Adsorption of apple polyphenols onto β-glucan

Czech J. Food Sci., 35, 2017 (6): 476–482 Food Analysis, Food Quality and Nutrition doi: 10.17221/75/2017-CJFS Adsorption of Apple Polyphenols onto β-Glucan Lidija JAkobek 1*, Petra MAtić1, Vedrana krešić 1 and Andrew r. bArroN 2 1 Department of Applied Chemistry and ecology, Faculty of Food technology, J.J. Strossmayer University of osijek, osijek, Croatia; 2Department of Statistics, Yale University, New Haven, USA *Corresponding author: [email protected] Abstract Jakobek L., Matić P., Krešić V., Barron A.R. (2017): Adsorption of apple polyphenols onto β-glucan. Czech J. Food Sci., 35: 476–482. The adsorption of polyphenols from apples, a good source of polyphenols in the human diet, onto β-glucan, a soluble dietary fibre were studied. Polyphenols were extracted from the flesh and peel of two apple varieties (wild apple and Slavonska srčika) and adsorbed onto β-glucan for 16 hours. The adsorption capacities (mg/g) and equilibrium polyphenol concentrations (mg/l) were modelled with Freundlich and Langmuir isotherms. Polyphenols from the flesh and peel showed different behaviours – flesh polyphenols exhibited greater affinity and peel polyphenols greater theoretical adsorption capacity. The analysis of individual polyphenols with high-performance liquid chromatography revealed that the composition of the flesh and peel differed (flesh was rich in phenolic acids, peel in flavonols) which could explain the contrasting adsorption behaviour. This study shows that polyphenols from apples can be adsorbed onto β-glucan, that the flesh and peel exhibit distinct adsorption behaviours and that the polyphenol composition can affect the adsorption mechanism. Keywords: adsorption isotherms; Freundlich; interactions; Langmuir Polyphenols have been studied intensively because they have shown many potential benefits for human health. One important aspect of their bioactivity is the interaction with other food constituents such as carbohydrates, lipids, and proteins (Le Bourvellec & Renard 2012; Jakobek 2015), which can have potentially important consequences. One such consequence is that polyphenols might be ‘protected’ and pass to the lower parts of the digestive tract without being metabolised. Once there, they might exert positive effects in their intact forms (Gorelik et al. 2008; Kanner et al. 2012). Dietary fibres are especially interesting since they can arrive in the colon in a non-metabolised form. This makes them potential ‘carriers’ of polyphenols ( Jakobek 2015). Interactions between polyphenols and dietary fibre can be studied through adsorption processes in vitro (Renard et al. 2001; Wu et al. 2011; Gao et al. 2012; Wang et al. 2013). Adsorption is a process in which molecules from a solution adsorb onto the surface of an adsorbent and can be described through adsorption isotherms (Soto et al. 2011). β-Glucan, a water-soluble dietary fibre, can serve as a model in adsorption processes. It can be found in different cereals where it may come into contact with fruit polyphenols. Namely, breakfast cereals or any other cereal products can be consumed in combination with fruits or fruit products. In these situations, β-glucan and polyphenols can interact. Furthermore, β-glucan is produced in the form of a dietary supplement and as such can be a part of a regular diet. Apples are a good source of polyphenols present in the everyday diet (Wojdyło et al. 2008), and can be consumed with breakfast cereals Supported by the Adris foundation, J.J. Strossmayer University of Osijek (Department of Mathematics), and by the Croatian Science Foundation under, Projects No. IP-2016-06-6777 and IP-2016-06-6545. 476 Czech J. Food Sci., 35, 2017 (6): 476–482 Food Analysis, Food Quality and Nutrition doi: 10.17221/75/2017-CJFS or other foods containing dietary fibres. As far as we are aware, adsorption onto β-glucan was not previously studied for apple polyphenols. Isotherms of adsorption onto β-glucan have been studied for tea polyphenols (Wu et al. 2011; Gao et al. 2012) and some other adsorption properties of polyphenolic compounds onto β-glucan have been investigated (Wang et al. 2013). The aim of this work was to study the adsorption of polyphenols from the flesh and peel of apples onto β-glucan as a dietary fibre. Freundlich and Langmuir non-linear isotherm models were created and used for the interpretation of adsorption. Additionally, the polyphenol content of apple samples was determined using high-performance liquid chromatography with photo-diode array detection (HPLC-PDA) with the aim of characterising individual polyphenols and evaluating their influence on the process of adsorption. MATERIAL AND METHODS Chemicals. Gallic acid monohydrate, (+)-catechin hydrate, (–)-epicatechin, chlorogenic acid, p-coumaric acid, quercetin dihydrate, quercetin-3-β-d-glucoside and β-d-glucan from barley were purchased from Sigma-Aldrich (USA). Procyanidin B1, procyanidin B2, quercetin-3-o-galactoside, quercetin-3-o-rhamnoside, phloretin-2'-o-glucoside, and phloretin were from Extrasynthese (France). Orto-phosphoric acid (85% HPLC-grade) was from Fluka (Switzerland), HPLC-grade methanol was acquired from J.T. Baker (the Netherlands), and hydrochloric acid, sodium carbonate and Folin-Ciocalteau reagent were from Kemika (Croatia). Samples and sample preparation. Old apple varieties – Slavonska srčika (Malus domestica) and a wild variety (crab-apple) were harvested at maturity (orchard of M. Veić, Požega, Croatia). Approximately 1 kg of sampled apples were peeled. The peel was pooled and homogenised using a blender. The core and the seeds were removed from the flesh, and the flesh was then cut into smaller pieces, pooled and homogenised with a stick blender. Extracts were prepared immediately. Polyphenol extraction. For the adsorption study, three samples of flesh or peel were weighed (0.2 g) and extracted with 1.5 ml of extraction solvent (0.1% HCl in methanol for peel samples; 80% methanol in water for flesh samples) in accordance with our previously described protocol for polyphenol extraction from apples ( Jakobek et al. 2015). We have observed that acidified methanol was better for peel polyphenols, while 80% methanol was a good choice for flesh polyphenols ( Jakobek et al. 2015). The samples were vortexed (Grant Bio, UK), placed in an ultrasonic bath (Bandelin Sonorex RK 100; Bandelin electronic, Germany) for 15 min and then centrifuged (Minispin; Eppendorf, Germany). Three extracts were combined and used for the adsorption study. For the polyphenol characterisation with HPLC-PDA, polyphenols were extracted from the peel (0.1% HCl in methanol) and from the flesh (80% methanol in water). Samples were weighed (0.2 g of the flesh or peel), mixed with 1.5 ml of extraction solvents, vortexed, placed in an ultrasonic bath for 15 min and then centrifuged. The extract was removed and the residue was extracted once more in 0.5 ml of extraction solvent. These extracts were combined and filtered (0.45-µm PTFE syringe filter). Two parallel extracts were prepared for each peel or flesh sample and each was analysed once with the HPLC-PDA method. Spectrophotometric method for total polyphenol determination. Total polyphenols were determined by the Folin-Ciocalteu method (Waterhouse 2016). Distilled water (1580 µl) was mixed with extract (20 µl), Folin-Ciocalteu reagent (100 µl), and a sodium carbonate solution (200 g/l, 300 µl). After incubation (40°C, 30 min), the absorbance was read at 765 nm on a UV-Vis spectrophotometer (JP Selecta, Spain). The results were expressed in mg/l of extract as gallic acid equivalents. High-performance liquid chromatography with photodiode array detection. Individual polyphenols were determined on a Varian HPLC system (Varian Inc., USA); ProStar 230 solvent delivery module, ProStar 330 PDA detector, OmniSphere C18 column (250 × 4.6 mm, 5 µm), guard column (ChromSep 1 cm × 3 mm). Mobile phases were 0.1% phosphoric acid in water (A) and 100% methanol (B). The gradient was 5% B (0 min), 25% B (0–5 min), 34% B (5–14 min), 37% B (14–25 min), 40% B (25–30 min), 49% B (30–34 min), 50% B (34–35 min), 51% B (35– 58 min), 55% B (58–60 min), 80% B (60–62 min), maintained at 80% B (62–65 min), down to 5% B (65–67 min) and maintained at 5% B (67–72 min). The flow rate was 0.8 ml/min; injection volume 20 µl; spectra 190–600 nm. The limits of detection and quantification are presented in the tables. Polyphenols were identified by comparison of the 477 Czech J. Food Sci., 35, 2017 (6): 476–482 Food Analysis, Food Quality and Nutrition doi: 10.17221/75/2017-CJFS retention times and spectral data with the those of standards. Furthermore, p-coumaroylquinic acid, quercetin-xyloside, and phloretin-2'-xyloglucoside were tentatively identified (Tsao et al. 2003) and quantified using p-coumaric acid, quercetin and phloretin calibration curves, respectively. The results were expressed in mg/kg of the fresh weight (FW). Adsorption experiment. The β-glucan was dissolved (190 mg/l) in distilled water. Total polyphenols in extracts (initial polyphenol concentration) were determined using the Folin-Ciocalteu method. For the adsorption study, four different volumes of polyphenol extract (10, 200, 500, and 700 µl), β-glucan (53 µl) as an adsorbent, and a phosphate buffer (0.13 mol/l, pH 5.5) were combined in plastic cuvettes (total volume was 2 ml). Solutions were mixed in a laboratory shaker (IKA KS 130; IKA Werke, Germany; 16 h, room temperature) and filtered through 0.1-µm cellulose nitrate membranes (Whatman, GE Healthcare, Germany). Unadsorbed polyphenols (polyphenol concentration at equilibrium – ce) were determined with the Folin-Ciocalteu method. The adsorption capacity (mg of adsorbed polyphenols per g of β-glucan) was calculated (q e): qe = (c0 – ce) Vrs cβ-glucan × Vβ-glucan (1) where: c0 – initial polyphenol concentration in the reaction solution (mg/l); ce – equilibrium polyphenol concentration in the reaction solution (mg/l); Vrs – volume of reaction solution (1); cβ-glucan – concentration of β-glucan (g/l); Vβ-glucan – volume of β-glucan in the reaction solution (1) Freundlich (2) and Langmuir models (3) were constructed: qe = kFce1/n qe = qm ce 1/kL + ce (2) (3) where: ce – polyphenol concentration in the solution at equilibrium (mg/l); qe – amount of polyphenol adsorbed per g of β-glucan at equilibrium (mg/g); k F – Freundlich constant indicative of relative adsorption capacity of β-glucan (mg/g) × (mg/l)–1/n; 1/n – intensity of adsorption; kL – Langmuir equilibration constant of adsorption (l/mg) or apparent affinity constant; qm – apparent maximum adsorption capacity of β-glucan (mg/g) (Soto et al. 2011) The data (qe vs. ce) were fitted with nonlinear models in such a way that the sum of square differences is minimal, and adsorption parameters were determined 478 (k F and 1/n from the Freundlich isotherm, k L and q m from the Langmuir isotherm). Statistical analyses. For the adsorption experiments, total polyphenols were measured at four concentration levels two times each. Nonlinear regression was performed (Minitab, USA) on qe and ce means by minimising the sum of square errors. The root-mean-square error (RMSE) of nonlinear least squares regression was calculated: √ RMSE = 1/n∑ni=1 (qe.i – f(ce,i , a, b))2 (4) where: ce,i – ce mean values for the ith concentration level; qe,i – qe mean values for the ith concentration level; f (ce,i , a, b) – nonlinear model function with generic parameters a and b; n = 4 is number of concentration levels Two extracts from each flesh and peel were prepared for individual polyphenol characterisation, each was analysed once using HPLC-PDA (n = 2). Means and coefficients of variation were calculated. RESULTS AND DISCUSSION Adsorption. The adsorption of apple polyphenols onto β-glucan was described with Freundlich and Langmuir isotherms, in an approach which can be compared to that of earlier studies (Wu et al. 2011; Gao et al. 2012). Polyphenols from the flesh of the two types of apple showed similar behaviours as evidenced by their similar curve shapes (Figures 1A and C). The curve shapes for peel polyphenols differed between the two typed of apple (Figures 1B and D), suggesting different behaviour of peel polyphenols. Moreover, polyphenols from the wild apple peel adsorbed to a greater extent (larger q e) than polyphenols from Slavonska srčika peel. Adsorption parameters. Table 1 displays the RMSE of each model and the parameters of the Langmuir and Freundlich isotherms. Both isotherms could be equally applied for the description of the flesh polyphenol adsorption (errors were similar). Both isotherms could also be used for the peel polyphenol adsorption, but the Langmuir model was somewhat better for the wild apple peel (smaller RMSE), and the Freundlich for the Slavonska srčika peel (smaller RMSE). According to the k F value (Table 1), the relative adsorption capacity of β-glucan was similar for flesh polyphenols, and different for peel polyphenols from the two apple types (somewhat higher for the Slavonska srčika peel and lower for the wild apple Czech J. Food Sci., 35, 2017 (6): 476–482 Food Analysis, Food Quality and Nutrition doi: 10.17221/75/2017-CJFS (A) 5000 qe (mg/g) 4000 qe (mg/g) (B) 8000 3000 2000 6000 4000 Wild apple 1000 2000 Slavonska srčika 0 0 0 5000 10 20 (C) 30 40 c (mg/l) 50 60 0 70 50 100 150 200 250 300 50 100 150 200 ce (mg/l) 250 300 (D) 8000 qe (mg/g) qe (mg/g) 4000 3000 2000 6000 4000 2000 1000 0 0 10 20 30 40 ce (mg/l) 50 60 0 70 0 Figure 1. The adsorption isotherms representing the adsorption of apple polyphenols onto β-glucan (28°C, 16 h, nonlinear models): Freundlich isotherms of flesh polyphenols (A), Freundlich isotherms of peel polyphenols (B), Langmuir isotherm of flesh polyphenols (C), and Langmuir isotherm of peel polyphenols (D) qe – (polyphenols mg/g β-glucan) as a function of ce (polyphenols mg/l) peel polyphenols). According to the estimated q m values of maximum adsorption, β-glucan may have the capacity to adsorb more peel polyphenols onto its surface than flesh polyphenols (qm value 11 949 and 7254 mg/g for peel; 3927 and 3114 mg/g for flesh). The adsorption intensity was shown to be similar for peel and flesh polyphenols (1/n was similar) except for wild apple peel polyphenols which showed much higher adsorption capacity. The kL values showed that the apparent affinity of polyphenols for β-glucan was higher for the flesh polyphenols (0.29 and 0.14 l/mg) than for peel polyphenols (0.036 and 0.023 l/mg). In general, differences between polyphenols from the flesh and peel could be seen. Polyphenol composition in apples. Figure 2 shows the polyphenols identified in apples, Table 2 their levels and Figure 3 the percentages of polyphenolic subgroups. The identification and levels of different polyphenols are in accordance with earlier studies (Tsao et al. 2003; Jakobek et al. 2013). Differences between samples were found – higher flavonol content and proportion in the peel, and a much higher phenolic acid proportion in the flesh. Furthermore, the two flesh samples differed in their polyphenol content (higher in wild apple). There was a higher total polyphenol content in the Slavonska srčika peel compared to that of wild apple, while the former also contained phenolic acids in contrast to the latter. Table 1. Parameters of Freundlich and Langmuir isotherms obtained with nonlinear models Apple Freundlich isotherm Langmuir isotherm kF 1/n RMSE Wild apple flesh 1230.5 0.26 1059.6 0.137 3926.8 1066.7 Slavonska srčika flesh 1325.1 0.23 626.8 0.292 3114.4 636.5 Wild apple peel Slavonska srčika peel kL qm RMSE 635.6 0.57 590.5 0.023 11 949.0 375.0 1510.5 0.27 421.4 0.036 7254.0 639.9 kF – indicative constant of the relative adsorption capacity of β-glucan (mg/g)(mg/l)–1/n; 1/n – intensity of adsorption; kL – Langmuir equilibration constant of adsorption (l/mg), apparent affinity constant; qm – apparent maximum adsorption capacity of β-glucan (mg polyphenols/g β-glucan); RMSE – root mean square error 479 Czech J. Food Sci., 35, 2017 (6): 476–482 Food Analysis, Food Quality and Nutrition doi: 10.17221/75/2017-CJFS (A) (B) Figure 2. HPLC-PDA chromatogram of wild apple flesh (A) and Slavonska srčika peel (B) 1 – procyanidin B1; 2 – (+)-catechin; 3 – procyanidin B2; 4 – chlorogenic acid; 5 – (-)-epicatechin; 6 – p-coumaroylquinic acid; 7 – phloretin-2’-xyloglucoside; 8 – quercetin-3-galactoside; 9 – quercetin-3-glucoside; 10 – quercetin derivative 1; 11 – quercetin derivative 2; 12 – phloretin-2’-glucoside; 13 – quercetin-3-xyloside; 14 – quercetin-3-rhamnoside; 15 – quercetin Apple polyphenol – β-glucan adsorption. The contrasting behaviour of polyphenols from flesh and peel in terms of adsorption could be explained by their different polyphenol compositions, manifested as higher flavonol content in peel samples, higher phenolic acid portion in flesh samples and the pres- Table 2. The content of polyphenols in the flesh and peel of old apple varieties (mg/kg of fresh weight) Slavonska srčika Flavan-3-ols Procyanidin B1 (+)-Catechin Procyanidin B2 (–)-Epicatechin Total Phenolic acids Chlorogenic acid p-Coumaroylquinic acida total Flavonols Quercetin-3-galactoside Quercetin-3-glucoside Quercetin derivative 1 Quercetin derivative 2 Quercetin-3-xylosidea Quercetin-3-rhamnoside Quercetin Total Dihydrochalcones Phloretin-2’-xyloglucosidea Phloretin-2’-glucoside Total Total Wild flesh peel flesh peel 12.6 9.2 20.5 Nd 42.3 23.3 248.4 135.2 253.7 660.6 31.3 277.7 84.9 196.5 590.4 22.8 86.5 36.8 114.0 260.1 338.3 10.6 348.9 438.4 nd 438.4 855.0 17.8 872.8 nd nd nd nd 1.5 0.4 nd 0.3 2.1 nd 4.3 728.1 1182.1 164.7 22.7 224.5 404.2 21.3 2747.6 49.9 17.5 1.2 nd 4.9 nd nd 73.5 152.4 337.3 61.0 13.0 68.3 52.7 13.6 698.3 26.0 25.2 51.2 446.7 nd 207.1 207.1 4053.7 24.4 22.1 46.5 1583.2 nd 19.3 19.3 977.7 nd – not detected; LOD and LOQ were: (+)-catechin – 0.2 and 0.7; (–)-epicatechin – 0.3 and 1; procyanidin B1 – 0.3 and 0.9; procyanidin B2 – 1.2 and 3.9; p-coumaric acid – 0.1 and 0.3; chlorogenic acid – 0.14 and 0.4; phloretin – 0.15 and 0.5; phloretin-2’glucoside – 0.13 and 0.43; quercetin – 0.03 and 0.1; quercetin-3-rhamnoside – 0.3 and 1; quercetin-3-galactoside – 0.6 and 2.0; quercetin-3-glucoside – 0.08 and 0.3; *data based on two extracts, each measured once (n = 2); variation coefficient range 1–25% for flesh and 1 – 28% for peel; atentatively identified 480 2 Czech J. Food Sci., 35, 2017 (6): 476–482 Food Analysis, Food Quality and Nutrition doi: 10.17221/75/2017-CJFS flavan-3-ols phenolic acids flavonols dihydrochalcones 100 80 60 40 tivity. If apple polyphenols create associations with dietary fibre, there is a possibility that they can reach the colon which might influence their bioaccessibility, bioavailability and different beneficial activities in the lower parts of the digestive tract. Since apples are present in the everyday diet understanding their actual bioactivity is important. 20 0 CONCLUSIONS Slavonska srčika flesh Wild apple flesh Slavonska srčika peel Wild apple peel Figure 3. Percentage distribution of polyphenolic subgroups in apples ence of phenolic acids in Slavonska srčika peel but not in wild apple. This would be in agreement with earlier studies where it was shown that individual apple polyphenols have different affinities toward resin (Kammerer et al. 2007), that different tea polyphenols have different affinities for β-glucan (Gao et al. 2012) and procyanidins with different degrees of polyperisation towards polysaccharides (Le Bourvellec et al. 2005). It has been shown that flavonols adsorbed with higher adsorption capacity than phenolic acids onto β-glucan (Wang et al. 2013) and resins (Kammerer et al. 2007). In our study, peel polyphenols had higher flavonol content and showed higher maximum adsorption capacity (qm). Thus, in accordance with an earlier study (Wang et al. 2013), it appears that flavonols from the peel were adsorbed in higher amounts. Phenolic acids, on the other hand, predominated in flesh which might be the reason for the lower adsorption capacity of flesh polyphenols, in accordance with earlier studies (Kammerer et al. 2007; Wang et al. 2013). Moreover, Slavonska srčika peel contained phenolic acids, which might be the reason for its lower adsorption capacity in comparison to wild apple peel. The bonds created between polyphenols and β-glucan have been described to be non-covalent in nature, i.e., hydrogen bonds, Van der Waals forces and hydrophobic bonding (Wu et al. 2011; Veverka et al. 2014; Nguela et al. 2016). H-bonds and Van der Waals forces might be created between OH groups of polyphenols and β-glucan (Wu et al. 2011). Hydrophobic bonding is possible due to hydrophobic aromatic rings on polyphenols. The same type of bonding could be responsible for the adsorption in this study. Interactions of apple polyphenols with β-glucan (dietary fibres) might be important for apple bioac- We have here reported that polyphenols from the flesh and peel of apples adsorb onto the surface of β-glucan and that the adsorption could be described with Freundlich and Langmuir models. 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