alkali fraction. The data concerning the sugar composition is given in Table 1 and is in agreement with the previous study of Montella, Coïsson, Travaglia, Locatelli, Bordiga, et al. (2013), except for N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc) content. These components were established in Montella, Coïsson, Travaglia, Locatelli, Bordiga, et al. (2013) by GC in trimethylsilyl methyl glycoside derivatives. In our case, the HPAEC-PAD analysis (using GalNAc and GlcNAc as a standard) together with the nitrogen content, NMR, or FTIR results (described below) did not show any presence of these components. This could have been caused by using a marked shorter time of extraction, which was insufficient to release them into the solution. The parts non-precipitated in ethanol (1W/EtOH and 1A/EtOH) were also tested for their sugar composition. The major components were an easily-cleavable arabinose and a glucose unit. The glucose could have originated from the cellulose fragments as determined by Mandalari et al. (2010) they found pectin and arabinose-rich polysaccharides encased with microfibrils of cellulose in the extract of

2.3. General methods Composition analysis of polysaccharides is typically based on hydrolysis procedures, which use hydrochloric, sulfuric, or trifluoroacetic acid (TFA) at elevated temperatures. Due to the most resistance of β1, 4-glycosidic linkage of uronic acid amplified with a degree of esterification (Krall & McFeeters, 1998), the 2 or 6 M TFA did not properly hydrolyse the acid backbone of the isolated polysaccharides. The samples were hydrolysed using sulphuric acid (72% w/w) at 30 °C for 1 h, followed by a dilution with water to 2% w/w, and incubation at 120 °C for 4 h. Aliquots of hydrolysates were filtered (0.22 mm membrane filter), and the neutral and acidic monosaccharide components were determined using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The Dionex™ ICS-5000+ (Thermo Fisher Scientific Inc., Waltham, MA, USA), equipped with an ED 5000+ electrochemical detector and a gradient pump, was used. It was operated with a column CarboPac PA1 using the method according to Košťálová, Aguedo, and Hromádková (2016). Dionex Chromeleon 7.2 SR3 Build 7553 software (Sunnyvale, USA) was used for evaluation. The nitrogen content was estimated by elemental analysis using a FLASH 2000 Organic elemental analyser (CHNS-O; Thermo Fisher Scientific, Waltham, MA, USA). The FTIR-ATR spectra were measured by a Nicolet iS50 FTIR (Thermo Scientific, USA) spectrometer equipped with a DTGS detector and controlled by Omnic 9.0 software. The spectra were collected from 4000 to 400 cm−1 at a resolution of 4 cm−1. 128 scans were done. A diamond Smart Orbit ATR accessory was used for measurements in the solid state. The high-resolution NMR spectra of sample 1Wws were recorded by a Bruker AVANCE III HD 400 MHz with broad band BB-(H-F)-D-05-Z liquid N2 prodigy probe and there of sample 1Aws by a Bruker AVANCE 180

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Z. Košťálová and Z. Hromádková

Table 1 Chemical composition of the hazelnut skin. Sample

1W

1W/EtOH

1A

1A/EtOH

Residue

Yield (wt % of AIR)

2.4

2.9

26.4

24.4

41.5

0.8 ± 0.3a,A 8.6 ± 2.3b,B 15.3 ± 1.4c,A 17.8 ± 2.7c,B 27.7 ± 2.4d,C 2.5 ± 0.8a,AB 8.8 ± 1.1b,B 15.1 ± 1.8c,B 1.2 ± 0.3a,A 1.95

nd 10.8 ± 0.4b,B 31.4 ± 2.0c,B 10.1 ± 1.6b,A 30.3 ± 1.8c,C 5.4 ± 0.5a,C 3.3 ± 0.6a,A 5.5 ± 0.7a,A 3.2 ± 0.3a,B –

2.0 ± 0.3a,B 3.6 ± 0.4ab,A 11.4 ± 0.7c,A 7.1 ± 1.6bc,A 42.6 ± 3.7e,D 3.7 ± 1.0ab,BC 21.5 ± 2.2d,C 6.9 ± 1.6bc,A 1.1 ± 0.1a,A –

Monosaccharides composition (rel. wt % after 72% H2SO4 hydrolysis) Fuc 0.6 ± 0.1a,A 1.0 ± 0.7a,AB Rha 11.0 ± 0.7b,B 16.5 ± 1.4c,C Ara 11.6 ± 2.3b,A 42.7 ± 2.4d,C Gal 8.8 ± 0.1b,A 9.5 ± 2.1b,A Glc 3.7 ± 1.8a,A 20.2 ± 3.7c,B Man 0.7 ± 0.3a,A 4.4 ± 1.1a,BC Xyl 3.3 ± 2.0a,A 1.6 ± 0.1a,A GalUA 59.0 ± 3.2c,C 4.0 ± 2.2a,A GlcUA 1.3 ± 0.0a,A nd Nitrogen (wt %) 0.23 – Sample

1Wws

1Wwis

1Aws

1Awis

Proportion (wt %)*

96.7

3.3

89.2

10.8

nd 9.9 ± 0.7c,BC 14.7 ± 1.0e,AB 14.5 ± 0.7de,B 11.4 ± 1.8 cd,B 1.1 ± 0.1ab,A 3.9 ± 0.6b,AB 44.5 ± 3.5f,C nd

0.6 ± 0.1a,A 8.7 ± 0.7b,AB 24.0 ± 2.8d,C 18.0 ± 1.4c,C 17.2 ± 1.7c,C 2.2 ± 0.3a,B 11.3 ± 1.1b,C 17.9 ± 1.3c,A nd

nd 7.1 ± 0.4b,A 15.6 ± 0.9d,B 10.9 ± 0.7c,A 30.9 ± 2.0 e,D 1.2 ± 0.1a,A 5.2 ± 0.3b,B 29.1 ± 1.3e,B nd

Monosaccharides composition (rel. wt % after 72% H2SO4 hydrolysis) Fuc 0.6 ± 0.0a,A Rha 11.5 ± 0.7c,C Ara 11.1 ± 0.9c,A Gal 9.4 ± 0.6c,A Glc 5.7 ± 1.8b,A Man nd Xyl 3.0 ± 0.7ab,A GalUA 53.6 ± 3.7d,D GlcUA 5.1 ± 0.4b,A

ws-water soluble part; wis-water insoluble part; EtOH-ethanol not precipitated fraction. nd-not detected; * the wt% from 1W or 1A fraction. The values are given as mean ± SD (n = 2). The values followed by different superscripts (a–e) within the same column are significantly different (p < 0.0.5) from each other. The superscripts (A–D) different letters in each row indicate significant difference (p < 0.0.5).

almond skins. The residue of the extraction had a high glucose and xylose monosaccharide content, which could belong to the cellulose and xylan polysaccharides. The dark colour of the samples did not allow the use of colorimetric methods to determine phenolic substances, or proteins. Therefore, spectroscopic methods were used to estimate their presence. 3.2. Structural analyses 3.2.1. FTIR spectroscopy The monosaccharide analysis gives only a brief outlook on the possible sugar composition of the sample, whereas the FTIR spectroscopy (Fig. 1) helps reveal the main polysaccharides. In the FTIR spectrum, the polysaccharides clearly illustrated the typical signal pattern. In the ATR spectrum of 1W with pH ∼ 7, it is clear that all the non-esterified carboxylic groups are in the form of carboxylate ions. The dominant signals were 1602 cm−1, which contributed to the asymmetric stretching vibration of the carboxylate anion COO−. The corresponding symmetric stretching mode of COO− is presented as a small shoulder near 1416 cm−1. The signal at 1732 cm−1 was assigned to the stretching C]O vibrations of esters. The area of the ester band was used to rough estimation of the degree of methylesterification (DE) for the sample 1W (Gnanasambandam & Proctor, 2000). Because the band of COO− was overlapped with Amid I, phenolics and water bands, only the area of ester carbonyl band was used. The spectra were normalized to the band 1143 belonging to OeCeO asymmetric stretching of glycosidic linked pectin (Szymanska-Chargot & Zdunek, 2013). The pectin standards with a known DE were used for calibration curve formation. The DE of 1W was estimated to 47%. Polysaccharides produce specific bands in the region 1200–950 cm−1. The bands 1235, 1143, 1072, 1048 and 1015 cm−1 showed the pectin fingerprint overlapped with the bands of the arabinogalactans (Szymanska-Chargot & Zdunek, 2013). The presence of

Fig. 1. Fourier transform infrared spectrum of hazelnut polysaccharides.

phenolics was shown by the (C]C) signal at 1602 cm−1 overlapping with the COO− (see above) and the weaker signal of (CeC) at 1520 cm−1 and (OeC) at 1441 cm−1(Nogales-Bueno et al., 2017). Contrary to this, the spectrum of the alkali fraction 1A showed very intensive bands at 1602, 1520 and 1441 cm−1, which belong to the phenolics. The sugar signals in 1A were represented by a band of xylose at 1096 cm−1, and bands of the mixture of pectin and arabinogalactan at 1143, 1073 and 1043 cm−1. 3.2.2. The water soluble polysaccharides Because the whole fractions 1W and 1A do not have good swelling 181

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esterification was confirmed by the cross-peak at 3.81/166.2 ppm, which correspond to the correlation of H1 of the CH3Oe group and C6 of Δ6MeGalpA. Furthermore, the same HMBC spectrum showed the correlation between H1 of Δ6MeGalpA and C4 of 6MeGalpA, indicating the presence of the α-Δ6MeGalpA-(1 → 4)-α-6MeGalpA-(1 → … moiety, which 6MeGalpA had a H4/C4 cross-peak shifted to 4.59/81.6 ppm. Similar oligomers were generated by the chemical depolymeriazation of the rhamnogalacturonan by Deng, O’Neill, and York (2006). The unsaturated GalpA may be a product of β-elimination in alkaline media and higher temperature, but may also be a product of enzymatic depolymerization (Beck & Wieczorek, 2012; Bédouet, Courtois, & Courtois, 2005). In this case, the formation of Δ6MeGalpA was probably related to the hazelnut kernel processing method. The hazelnut skins were obtained as by-products after the roasting process, during which the temperature could have reached 170 °C for 10–45 min (Schlörmann et al., 2015). This could have led to the dehydration, and to the complex of reactions. The anomeric region of the HSQC spectrum of 1Wws contained an intensive signal H1/C1 of 1,2-α-rhamnopyranose (Rhap) at 5.26/ 101.3 ppm, which indicated the existence of rhamnogalacturon I blocks. Besides the 1,2-α-Rhap, the substituted 1,2,4-α-Rhap was also identified with characteristic H4/C4 at 3.72/82.9 ppm. According to Dourado, Cardoso, Silva, Gama, and Coimbra (2006), the two terminal α-L-arabinofuranoses (Araf) at 5.14/109.8 and 5.24/ 111.8 ppm, together with the 1,5 linked α-L-Araf at 5.07/110.3 ppm and the 3,5-di-O-substituted α-L-Araf at 5.07/110.3 ppm were observed in the HSQC of 1Wws. The HMBC spectrum showed interglycosidic linkage between the T-Araf and 1,2,4-α-Rhap with a cross-peak H1/C4 at 5.24/82.9 ppm. β-galactopyranose (Galp) and 4-O-Me-glucuronopyranosyluronic acid (GlcpA) were also observed. The NMR spectra showed the presence of O-acetyl groups, which were probably located on some of the Rhap and GalpA residues. Ethanol contamination was also visible in the spectra with chemical shifts 1.18/18.3 and 3.67/ 60.4 ppm. As shown in the HSQC spectrum (Fig. 3) of the alkali extracted fraction 1Aws, the Araf was the dominant component. The 1,5 linked αL-Araf at δ 5.08/110.3 ppm, the 3,5-di-O-substituted α-L-Araf at δ 5.07/ 110.3 ppm, and the terminal α-L-Araf at 5.23/112.1 ppm were identified (Dourado et al., 2006). In comparison to the pectin sample 1Wws, the NMR analysis of the fraction 1Aws revealed the presence of a 1,4-βD-xylopyranosyl unit (Xylp, Fig. 3). The characteristic cross-peaks of the Xylp unit (Table 3), along with H4/C4 of GlcpA at 3.21/85.1 ppm and eOCH3 at 3.44/62.7 ppm, indicated the existence of a4-O-Me-glucuronoxylan moiety (Wen et al., 2011). The high amount of the xyloses in the hazelnut shells was also detected by Surek and Buyukkileci (2017). The GalpA in the 1Aws spectrum was deesterified because of the alkali extraction condition. Therefore, the shifts of all GalpA moieties were shifted in comparison to the spectra of 1Wws. The deesterified unsaturated GalpA, with a characteristic cross-peak of H4/C4 at 5.77/ 109.5 ppm, had a very weak intensity. The polygalacturonan units, … → 4)-α-GalpA-(1 → …, had H4/C4 at 4.41/80.6 ppm. And the GalpA of the rhamnogalacturonan I block units, … → 2)-α-Rhap-(1 → 4)-αGalpA-(1 → …, had H4/C4 at 4.39/80.0 ppm. The HMBC correlation of H1 of Rhap and C4 of GalpA at 5.25/80.0 ppm confirmed the glycosidic linkages (Makarova, Shakhmatov, & Belyy, 2016). Surprisingly, the rhamnogalacturonan moiety was the dominant acid component, with signals at 4.99/100.2 (H1/C1), 3.89/70.7 (H2/C2), 4.10/73.1 (H3/C3), 4.39/80.0 (H4/C4), 4.65/74.1 ppm (H5/C5), respectively. The ratio between the … → 4)-α-GalpA-1 → … and … → 2)-α-Rhap-(1 → 4)-αGalpA-(1 → … was 0.27:1, calculated from the H4/C4 cross-peaks, and 0.34:1 from cross-peak H5/C5. This calculation pointed out that rhamnogalacturonan blocks formed ∼75% of the pectin backbone. Moreover, inspection of the spin systems of rhamnose residues revealed an additional substitution at O4. The ratio of 2- to 2,4-substituted rhamnose, as deduced from the C6/H6 signals in the HSQC spectrum, is 1:0.3. The ∼15% of the rhamnose branching was also confirmed by a

properties in water, the samples were split into water soluble and water insoluble parts. This was essential for the detailed NMR characterisation and for the detection of the molecular properties. The water insoluble parts were hydrolysed with sulfuric acid and characterised by monosaccharides analysis (Table 1). The insoluble parts of both samples, 1Wwis and 1Awis, still contained ∼44 or 29% of galacturonic acid, which indicated the isolation of the water-insoluble, weakly-hydrolyzable pectin substances called protopectin (Maclay & Nielsen, 1945). The HPAEC-PAD analyses of the water soluble parts (Table 1) revealed the major components. In the water extract 1Wws, the galacturonic acid formed 53% and, together with 11.5% of rhamnose and 11.1% of arabinose indicate the pectin pattern. However, the water soluble part of the alkali fraction 1Aws consisted of comparable amounts of arabinose (24%), galacturonic acid (18%), galactose (18%) and glucose (17%). The xylose residues were also presented in significant amounts. The prevalence of neutral polysaccharides was evident in this sample. 3.2.3. The NMR analyses 1D and 2D NMR spectroscopy, including 1H, 1H-13C HSQC and 1 H-13C HMBC techniques, were used to reveal the detailed structure of the polysaccharides of the hazelnut skins. A structural analysis which uses only 1H NMR spectroscopy (Fig. S1) is limited not only because of the structural diversity of the different protons along the chain and their coupling patterns, but also because of overlapping proton shifts among the individual polysaccharides. For a more precise analysis, the 1 H was supplemented by 1H-13C HSQC and HMBC techniques. In the water-extracted fraction 1Wws, the strong signals of 1,4-α-Dgalactopyranosyluronic acid (GalpA) were detected (Fig. 2, Table 2). In the area of the cross-peaks H4/C4 of galacturonic acid (∼4.44/ 80.0 ppm), at least three moieties were detected. The first and main fragment was 6-O-methylesterified GalpA, … → 4)-α-6MeGalpA-(1 → 4)-α-6MeGalpA-(1 → …, with its corresponding cross-peaks, as showed in Table 2. Its H4/C4 cross-peak was at 4.46/81.4 ppm. The esterification was confirmed by an intensive cross-peak at 3.81/173.4 ppm in the HMBC spectrum, which belongs to the correlation of H1 of CH3O and C6 of GalpA. The second fragment was non-esterified GalpA, … → 4)-α-GalpA-(1 → … . Its interglycosidic linkages were not possible to determine, but its H4/C4 cross-peak was at 4.44/80.0 ppm. The third fragment started with an unsaturated methylesterified galacturonic acid (mehtyl 4-deoxy-β-L-threo-hex-4-enopyranosyluronate, Δ6MeGalpA) with the specific cross-peaks H1/C1, H2/C2, H3/C3, H4/C4, and C5, C6 at 5.02/102.8, 3.72/72.4, 4.34/68.3, 6.10/114.8, and 143.1, 166.2 ppm, respectively. In the HMBC spectrum, the methyl-

Fig.2. The selected region of HSQC spectrum of the polysaccharide 1Wws. See Table 2 for signal assignment and abbreviations. 182

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Z. Košťálová and Z. Hromádková

Table 2 Chemical shift assignments δ (ppm) of the polysaccharide 1Wws, referenced to DSS. Sugar residue

Abb.

H1 C1

H2 C2

H3 C3

IH4 C4

H5 C5

→4)-α-6MeGalpA-(1→

MGA

→4)-α-GalpA-(1→

GA

β-Δ4,56MeHexpA-(1→

MΔ

β-Δ4,56MeHexpA-(1 → 4)-α-6MeGalpA-(1→

MGA*

→2)-α-Rhap-(1→

R

→2,4)-α-Rhap-(1→

R*

→5)-α-Araf-(1→

A1,5

α-Araf-(1 → 5)-α-Araf-(1→

At

α-Araf-(1 → 2)-α-Rhap-(1→

At*

→3,5)-α-Araf-(1→

A3,5

β-Galp-(1→

G

4-O-Me-β-GlcpA-(1→

GlA

4.96 102.8 5.12 102.1 5.02 102.8 – – 5.26 101.3 5.26 101.3 5.07 110.3 5.14 109.8 5.24 111.8 5.07 110.3 4.59 105.8 – –

3.73 70.7 – – 3.72 72.4 – – 4.11 79.2 4.11 79.2 4.12 83.7 4.12 84.6 – – 4.28 82.0 3.55 74.2 – –

3.98 70.9 – – 4.34 68.3 4.34 70.7 – – – – 3.99 79.5 3.95 79.3 3.95 79.1 4.06 – 3.65 75.4 3.50 78.5

4.46 81.4 4.44 80.0 6.10 114.8 4.58 81.6 3.43 74.5 3.72 82.9 4.19 84.9 4.08 86.4 4.13 86.66 4.29 84.11 3.91 – 3.24 83.6

5.05 73.3 4.78 73.6 – 143.1 – – – 71.9 – – 3.86/3.80 69.7 3.80/3.76 64.0 3.91/3.74 – – – 3.68 77.7 – –

H6 C6 (6a,6b)

173.4 – – 166.2 – – 1.25 19.32 1.29 19.32

3.79 63.9 – –

– not detected; Abb.-abbreviations; eOCH3 at 3.81/55.6 ppm from → 4)-α-6MeGalpA-(1 → and β-Δ4,56MeHexpA-(1→; eOCH3 at 3.49/60.4 ppm from 4-O-Me-βGlcpA-(1 → .

3.3. Molecular weight The HPSEC was used to evaluate the number-average (Mn) and weight-average (Mw) molecular weights of all water soluble parts. The polydispersity index as a measurement of the broadness of molecular weight distribution was calculated by Mw/Mn. The larger value of the PDI indicates the broader molecular weight distribution as it was in the sample 1Wws (Fig. S2). Based on the dextran calibration, the major peak of 1Wws was determined as 22 kg·mol−1, the PDI was 2.26, which means that the curve of this peak overlapped the Mw from 113 to 1.2 kg·mol−1. The Mw of the major peaks (72 area%) of sample 1Aws was slightly higher (39 kg·mol−1) and narrower (PDI was 1.61) than sample 1Wws. 20.9% of the sample had Mw 3.6 kg·mol−1, with PDI 1.2. Chromatographs of both samples also showed peaks in the void volume (2–6 area%), which could have been formed with self-association of the polysaccharides.

Fig. 3. The selected region of HSQC spectrum of the polysaccharide 1Aws. See Table 3 for signal assignment and abbreviations.

4. Conclusion

ratio of the C4/H4 cross-peaks of 2- and 2,4-substituted rhamnose. The characteristic H4/C4 cross-peaks of two 4-O-MeGlcpA moieties were noticeable in the spectrum when the intensity was increased. One of them (3.21/85.1 ppm) could have been attached to the xylose unit, and have formed the 4-O-Me-glucuronoxylan. And the second (3.21/ 83.4 ppm), which was also shown in the pectin spectrum 1Wws, could have been attached to the GalpA backbone of the pectin. The high amount of phenolic components is evident in the 1H spectra of both samples (Fig. S1). The presence of the GalNAc and the GlcNAc, found in the nut skins by Montella, Coïsson, Travaglia, Locatelli, Bordiga, et al. (2013), was not confirmed by any NMR methods of water-soluble parts of our samples. Nevertheless, galacturonic acid, arabinose and xylose were confirmed by our research and structurally characterized in detail by NMR.

The two samples of polysaccharides were isolated from the hazelnut skins in a two-step extraction. Their detailed structure was investigated using HPAEC-PAD, FTIR, and NMR analysis. Firstly, the mixture of high-esterified pectin polysaccharide, arabinan, and arabinogalactan with Mw around 22 kg·mol−1, was released by water. Secondly, the mixture of deesterified rhamnogalacturonan with arabinan, and xylans with Mw between 3.6 and 39 kg·mol−1, was extracted by alkali. The identification of the unsaturated galacturonic acid in both samples and the considerable molecular heterogeneity confirmed the pectin degradation during the roasting process. Information on degradation of the polysaccharides in the hazelnut skins, during which the double bonds are formed, is essential for their future manipulation and application. The results obtained fill a gap in the research concerning the composition of hazelnut skins, and supplement current knowledge on the influence of the roasting process on the structure of polysaccharides.

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Z. Košťálová and Z. Hromádková

Table 3 Chemical shift assignments δ (ppm) of the polysaccharide 1Aws, referenced to DSS. Sugar residue

Abb.

H1 C1

H2 C2

H3 C3

H4 C4

H5 C5

H6 C6 (6a,6b)

→4)-α-GalpA(1→

GA

→2)-α-Rhap-(1 → 4)-α-GalpA-(1→

GA* Δ

→2)-α-Rhap-(1 → 4)-α-GalpA-(1→

R

3.76 70.9 3.89 70.7 – – 4.11 78.9 – – 4.12 83.7 4.28 82.0 4.12 83.7 3.55 74.2 3.27 75.5 – – – –

3.98 71.6 4.10 73.1 4.25 68.7 3.88 72.2 – – 3.99 79.5 4.07 86.7 3.95 79.4 3.65 75.4 3.54 76.4 – – – –

4.41 80.6 4.39 80.0 5.77 109.5 3.40 74.7 3.70 83.1 4.19 85.1 4.20 84.10 4.11 86.66 3.91 71.2 3.76 79.1 3.21 85.1 3.24 83.4

4.75 74.2 4.65 74.1 – – – 71.8 – – 3.86/3.77 69.7 3.70 – 3.91–3.72 – 3.68 77.7 4.08/3.35 65.7 – – – –

– – – –

β-Δ4,5HexpA-(1→

5.09 101.7 4.99 100.2 4.99 100.2 5.25 101.2 5.25 101.3 5.08 110.3 5.07 110.3 5.23 112.1 4.51 105.8 4.46 104.5 – – – –

→2,4)-α-Rhap-(1→

R

→5)-α-Araf-(1→

A1,5

→3,5)-α-Araf-(1→

A3,5

α-Araf-(1→

At

β-Galp-(1→

G

→4)-β-Xylp-(1→

X

4-O-Me-β-GlcpA-(1→

GlA

4-O-Me-β-GlcpA-(1→

GlA

*

eOCH3

1.24 19.4 1.28 19.6

3.79 63.9

– – – –

3.44 62.7 3.48 60.4

– not detected; Abb.-abbreviations.

Acknowledgements

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