Juices, Fibres and Skin Waste Extracts from White ... - Springer Link

Food Bioprocess Technol (2013) 6:377–390 DOI 10.1007/s11947-011-0692-6

ORIGINAL PAPER

Juices, Fibres and Skin Waste Extracts from White, Pink or Red-Fleshed Apple Genotypes as Potential Food Ingredients A Comparative Study Dongxiao Sun-Waterhouse & Cecile Luberriaga & David Jin & Reginald Wibisono & Sandhya S. Wadhwa & Geoffrey Ivan Neil Waterhouse

Received: 4 June 2011 / Accepted: 13 September 2011 / Published online: 28 September 2011 # Springer Science+Business Media, LLC 2011

Abstract This study measures and compares the bioactive content and appearance attributes of juices, dietary fibres (DFs) and skin wastes of three apple genotypes (white fleshed (WF), pink fleshed (PF) and red fleshed (RF)). The juices of the PF and RF apples had more appealing and stable colours and much greater total extractable polyphenol content (TEPC) (RF had the highest, 3.40 mg catechin equivalent/mL juice) and vitamin C (PF had the highest, 14.2 mg/100 mL juice), compared with the WF apple. DFs isolated from the three apples using aqueous and ethanolic methods varied in bioactive profiles as a function of genotype. The TEPC and antioxidant activity (AA) of the fibres decreased in the order of PF > RF > WF. The total DF (TDF) in the fibre obtained using the aqueous method decreased in the order of RF>PF>WF. The ethanolic method yielded higher neutral monosaccharide (NM) and slightly greater TDF contents than the aqueous method. More polyphenol species were detected in the PF fibres, D. Sun-Waterhouse (*) : C. Luberriaga : D. Jin : R. Wibisono : S. S. Wadhwa The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland 1142, New Zealand e-mail: [email protected] C. Luberriaga Graduate School of Chemistry, Biology and Physics, Bordeaux Institute of Technology, Bordeaux, France G. I. N. Waterhouse School of Chemical Sciences, The University of Auckland, Auckland, New Zealand

especially those obtained using the aqueous method. The polyphenol content in the apple skin decreased in the order of RF > WF > PF, with PF having slightly more pectic polysaccharides. As a whole, the RF apple appeared to be the best genotype as the potential source for juice, fibre and skin waste extract (SWE) ingredients. The PF apple would be the second best genotype for juice and fibre ingredients. The skin of the RF and WF genotypes would provide a good source of polyphenols. There is potential for promoting RF and PF apple genotypes because of their excellent nutritional values. The aqueous fibre preparation method used herein containing no solvent treatment and freezing steps represents an industrial-scale cost-effective alternative to the conventional ethanolic methods used for producing DFs whilst retaining polyphenols. Keywords Apple genotypes . Flesh colour . Industryrelevant fibre preparation methods . Natural dietary fibres . Novel fruit juice . Polyphenols . Skin waste utilisation

Introduction Apples are one of the most widely consumed fruits, due in part to their wide-ranging positive health benefits. A high intake of apples has been shown to prevent a variety of chronic diseases and reduce the risk of lung cancer, asthma, type-2 diabetes, thrombotic stroke, and ischaemic heart disease (Boyer and Liu 2004; Okoko et al. 2007; Hansen et al. 2009; Chai et al. 2011). These health benefits are associated with the large amount of structural cell walls and fibre polysaccharides of apple (Sun-Waterhouse et al.

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2008a, b), as well as various intrinsic phytochemical antioxidants (Lee et al. 2003; McGhie et al. 2005; Devic et al. 2010). Dietary fibres (DFs) have beneficial physiological effects on regulation of energy intake, satiety and digestive health, as well as reduction in the incidence of cancer, heart disease, obesity and diabetes (Schulze et al. 2004; Slavin and Green 2007; Scott et al. 2008). Globally, the DFs are defined (the Codex Alimentarius, ALINORM 09/32/26) as carbohydrate polymers with 10 or more monomeric units that are not hydrolysed by endogenous enzymes in the small intestine of humans. Plant cell walls, a major source of DF, contain cellulose and non-cellulosic polysaccharides such as pectic polysaccharides, xyloglucans, glucomannans and galactoglucomannans (Fry 1988). The fruit cell wall consists of balanced soluble and insoluble fibre fractions that complement each other in terms of physical properties and physiological effects (Schneeman 1987; Bingham et al. 2003). The composition of apple fibre varies with genotype (Percy et al. 1997; Watt et al. 2000; Sun-Waterhouse et al. 2008a, b). The structure and composition of a DF preparation depend largely on their material origin and also the extraction method used (Fry 1988; Sun-Waterhouse et al. 2008a, b). Different fibre preparation procedures result in variations in fibre composition, microstructure and food processing functionality, due to the complex nature of tissues and occurrence of polysaccharide degradation (Fry 1988). The polyphenol (PP) content of apples also varies with genotype (Lee et al. 2003; Tsao et al. 2003; McGhie et al. 2005) and includes flavonols (quercetin glycosides), cinnamic acids (chlorogenic and caffeic acids), flavanols (catechin and epicatechin), procyanidins, dihydrochalcones (phloridzin) and anthocyanins (cyanidin glycosides). Apple flesh is normally white, but some genotypes have red or pink flesh due to the presence of anthocyanin pigments (Mazza and Velioglu 1992; Volz et al. 2009). Accordingly, apple juices can have a range of natural colours depending on the genotype from which they are derived, and this offers additional advantages other than fresh dessert use for the development of novel beverages. Due to the large quantity of health-promoting components in apples and the growing consumer demand for healthy and natural foods, apples are also suitable raw materials for producing functional fibre ingredients on an industrial scale. This study evaluates the PP and/or DF compositions of the juices, fibre and skin waste extract (SWE) ingredients generated from three apple genotypes with white, pink or red flesh. The main objective is to demonstrate which genotype is preferable in terms of its PP, DFs, and juice stability and attractiveness for the development of functional foods or ingredients and thus should be selected for commercial-scale crop production.

Food Bioprocess Technol (2013) 6:377–390

Materials and Methods Materials and Chemicals Three selections of pink-fleshed (PF), red-fleshed (RF) and white-fleshed (WF) apples from Plant & Food Research’s breeding programme were harvested between March and April 2010 in Hawke’s Bay, New Zealand, and delivered to Plant & Food Research in Auckland, New Zealand the morning following picking. The apples were stored at 2 °C prior to juice, fibre and SWE preparation. For each apple genotype, 15 fruits were selected and divided into three replicate groups of five apples. Food-grade ethanol (96%, SEA ethanol 220 L, produced by Anchor Ethanol Ltd, Tirau, New Zealand) was purchased from PolyChem Marketing, Auckland, New Zealand. The total dietary fibre test kit (including alpha amylase, protease and amyloglucosidase enzymes) was obtained from Megazyme International, Wicklow, Ireland. Sodium carbonate was purchased from Merck (Darmstadt, Germany). Sulfamic acid and sulphuric acid were from Ajax Chemical Ltd, Sydney. Celite™ was from Maville Service Corporation, USA. All the other chemicals including catechin, epicatechin, phloridzin, p-coumaric acid, phloretin, quercetin, ferulic acid, D-galacturonic acid, m-hydroxydiphenyl and Folin-Ciocalteu phenol reagent were purchased from Sigma-Aldrich (St Louis, MO, USA). Milli-QPLUS water was used for all reagent preparation. All the other solvents were purchased from Ajax Finechem Pty Ltd (Auckland, New Zealand). Apple Juice Preparation and Analyses Measurements of the Total Soluble Solid, pH and UV–vis Absorption of Juice Apple juices were generated from each replicate group of three apple genotypes using a Juice Fountain juicer (Model BJE 200 C; Breville, Sydney, New South Wales, Australia). Approximately 108, 93 or 117 mL juice was generated from each PF, RF or WF apple, respectively. A portion of freshly prepared juice was subjected to total soluble solid content (TSSC) and pH measurements, and the remaining portion was stored at −20 °C until further analysis. The TSSC (°Brix) was measured in triplicate using a handheld refractometer (Pocket Pal-1, Atago, Tokyo, Japan) at 20 °C. The pH was determined in triplicate using a pH meter (CG837, Schott Instruments, Germany) equipped with a glass electrode (850, Schott Instruments, Mainz, Germany). The UV–vis absorption characteristics of the apple juices were examined. The frozen juices were thawed, stirred and transferred to 50-mL polypropylene Falcon tubes and centrifuged at 3000 rpm (rcf 1400) for 10 min in an Eppendorf 5702 centrifuge (Eppendorf AG, Hamburg,


Germany) to separate the juice and insoluble apple solids (typically around 2–4 mg/mL juice). UV–vis spectra of the obtained clear juices were recorded from 300 to 900 nm using a Shimadzu Pharmaspec UV-1700 UV–vis spectrophotometer (Scientific Instruments, Portland, Oregon, USA) using disposable plastic cuvettes (Labserv). For the RF sample, the juice was diluted 2-fold with Milli-Q water to obtain an acceptable absorbance reading.

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using the titration method of the Association of Official Analytical Chemists (AOAC 1990). L-Ascorbic acid was extracted from 0.2-mL juice sample using 6.8 mL of extraction solution and then titrated using standardized dichlorophenol indophenol. Apple dietary Fibre Preparation and Analyses Apple Dietary Fibre Preparation

Polyphenol (PP) Analyses of Apple Juices A Solid Phase Extraction (SPE) column was used to pre-treat the apple juices (3 mL per juice sample) prior to the TEPC analysis to eliminate the potential interference of sugar and ascorbic acid with the Folin-Ciocalteu assay. The first eluate from SPE was used for the analysis of vitamin C (see “Determination of L-ascorbic Acid in Apple Juice”). The subsequent eluates resulting from each flush with 95% methanol (in an aliquot of 5 mL) were collected for TEPC, AA and HPLC analysis. The eluates resulting from SPE were subjected to a series of dilutions (5×, 10×, 20× and 50×). The TEPC of each diluted eluate (200 μL per test sample) from each replicate group of three apple genotypes was analysed (in duplicate) following the method of Singleton et al. (1997) using a microplate reader (SpectraMax Plus 384, Molecular Devices, Sunnyvale, USA) and expressed as milligrams catechin equivalent (CtE)/ milliliter juice. The AA of the each diluted eluate (10 μL per test sample) was determined (in duplicate) using the ferric reducing antioxidant power (FRAP) assay (Benzie et al. 1999) and expressed as milligrams Trolox equivalent/milliliter juice. Individual PPs of the resultant eluates (120 μL per test sample) were analysed in duplicate by HPLC following the method of Stevenson et al. (2006). The HPLC analysis was run (at 280 and 530 nm) using a Shimadzu analytical HPLC with a column oven (C40-10ASVP), auto-sampler (SIL10AF), vacuum solvent degas module and diode-array detector (SPD-M10AVP), fitted with a Synergi® Polar-RP ether-linked column (250×4.6 mm, 4-μm particle size, 80-Å ether-linked column, Phenomenex, Auckland, New Zealand). The mobile phases (A) acetonitrile+0.1% formic acid and (B) acetonitrile/water/formic acid (5:92:3) were pumped at 1.5 mL/min at 45 °C. The injection volume was 40 μL. Individual PPs were identified based on their retention time and absorbance maximum (λmax). External standards, catechin, epicatechin, quercetin, caffeic acid, protocatechuic acid, ferulic acid, p-coumaric acid, chlorogenic acid, phloridzin and rutin were used to assist with PP identification. Determination of L-ascorbic Acid in Apple Juice The vitamin C content in the juice from each replicate group of three apple genotypes was analysed (in duplicate)

Natural apple DF was prepared by modifying the aqueous or ethanolic method of Sun-Waterhouse et al. (2008a). Apples from each replicate group of three apple genotypes were peeled and cored to remove the carpel, seed, pith tissue, skin and epidermal layer and then cut into cubes (~1 cm3). The apple skin was collected, stored in a freezer (−80±3 °C, Thermo Electron Corp., Revco, Cambridge, UK) and freeze dried (Telstar Cryodos-80 Freeze Drier, Telstar Industrial, SL, Terrassa, Spain). Approximately 3.6, 3.4 or 3.9 g dried skin was generated from each PF, RF or WF apple, respectively. The apple cubes were soaked in cold water (4 °C) until 400 g of cubes had been prepared. The apple cubes were then removed from the cold water and divided into two equal portions (~200 g each portion). One-half was further processed using an aqueous method and the other using an ethanolic method to obtain fibre ingredients. The apple cubes (~200 g) were put in a bath (aqueous method: boiled water cooled to 50 °C; ethanolic method: 96%v/v food-grade ethanol) for 1 min, removed from the bath and ground into coarse apple particles using a coffee grinder (CG 2B, Breville, NSW, Australia). The coarse apple particles were placed onto a 53-μm nylon mesh which was seated on top of a sieve (100-μm nylon mesh) and immersed briefly (1 min) in a bath (aqueous method: boiled water at 50 °C; ethanolic method: 96% ethanol). This step was repeated once further in another bath. The treated coarse apple particles were then homogenised at room temperature in 800 mL water (aqueous method) or in 800 mL ethanol (ethanolic method) using a mixer (L5T, Silverson Machines Inc., East Longmeadow, MA, USA; emulsifying screen, 6600 rpm, 1-min ×2 bursts). The obtained homogenate was then filtered through a 53-μm nylon mesh, and the fibre was collected. The obtained fibres were weighed and stored in a −80 °C freezer, and a portion was examined by optical microscopy. A portion of freeze-dried fibres was used for an informal taste evaluation, field emission gun scanning electron microscopy (FEGSEM) examination and chemical analyses. For the ethanolic method, approximately 2.4, 1.0 or 1.2 g dried fibre was generated from each PF, RF or WF apple, respectively. For the aqueous method, approximately 4.2, 2.5 or 2.8 g dried fibre was generated from each PF, RF or WF apple, respectively.

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Fibre Examinations by Optical and Field Emission Gun Scanning Electron Microscopy (FEGSEM) Frozen fibre was thawed and examined by optical microscopy using a Nikon Eclipse E600 microscope (Nikon Corporation, Chiyoda-ku, Tokyo, Japan) equipped with a Nikon Coolpix 995 3.34-megapixel camera (40×, Nikon corporation, Chiyoda-ku, Tokyo, Japan). Freeze-dried apple fibres prepared by the aqueous or ethanolic method were examined using a Philips XL-80 field emission gun scanning electron microscope (FEGSEM) (Eindhoven, Holland). Samples were Platinum-sputter coated (sputter coater, Oxford Instruments, England) for 5 min before analysis to minimise specimen charging under the electron beam. Chemical Analyses of Apple Fibres Uronic Acid (UA), Neutral Monosaccharide (NM), Total Dietary Fibre (TDF) Contents The UA and TDF contents of the fibre from each replicate group of three apple genotypes were determined (in duplicate) following the procedures of Sun-Waterhouse et al. (2010) derived from the basic method of Filisetti-Cozzi and Carpita (1991). The NM content of these fibres was analysed by gas chromatography (GC, HP 6890 series, injector: HP 7683 series, Hewlett Packard, USA), following the method of Albersheim et al. (1967). Rhamnose, fucose, arabinose, xylose, mannose, glucose and galactose were used as standards. A quantity of 5-mg, 1-g or 2-mg test sample was used for UA, TDF and NM analyses, respectively.


loss during measurement. A 1-mm measurement gap was set. The viscosity was determined at 37 °C (because this is the temperature of normal human oral temperature). The shear rates in the range of 30–60 s−1 were close to the effective shear rate range (40–50 s−1) in the mouth (Wood and Goff 1973). The viscosity of each sample (in duplicate) was measured (30 measurements, in rotational mode) over the shear rate range of 0.001 to 100 s−1. Data were acquired and analysed with Rheoplus V3.4 software (Anton Paar GMBH, Graz, Austria). Preparation and Analysis of SWE The freeze-dried apple skin from each replicate group of three apple genotypes was ground to a fine powder and subjected to extractions following the alkali treatment method of Sun-Waterhouse et al. (2009). The ground skin (5 g) was treated with sodium hydroxide (10 mL, 0.1 or 0.5 M) under a constant stream of nitrogen gas, subjected to pH adjustment with 1 M hydrogen chloride (to pH 3), extracted with 96%v/v food-grade ethanol (40 mL) in an orbital shaker (Kika Labortechnik KS250, Staufen, Germany) at 70 rpm for 1 h and 140 rpm for 1 h and centrifuged at 3000g (relative centrifugal force) for 5 min. The supernatants were then subjected to concentration using a Labconco CentriVap® concentrator (Model 78100–01, Ultra-Low Cold Trap, Kansas, USA) and freeze-drying (the obtained dried extract weighed from 1.6 to 2.1 g). A quantity of 30, 15, or 10 mg dried extract was used for TEPC, HPLC and UA analyses, respectively. Statistical Analysis

Polyphenol (PP) Analysis Extraction and analysis of PPs in fibre were performed following the method of SunWaterhouse et al. (2008a). Freeze-dried fibres (100 mg) were treated with 0.5 M NaOH, acidified to pH 3 with 1 M HCl, and then extracted using ethyl acetate (at the ratio of 1:1). Three batches of extractions were conducted. The obtained organic extract, ranging from 11 to 15 mg, was dried and re-dissolved in 25% methanol (2 mL) and used for TEPC determination (Singleton et al. 1997) and HPLC analysis (Stevenson et al. 2006). Viscosity of Fibre Suspensions The dried fibre from each replicate group of three apple genotypes was reconstituted in water with gentle stirring (at a final concentration of 2%w/w). The viscosity of the fibre suspensions was examined at 37 °C using a stresscontrolled rheometer (Anton Parr Physica MCR301, Anton Parr GmbH, Graz, Austria) equipped with a Peltier temperature control device and flat plate geometry (diameter 49.954 mm). A humidity chamber was used to prevent water

At least three observations per analysis were performed. Data were analysed using repeated-measures ANOVA (Minitab 15).

Results and Discussion Appearance and Attributes of the Juice and Fibre Preparations from the Apples The three apple genotypes have different skin and flesh colours (Fig. 1a): PF apple has a pink-yellow skin colour, pink flesh, red cortex and white core, RF apple has a dark red skin colour, mostly red flesh with some white near the core and a red core, and WF apple has a red skin colour and white flesh. Polyphenoloxidase (PPO)-catalysed oxidative browning occurs commonly when apple is sliced (Yemenivioğlu et al. 1997; Falguera et al. 2011). During processing, the PF and RF apples did not turn brown when sliced or peeled, and their juices did not turn brown during juicing, compared with the

Food Bioprocess Technol (2013) 6:377–390 Fig. 1 Digital photographs of the a fruits, b juices and c fibres prepared using an aqueous (H2O) or ethanolic method (EtOH) for the pink-fleshed (PF), red-fleshed (RF) and white-fleshed (WF) apple genotypes

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RF

PF

A

WF

B PF

RF

WF

C

WF genotype. These phenomena suggest the presence of inhibitor(s) for PPO-catalysed oxidative browning in the PF and RF apples. The pH values of the juices from the three genotypes were different (Table 1). The lower pH of the PF and RF juices explains why these juices were less susceptible to PPO-catalysed browning than WF (PPOs have an optimal pH range in 5.5–6). The PF and RF juices had a greater total soluble solid content (TSSC) than the WF juice. Different amounts of insoluble solids were found in the juices of PF (3.77 mg dry solids/mL juice), RF (2.08 mg dry solids/mL juice) and WF (3.96 mg dry solids/mL juice) apples. An informal and preliminary evaluation on the juices from the three apple genotypes (Fig. 1b) was conducted in terms of the intensity of fruit aroma and

colour, observed foam/precipitate, taste and mouthfeel. Results showed that PF juice had a pleasant pink colour, appeared more watery and least cloudy and tasted sugary sweet with detected sourness and minimal texture/mouthfeel. RF juice had a dark red colour, thick texture and good mouthfeel and tasted sweet with a very strong apple flavour. WF juice had a dark yellow brown colour, with a normal apple flavour, thick texture and a lot of foam. Juice clarity decreased in the order PF > RF > WF, while the trend of juice thickness was reversed. The supernatant of the PF juice was still pinkish red in colour, and its UV spectra showed an intense feature at ~515 nm (Fig. 2), characteristic of monomeric anthocyanin-glucosides. The supernatants of the RF and WF juices, especially the former, showed very intense absorption in the region of

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Table 1 Chemical analyses of the juice and skin extract samples from the pink-fleshed, red-fleshed and white-fleshed apples Sample

Analysis

Genotype Pink fleshed

Red fleshed

White fleshed

Juice

pH TSSC (°Brix) TEPC (mg CtE/mL juice) Vitamin C (mg/100 mL juice) AA (mg TroloxE/mL juice)

3.50±0.02a 14.8±0.0b 1.90±0.03b 14.2±0.02c 1.00±0.05b

4.10±0.01b 14.8±0.0b 3.40±0.09a 6.71±0.03b 6.71±0.03b

4.40±0.01c 14.0±0.1a 0.50±0.03c 3.89±0.03a 3.89±0.03a

Skin

TEPC (mg CtE/g dried extract) UA (GalA, %w/w)

1.79±0.10a 10.4±0.36b

4.04±0.07c 9.36±0.45a

2.83±0.10b 9.64±0.30a

Data are expressed as mean ± standard deviation. Different lowercase superscript letters (within the same row) indicate statistically significant differences at P RF juice (6.7 mg/100 mL) > WF juice (3.9 mg/100 mL). Vitamin C is a PP degradation or oxidation inhibitor which explains in part why the PF juice was the least susceptible juice to enzymatic browning (Liao and Seib 1988; Almeida and Nogueira 1995; Evans 1997; Soliva et al. 2001; Lamikanra 2002; Guerrero et al. 2005).

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Table 2 Chemical analyses of the fibres prepared using the aqueous or ethanolic method from the pink-fleshed, red-fleshed and white fleshed apples Analysis

TEPC (mg CtE/g dried fibre) AA (mg TroloxE/g dried fibre) TDF (%) UA (GalA, %)

Aqueous preparation method

Ethanolic preparation method

PF

RF

WF

PF

RF

WF

2.54±0.16b 3.17±0.19c 84.7±1.02b 13.3±0.08a

0.49±0.04c 0.47±0.01b 95.9±1.89d 16.8±0.12b

0.24±0.05b 0.10±0.00a 81.8±1.00a 32.6±0.09e

1.90±0.06d 3.60±0.13c 88.9±1.93c 19.9±0.10c

0.23±0.01b 0.51±0.02b 83.5±1.68b 24.1±0.09d

0.13±0.01a 0.09±0.01a 83.8±0.69b 36.8±0.39f

Data are expressed as mean ± standard deviation. Different lowercase superscript letters (within the same row) indicate statistically significant differences at P RF > WF) for both the aqueous (i.e. values 10.6:2.0:1) and ethanolic methods (14.6:1.8:1) respectively. Interestingly, the differences in the AA values between the fibres from the same genotype using the two different preparation methods were insignificant (P ethanolic). For the fibre made using the aqueous method, the TDF decreased in this order: RF > PF > WF. For the fibre made with the ethanolic method, the TDF content of

1

0 0.001

0.01

0.1

1

10

100

Shear rate (s-1)

Fig. 4 Viscosity as a function of shear rate fibres prepared from the pink-fleshed (PF), red-fleshed (RF) and white-fleshed (WF) apple genotypes using the aqueous or ethanolic method, and then reconstituted in water 2%w/w


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ethanolic methods. The PF fibre prepared using the aqueous or ethanolic method contained galactose 70.4 and 89.5, arabinose 43.7 and 54.1, xylose 26.7 and 35.7, glucose 26.3 and 33.5, rhamnose 8.6 and 11.5, mannose 6.4 and 8.7, and fucose 4.7 and 6.0 μg/mg dried fibre, respectively. The PF fibres prepared using the ethanolic method were found to have higher amounts of each NM, especially galactose, arabinose, xylose and glucose. The aqueous method may potentially cause hydrolysis of NM side chains from fibre polysaccharides. The monosaccharides identified were those typically found in apples (Fry 1988;

apple genotypes contained significant amounts (P