Orosensory contributing compounds in crowberry (Empetrum nigrum) press-byproducts

Food Chemistry 124 (2011) 1514–1524

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Orosensory contributing compounds in crowberry (Empetrum nigrum) press-byproducts Oskar Laaksonen a,*, Mari Sandell a,b, Riikka Järvinen a, Heikki Kallio a,c a

Department of Biochemistry and Food Chemistry, University of Turku, FI-20014 Turku, Finland Functional Foods Forum, University of Turku, FI-20014 Turku, Finland c The Kevo Subarctic Research Institute, University of Turku, FI-20014 Turku, Finland b

a r t i c l e

i n f o

Article history: Received 28 January 2010 Received in revised form 6 June 2010 Accepted 3 August 2010

Keywords: Astringency Bitterness Crowberry Ethanol extraction Phenolic compounds

a b s t r a c t Berries of crowberry (Empetrum nigrum) were fractionated by juice pressing, ethanol extraction, solvent evaporation and supercritical fluid extraction. Phenolic compounds, sugars and acids in the fractions were analysed by high-performance liquid chromatography and gas chromatography. The sensory characteristics of the fractions were studied by using generic descriptive analysis. Most of the sugars were located in the juice and this was perceived as the sweetest of the fractions. The majority of the phenolic compounds were anthocyanins, located in the press residue. Ethanol extracted nearly all the phenolic compounds from the press residue. The extracts were the most bitter and astringent of the fractions. Eight flavonol glycosides and two flavonol aglycones were discovered to contribute particularly to bitterness and astringency. After ethanol extraction, only fibres and seeds were left, and the supercritical fluid extraction removed only a small amount of compounds from this fraction, which did not have any impact on sensory properties. This study shows that crowberries are rich in different nutrients and some of them are contributing to orosensory properties. The sequential fractionation by ethanol ended up in products with substantial differences in their orosensory characteristics and nutrient composition. Stepwise fractionation empowers versatile and beneficial ways to exploit the berries in food industry. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Crowberry (Empetrum nigrum L.) is a small evergreen shrub with edible berries found commonly in the northern hemisphere, especially in Scandinavia, Russia and Canada. These berries are used in juices and jams, and often in mixtures with other berries; however the crowberry is not effectively exploited in commercial products. In Finland the crowberry is the third largest wild berry crop after lingonberry (Vaccinium vitis-idaea L.) and bilberry (Vaccinium myrtillus L.). Nordic berries contain many compounds known to contribute to human health; in particular they contain anthocyanins and other flavonoids that are known antioxidants with several health-giving properties (Boots, Haenen, & Bast, 2008; Erlund et al., 2008; Galvano et al., 2004). Berries are also a source of dietary fibre (Järvinen, Kaimainen, & Kallio, 2010; Kallio, Nieminen, Tuomasjukka, & Hakala, 2006; Plaami, Kumpulainen, & Tahvonen, 1992). Most of the phenolic compounds are located and concentrated in the skin fractions of the berries (Riihinen, Jaakola, Kärenlampi, & Hohtola, 2008; Sandell et al., 2009) that are often discarded in the food industry. * Corresponding author. Tel.: +358 2 333 6816; fax: +358 2 333 6860. E-mail addresses: osanla@utu.fi (O. Laaksonen), mari.sandell@utu.fi (M. Sandell), rileni@utu.fi (R. Järvinen), heikki.kallio@utu.fi (H. Kallio). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.08.005

Bitterness and astringency might be considered as unpleasant characteristics in foods, and are caused by many different chemical components, including phenolic compounds (Bajec & Pickering, 2008; Drewnovski & Gomez-Carneros, 2000). Some phenolic compounds are known to produce these sensory properties in wine (Hufnagel & Hofmann, 2008), tea (Scharbert, Holzmann, & Hofmann, 2004), berries (Sandell et al., 2009; Schwarz & Hofmann, 2007a), grapefruit and chocolate (Drewnovski & Gomez-Carneros, 2000). In many foods, such as wines and teas, astringency and bitterness are desirable to a certain extent, but in some cases they may be a limiting factor in increasing consumption. By knowing the chemical factors behind these sensory properties, foods could be made more easily accepted by consumers and utilised in the food industry. The aim of this study was to isolate and identify compounds contributing to the orosensory properties of northern crowberry (E. nigrum ssp. hermafroditum) juice and the skin-rich press residue. Especially, we were focused on the flavonols and other phenolic compounds as they might have a key role in the sensory profiles of fractions. The contents of sugars and organic fruit acids in the fractions were investigated also. Berries were fractionated using juice pressing, ethanol extraction and supercritical carbon dioxide extraction (SFE), as previously applied to blackcurrant (Sandell et al., 2009). Fractionation was conducted without enzymes or sol-

O. Laaksonen et al. / Food Chemistry 124 (2011) 1514–1524

vents other than ethanol and CO2 to make the process safe and relatively simple. 2. Materials and methods 2.1. Berries Crowberries were collected in Lapland, northern Finland in 2006. They were stored frozen at 20 °C in polyethylene bags for processing and analyses. 2.2. Chemicals Myricetin, quercetin and p-coumaric acid were obtained from Sigma (St. Louis, MO). Quercetin-3-O-rutinoside, quercetin-3-Ogalactoside, quercetin-3-O-glucoside, delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, cyanidin-3-O-galactoside and malvidin-3O-galactoside were obtained from Extrasynthese (Genay, France). Sorbitol and tartaric acid were obtained from Merck (Darmstadt, Germany). Acetonitrile, ethyl acetate, methanol, formic acid, potassium hydrochloride and hydrochloric acid were HPLC-grade or the highest purity available. Activated carbon-filtered tap water was used to prepare the samples for sensory analysis. The ethanol used in sample preparation was 96% ETAX A (Altia, Helsinki, Finland). The reference compounds for sensory analysis are described in Table 5. 2.3. Sample preparation for sensory analyses Fractionation of crowberries was implemented using the previously reported methods of Sandell et al. (2009) starting with coldpressing of the crushed berries to produce juice and a press residue (Residue I). Four consecutive ethanol extractions (from Extract-1 to Extract-4) of Residue I were carried out with aqueous ethanol (90%), removing ethanol with a rotary evaporator. Finally, supercritical CO2-extraction of the residue obtained from the EtOH-extractions (Residue II) produced Residue III. The extracts were dissolved in filtered water at a concentration of 25 g/l after ethanol removal. The filtrated ethanol extracts were used for chemical analyses. 2.4. Supercritical fluid extraction of Residue II Residue II was extracted with supercritical CO2 using a pilot scale manufacturing procedure by a batch process without crushing the seeds (Aromtech Ltd., Tornio, Finland) to produce Residue III. 2.5. Dry matter For the gravimetric dry matter measurement of the berries, juice and Residue I, the samples were kept at + 110 °C overnight before weighing. The weights of Extract-1 to Extract-4 were measured after evaporation of ethanol. 2.6. Total anthocyanins The intensity of colour of the samples was analysed by spectrophotometry as previously described for blackcurrant (Sandell et al., 2009). The total content of anthocyanins was quantified using a reference compound mixture prepared according to the proportions of cyanidin-3-O-galactoside and malvidin-3-O-galactoside (ratio 50/50). 2.7. HPLC-DAD analyses of anthocyanins Anthocyanins of the samples (berry, juice, Residue I, Residue II and Extract-1 to Extract-4) were isolated and analysed in duplicate

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according to the method previously used for blackcurrant (Sandell et al., 2009). The HPLC-DAD system used was a Shimadzu LC10AVP (Shimadzu, Kyoto, Japan) with an LC-10AT pump, a SIL10A auto-sampler and a SPD-M10AVP diode array detector linked to an SCL-M10AVP data handling station. Samples were separated on a 250  4.60 mm i.d., 5-lm Phenomenex Prodigy RP-18 ODS-3 column (Torrance, CA) with a 30  4.60 mm i.d., 5-lm Phenomenex Prodigy pre-column, and anthocyanins were detected at 520 nm. Quantitative analysis was carried out after identification of the compounds using cyanidin-3-O-galactoside as an external standard for all anthocyanins. For berries, juice, Residue I and Residue II the concentrations were expressed as mg/100 g of fresh weight, and for the EtOH-extracts as mg/100 ml of water-diluted extract. Concentrations were also expressed as fractions of 1 kg of berries. In addition, the total content of each anthocyanin in Residue I (CR in Table 3) was calculated mathematically using Origin 8 software (Originlab Corporation, Northampton, MA, USA), using the previously reported method (Sandell et al., 2009). 2.8. HPLC-DAD analyses of other phenolic compounds Flavonols, their aglycones, and hydroxycinnamic acid conjugates were isolated and analysed in duplicate with a modified method previously applied to blackcurrant (Sandell et al., 2009) using ethyl acetate extractions and acid hydrolysis for aglycones. Analyses were performed using the same HPLC-DAD apparatus as described above. Flavonols and their aglycones were detected at 360 nm and hydroxycinnamic acid conjugates at 320 nm. Quantitative analysis was carried out using quercetin galactoside as the external standard for flavonol glycosides. Quercetin was used as the external standard for flavonol aglycones after acid hydrolysis, and p-coumaric acid and ferulic acid for hydroxycinnamic acid derivatives. For berries, juice, Residue I and Residue II, the concentrations were expressed as mg/100 g of fresh weight, and for Extract-1 to Extract-4 in mg/100 ml of the water-diluted extract. Concentrations were also expressed as fractions of 1 kg of berries. In addition, the total content of each compound was calculated using Origin 8 similarly to that of anthocyanins. 2.9. Identification of phenolic compounds by uHPLC-MS HPLC conditions were as described above and the apparatus was an Acquity™ Ultra Performance LC (Waters, Milford, MA) interfaced to a Waters Quattro Premier quadruple mass spectrometer. ESI-MS analysis for anthocyanins was performed in positive ion mode using a capillary voltage of 3.5 kV, a cone voltage of 40 V and an extractor voltage of 3 V; and for flavonols, a capillary voltage of 5 kV, cone voltage of 20 V and an extractor voltage of 3 V. In both cases, the source temperature was 120 °C and the desolvation temperature was 300 °C. In the MS analysis (full scan), data were acquired over a mass range of m/z 250–700. The UV–Vis spectra, retention times, available reference compounds, mass spectra and published data (Anttonen & Karjalainen, 2006; Buchert et al., 2005; Kärppä, Kallio, Peltonen, & Linko, 1984; Lätti, Riihinen, & Kainulainen, 2008; Määttä, Kamal-Eldin, & Törrönen, 2001; Määttä, Kamal-Eldin, & Törrönen, 2003; Määttä-Riihinen, Kamal-Eldin, Mattila, GonzálesParamás, & Törrönen, 2004; Ogawa et al., 2008; Sandell et al., 2009; Wu, Gu, Prior, & McKay, 2004) were used for identification. 2.10. Analyses of sugars and fruit acids Sugars and acids were analysed in duplicate by gas chromatography as trimethylsilyl (TMS) derivatives of berry, juice, Residue I, and Extract-1 according to the method of Sandell et al. (2009). Compounds were identified according to Sandell et al. (2009) and Tiitinen, Hakala, and Kallio (2005).

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2.11. Sensory evaluation

3. Results and discussion

The general guidelines (ISO 8586-1&2, 1988) for selection, training and monitoring of assessors were used. Sensory descriptive profiling was applied using 12 voluntary panellists, of whom 8 were women and 4 were men (ages 21–57 years). The assessors were selected according to their willingness, good health (self-reported), and availability. The descriptors were generated following an ISO/DIS standard (ISO/DIS 11035, 1992) during four independent sessions. During the training sessions the descriptors were created and the assessors were familiarised with the usage of the attributes, the intensity scale, and the Compusense-five data collection software (version 4.6, Compusense, Guelph, Canada). The intensity of the attributes was rated on a line scale anchored from 0 (none) to 10 (very strong) with the help of the references (Table 5), and each assessor evaluated all the samples in triplicate during separate sessions. All attributes of a given sample were evaluated at a session in a non-randomized order. Sensory evaluations consisted of two parts with three parallel sample evaluation sessions. In the first part, juice, Residue I, Residue II, Residue III, and a combination of the four ethanol extracts (Combined Extract) were evaluated. The juice and the combined ethanol extract evaporated and dissolved in water were liquid and the other three samples were solid. In the second part, the samples comprised the four consecutive ethanol extracts and the Combined Extract again. To determine the effect of SFE technology, we used the triangle test (ISO 4120, 2004) to evaluate the difference between Residue II and Residue III (12 assessors, 3 replicates, n = 36). All sensory analyses were performed at the sensory laboratory in accordance with ISO 8589 standard (1988).

3.1. Fractionation of the berries

2.12. Statistical analyses Differences between samples were analysed by a one-way analysis of variance (ANOVA) together with Tukey’s t-test and the Tamhane test (p < 0.05). The results of Extract-1 to Extract-4 were analysed with the Friedman test (p < 0.05). To interpret the results for the nine sensory attributes, principal component analysis (PCA) was applied. To find the relations between the two data matrices, the partial least squares regression (PLS) method was applied for standardised data. The X-variables (predictors) were the chemical compounds and the Y-variables (responses) were the sensory properties. Cross-validation was used to estimate the number of principal components for a statistically reliable model. Statistical analyses were performed using SPSS 14.0 (SPSS Inc. H, Chicago, IL), SAS 6.11 (SAS Institute Inc., Cary, NC) and Unscrambler 9.8 (Camo Process AS, Oslo, Norway).

The berries were fractionated using cold-pressing, ethanol extraction, and evaporation of ethanol followed by dissolving in water and supercritical CO2-extraction. Table 1 shows the distribution of the fractions and the compounds analysed. The yield of Residue I in the first six juice pressings was 14 ± 3.3%. The four consecutive ethanol extractions removed roughly 40% of the dry weight of Residue I, leading to a total of 21 g of EtOH-solubles from 1 kg of berries. Supercritical CO2-extraction removed less than 1% of Residue II. The SFE-extract did not contain significant amounts of seed oil, because the seeds were not cut and the composition of the seed oil was not analysed. 3.2. Identification of phenolic compounds Phenolic compounds were first identified as flavonol glycosides, hydroxycinnamic acid derivatives or anthocyanins according to their UV–vis spectra and reference compounds from HPLC-DAD. The mass spectra of these compounds were further analysed with uHPLC-MS. Fig. 1 shows the HPLC-DAD chromatograms of Extract1; the chromatograms of other samples were quite similar. All anthocyanins identified are shown in Table 2 with their UV– Vis kmax, [M+1]+ ions and fragments. Fifteen anthocyanins were detected and identified from Extract-1 (Fig. 1A); these compounds consisted of galactosides, glucosides and arabinosides of delphinidin, cyanidin, petunidin, peonidin and malvidin according to their [M+1]+ ions and fragments. The anthocyanins identified in our samples were the same as previously reported in crowberry (Kärppä et al., 1984; Määttä-Riihinen et al., 2004; Ogawa et al., 2008). The major peaks were galactosides and arabinosides whilst minor peaks were glucosides. Cyanidin-3-O-galactoside (peak 3) eluted before cyanidin-3-O-glucoside (peak 5) and a similar pattern was found in other anthocyanidins and in the standard compounds. Peaks 4, 7, 10, 14 and 15 were identified as arabinoses according to a mass loss of m/z 132 and published data (Ogawa et al., 2008). Traces of free aglycones were also detected, which might be due to either the pre-treatment of the sample or their existence as natural compounds. In addition to anthocyanins, crowberries have been reported to contain mostly flavonols and hydroxycinnamic acid derivatives (Häkkinen et al., 1999; Kähkönen, Hopia, & Heinonen, 2001; Määttä-Riihinen et al., 2004). A total of 35 flavonol glycosides and hydroxycinnamic acid derivatives were identified in Extract1 (Fig. 1 and Table 2). The identified flavonol glycosides were galac-

Table 1 Distribution of the dry matter and phenolic compounds in whole crowberries and their fractionsa. Fraction

Fresh weight (g)

Dry matter (g)

Total anthocyanins (mg)

Anthocyanins HPLC (mg)

Flavonol glycosides HPLC (mg)

Flavonol aglycons HPLC (mg)

Berry

1000

150

4800

5500

210

180

Juice Residue I

850 150

58 77

1100 3700

470 3700

76 63

27 75

Extract-1 Extract-2 Extract-3 Extract-4 Residue II

13 8 3 2 45

2900 1100 210 120 150

3300 930 170 120 30

150 57 15 6 *

100 51 12 8 1

Residue III SFE-extract

45 0.4

– –

– –

– –

– –

a Juice and Residue I are the fractions of the whole berry. Extract-1 to Extract-4 and Residue II are the fractions of Residue I. Residue IV and SFE-extract are the fractions of Residue II. *below the detection limit S/N > 3, – indicates that analysis was not carried out.

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Fig. 1. HPLC-DAD chromatograms of anthocyanins (A, 520 nm), flavonol glycosides and hydroxycinnamic acid conjugates (B, 360 nm, hydroxycinnamic acid conjugates were quantified at 320 nm) and flavonols (C, 360 nm, hydroxycinnamic acid conjugates were quantified at 320 nm) in first ethanol extract (Extract-1) of crowberry press residue. For the numbers and abbreviations of peaks refer to Table 2.

tosides, glucosides, arabinosides and xylosides of myricetin (mass m/z 319), quercetin (m/z 303), laricitrin (m/z 333), isorhamnetin (m/z 317) and syringetin (m/z 347). According to reference com-

pounds and their retention times, peaks 28 and 30 were identified as quercetin-3-O-galactoside and quercetin-3-O-glucoside. Similarly, some of the peaks were identified as galactosides and gluco-

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O. Laaksonen et al. / Food Chemistry 124 (2011) 1514–1524 Table 2 Identification of phenolic compoundsa in crowberry. No.

Tentative identification

UPLC-MSb +

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 My Caff Qu La p-Co Sy Is

Delphinidin-3-O-galactoside Delphinidin-3-O-glucoside Cyanidin-3-O-galactoside Delphinidin-3-O-arabinoside Cyanidin-3-O-glucoside Petunidin-3-O-galactoside Cyanidin-3-O-arabinoside Petunidin-3-O-glucoside Peonidin-3-O-galactoside Petunidin-3-O-arabinoside Malvidin-3-O-galactoside Peonidin-3-O-glucoside Malvidin-3-O-glucoside Peonidin-3-O-arabinoside Malvidin-3-O-arabinoside Caffeic acid derivative p-Coumaric acid conjugate p-Coumaric acid conjugate Unknown flavonol glycoside Unknown flavonol glycoside Myricetin-3-O-galactoside Myricetin-3-O-glucoside Unknown quercetin glycoside Unknown quercetin glycoside Myricetin-3-O-arabinoside Myricetin-3-O-xyloside Laricitrin-3-O-galactoside Quercetin-3-O-galactoside Laricitrin-3-O-glucoside Quercetin-3-O-glucoside Quercetin-3-O-glucuronide Laricitrin-3-O-arabinoside Quercetin-3-O-arabinoside Syringetin-3-O-galactoside Isorhamnetin-3-O-galactoside Quercetin-3-O-xyloside Syringetin-3-O-glucoside Isorhamnetin-3-O-glucoside Laricitrin-3-O-xyloside Isorhamnetin-3-O-arabinoside Syringetin-3-O-arabinoside Syringetin-3-O-xyloside Isorhamnetin-3-O-xyloside Free myricetin Unknown myricetin glycoside Unknown laricitrin glycoside Unknown quercetin glycoside Free laricitrin Free quercetin Unknown syringetin glycoside Unknown isorhamnetin glycoside Free syringetin free isorhamnetin Myricetin aglycon Caffeic acid derivative Quercetin aglycon Laricitrin aglycon p-Coumaric acid Syringetin aglycon Isorhamnetin aglycon

HPLC-DAD

[M+H] (m/z)

Fragment ions (m/z)

465 465 449 435 449 479 419 479 463 449 493 463 493 433 463 – – – – – 481 481 533 533 451 451 495 465 495 465 479 465 435 509 479 435 509 479 465 449 479 479 449 319 585 599 569 333 303 613 583 347 317 319 – 303 333 – 347 317

303 303 287 303 287 317 287 317 301 317 331 301 331 301 331 – – – 441, 441, 319 319 303, 303, 319 319 333 303 333 303 303 333 303 347 317 303 347 317 333 317 347 347 317 – 319 333 303 – – 347 317 – – – – – – – – –

Ref.c   



593, 301, 303, 898 593, 303, 453

465 465

 





  

HPLCd kmax (nm) 524 524 517 524 518 526 518 518 525 529 – 530 518 530 325 310 310 342 340 356  346 344  352 – 354    356   356   354  356   370 358  356 – 368     372 325 372 – 310 372 373

a

Numbering of the peaks is used throughout the figures and other tables. Mass spectral comparison, positive ion mode. Retention time and UV–Vis spectrum compared to reference compound. d UV–vis spectra of the analytes. kmax (nm) is shown,  indicates identification as a flavonol compound according to UV–Vis spectra, – indicates UV–Vis spectra were not detected. b

c

sides of other flavonols. Arabinosides and xylosides of flavonols were differentiated according to the literature (Koponen et al., 2008), with arabinoside eluting before xylosides. In berries, laricitrin glycosides have previously been reported only in bilberry

(Koponen et al., 2008) and now also in crowberry. Laricitrin galactoside (peak 27) co-eluted with quercetin galactoside (peak 28), while other laricitrin glycosides eluted close to the corresponding quercetin glycosides as well. Free aglycones of the five flavonols

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O. Laaksonen et al. / Food Chemistry 124 (2011) 1514–1524 Table 3 Anthocyanins and other phenolic compounds in fractionsB and in sensory samplesC. No.A

Berry

Juice

Residue I

CRD

Extract-1

Extract-2

Extract-3

Extract-4

Residue II

83 ± 5.1 10 ± 1.0 88 ± 6.3 * 11 ± 1.1 44 ± 3.4 29 ± 2.6 42 ± 2.9 110 ± 6.6 * 21 ± 1.9 23 ± 1.6 3.2 ± 0.0 16.6 ± 0.3 3.7 ± 0.0

920 34 840 150 44 450 260 400 1100 41 130 190

640 ± 11 26 ± 2.2 620 ± 6.6 110 ± 7.6 32 ± 1.2 320 ± 4.9 190 ± 3.1 290 ± 4.5 820 ± 12 30 ± 0.7 140 ± 3.4 95 ± 1.8 2.2 ± 0.4 13 ± 2.8 3.3 ± 0.8 4.0 ± 0.5 9.4 ± 1.4 25 ± 7.2 6.1 ± 1.5 4.1 ± 0.7 6.8 ± 1.0 0.4 ± 0.1 8.0 ± 1.9 29 ± 7.0 9.2 ± 2.1 2.3 ± 0.5 9.0 ± 2.2 7.0 ± 1.8 5.4 ± 1.4 1.1 ± 0.3 1.1 ± 0.2 3.5 ± 0.9 3.9 ± 1.1 2.7 ± 0.3 0.9 3.3 ± 0.7 2.2 ± 0.5 0.8 1.2 38 ± 7.0 46 ± 7.0 11 ± 3.6 8.6 ± 3.0 21 5.1

200 ± 1.2 6.7 ± 0.9 170 ± 2.0 33 ± 0.0 8.8 ± 0.9 96 ± 1.8 51 ± 0.6 78 ± 1.2 210 ± 3.6 7.9 ± 0.9 25 ± 0.5 37 ± 0.7 1.7 ± 0.1 6.0 ± 0.5 2.0 ± 0.1 1.1 2.7 ± 0.1 12 ± 1.2 2.5 ± 0.1 1.1 ± 0.0 2.3 ± 0.1 * 3.1 ± 0.1 12 ± 0.6 4.7 ± 0.7 1.2 ± 0.1 4.2 ± 0.0 2.9 ± 0.6 1.9 ± 0.0 * * 1.5 ± 0.1 1.7 ± 0.0 * * 2.0 ± 0.5 2.2 * * 18 ± 3.0 25 ± 5.0 4.4 ± 0.7 3.9 ± 0.8 13 ± 2.7 2.6 ± 0.5

42 ± 1.9 1.1 ± 0.4 31 ± 1.2 6.6 ± 1.0 1.6 ± 0.0 19 ± 1.0 9.4 ± 0.5 14 ± 0.8 37 ± 1.9 1.6 ± 0.2 4.5 ± 0.1 6.5 ± 0.4 0.7 ± 0.2 1.9 ± 0.1 0.7 ± 0.1 0.5 1.4 ± 1.1 3.3 ± 0.2 0.8 ± 0.0 * 0.7 ± 0.3 * 0.8 ± 0.1 3.4 ± 0.2 1.3 ± 0.1 * 1.3 ± 0.0 0.8 ± 0.1 0.5 * * 0.4 0.5 * * 0.5 0.7 * * 4.2 ± 0.2 5.8 ± 0.1 1.1 ± 0.0 1.0 ± 0.1 3.8 ± 0.1 0.7 ± 0.1

37 ± 1.1 1.1 ± 0.0 21 ± 0.5 5.2 ± 0.3 1.0 ± 0.0 14 ± 0.4 6.1 ± 0.2 8.4 ± 0.3 22 ± 0.8 0.9 ± 0.1 2.6 ± 0.0 3.9 ± 0.2 * 0.9 ± 0.0 0.4 ± 0.0 * * 1.4 ± 0.0 0.4 ± 0.0 * * * 0.4 ± 0.0 1.5 ± 0.0 0.8 ± 0.0 * 0.7 ± 0.0 0.4 ± 0.0 * * * * * * * 0.5 ± 0.0 * * * 2.3 ± 0.6 4.2 ± 1.1 0.7 ± 0.2 0.6 ± 0.2 3.8 0.4

10 ± 0.9 0.7 ± 0.1 5.0 ± 0.5 * 0.6 ± 0.0 3.1 ± 0.2 1.6 ± 0.1 2.4 ± 0.2 4.2 ± 0.3 * 1.2 ± 0.1 1.4 ± 0.0 * * * * * * * * * * * * * * * * * * * * * * * * * * * 0.4 0.5 * * 3.0 *

120 ± 2.2b 4.9 ± 0.4c 120 ± 1.3b 21 ± 1.5a 6.2 ± 0.2c 62 ± 0.9b 37 ± 0.6b 57 ± 0.9b 160 ± 2.2b 5.8 ± 0.1 a 18 ± 0.3bc 27 ± 0.7b 0.4 ± 0.0c 2.4 ± 0.5c 0.6 ± 0.2bc 0.8 ± 0.1 1.8 ± 0.3a 4.8 ± 1.4b 1.2 ± 0.3bc 0.8 ± 0.1a 1.3 ± 0.2a 0.3 ± 0.0 1.5 ± 0.4b 5.7 ± 1.3b 1.8 ± 0.4bc 0.4 ± 0.1b 1.7 ± 0.4bc

63 ± 0.4b 2.1 ± 0.3d 52 ± 0.6b 10 ± 0.0b 2.8 ± 0.3d 30 ± 0.6bcd 16 ± 0.2bc 24 ± 0.4c 67 ± 1.1cd 2.5 ± 0.3 b 8.0 ± 0.2cd 12 ± 0.2c 0.5 ± 0.0c 1.9 ± 0.2cd 0.6 ± 0.0bc 0.3 0.8 ± 0.0ab 3.6 ± 0.4bcd 0.8 ± 0.0bcd 0.3 ± 0.0b 0.7 ± 0.0b * 1.0 ± 0.0cd 3.7 ± 0.2cd 1.5 ± 0.2bcd 0.4 ± 0.0b 1.3 ± 0.0cd

35 ± 1.6b 0.9 ± 0.3d 26 ± 1.0b 5.5 ± 0.8c 1.3 ± 0.0d 15 ± 0.8d 7.8 ± 0.4c 11 ± 0.6c 31 ± 1.6d 1.3 ± 0.2 c 3.8 ± 0.1d 5.4 ± 0.3c 0.6 ± 0.1c 1.5 ± 0.1d 0.6 ± 0.1c 0.4 1.2 ± 0.0ab 2.7 ± 0.4cd 0.7 ± 0.0bcd * 0.6 ± 0.0b * 0.7 ± 0.0c 2.8 ± 0.2c 1.1 ± 0.2cd * 1.1 ± 0.0d

46 ± 1.3b 1.3 ± 0.1d 26 ± 0.6b 6.5 ± 0.3c 1.3 ± 0.0d 17 ± 0.6cd 7.7 ± 0.2c 10 ± 0.3c 28 ± 0.9d 1.1 ± 0.1 c 3.3 ± 0.0d 4.9 ± 0.2c * 1.1 ± 0.0d 0.5 ± 0.0c * * 1.7 ± 0.0d 0.4 ± 0.0d * * * 0.5 ± 0.0c 1.9 ± 0.0c 1.0 ± 0.0d * 0.8 ± 0.0d

23 ± 2.1b 1.5 ± 0.2d 11 ± 1.0b * 1.3 ± 0.1d 6.8 ± 0.5d 3.6 ± 0.3c 5.4 ± 0.4c 9.2 ± 0.6d * 2.7 ± 0.1d 3.1 ± 0.1c * * * * * * * * * * * * * * *

Fractions (g/1 kg of berry) 1 1100 ± 290 2 97 ± 3.6 3 1100 ± 270 * 4 5 96 ± 4.4 6 550 ± 130 7+8 320 ± 66 9 + 10 490 ± 110 11 + 12 1200 ± 280 * 13 14 230 ± 37 15 250 ± 45 16 8.4 ± 0.2 17 40 ± 0.7 18 9.0 ± 0.1 * 19 20 4.0 ± 0.8 21 39 ± 2.3 22 13 ± 2.0 * 23 * 24 * 25 26 14 ± 0.5 27 + 28 51 ± 2.6 29 + 30 21 ± 0.9 32 + 33 6.3 ± 1.7 34 + 35 21 ± 1.1 36 + 37 8.8 ± 0.8 38 + 39 9.1 ± 0.7 * 40 * 41 42 + 43 6.1 ± 0.8 44 6.7 ± 0.2 45 4.1 ± 0.1 46 47 6.0 ± 0.4 48 + 49 3.3 * 52 * 53 My 60 ± 1.0 Qu + La 95 ± 0.5 Sy 18 ± 0.1 Is 11 ± 0.1 p-Co 94 ± 0.6 Caff 28 ± 0.0

16 ± 1.4 4.5 ± 0.4 * * * 5.3 ± 0.5 21 ± 0.0 8.3 ± 0.9 1.5 6.0 ± 0.1 * 3.8 ± 0.2 * * 2.1 ± 0.5 2.8 ± 0.7 * * 2.4 ± 0.2 * * * 11 ± 0.5 12 ± 0.6 2.1 ± 0.1 * 23 ± 1.2 8.2 ± 0.4

820 ± 120 47 ± 2.0 840 ± 130 * 45 ± 3.5 360 ± 37 220 ± 22 330 ± 17 830 ± 82 * 130 ± 12 140 ± 11 2.1 ± 0.0 8.3 ± 0.0 2.2 ± 0.0 * 2.3 ± 0.6 15 ± 0.0 4.7 ± 0.5 * 1.4 * 4.5 ± 0.2 17 ± 0.3 7.3 ± 0.3 1.7 ± 0.0 5.9 ± 0.2 3.9 ± 0.1 3.6 ± 0.1 * * 1.9 ± 0.0 2.1 ± 0.6 1.6 ± 0.0 * 2.0 ± 0.2 1.7 ± 0.6 * * 34 ± 14 46 ± 16 9.2 ± 3.0 7.1 ± 3.0 36 6.3

Sensory samples (mg/100 g) 1 110 ± 29b 2 9.7 ± 0.4b 3 110 ± 27b * 4 5 9.6 ± 0.4b 6 55 ± 13bc 7+8 33 ± 6.6b 9 + 10 49 ± 11b 11 + 12 120 ± 28bc * 13 14 23 ± 3.7b 15 26 ± 4.5b 16 0.8 ± 0.0b 17 4.0 ± 0.1b 18 0.9 ± 0.0b * 19 20 0.4 ± 0.1b 21 3.9 ± 0.2bc 22 1.3 ± 0.2b * 23 * 24 * 25 26 1.4 ± 0.0bc 27 + 28 5.1 ± 0.3bc 29 + 30 2.1 ± 0.1b 32 + 33 0.6 ± 0.2ab 34 + 35 2.1 ± 0.1b

10 ± 0.6b 1.2 ± 0.6d 11 ± 0.8b * 1.3 ± 0.1d 5.3 ± 0.4d 3.5 ± 0.3c 5.1 ± 0.4c 13 ± 0.8d * 2.6 ± 0.2d 2.8 ± 0.2c 0.4 ± 0.0c 2.0 ± 0.0cd 0.4 ± 0.0c * * 1.9 ± 0.2d 0.6 ± 0.1cd * * * 0.6 ± 0.1c 2.6 ± 0.0c 1.0 ± 0.1d 0.4b 0.7 ± 0.0d

550 ± 81a 31 ± 1.3a 560 ± 88a * 30 ± 1.3a 240 ± 25a 150 ± 14a 220 ± 11a 550 ± 55a * 86 ± 1.3a 91 ± 7.6a 1.4 ± 0.0a 5.5 ± 0.1a 1.5 ± 0.0a * 1.3 ± 0.3ab 8.5 ± 0.0a 2.6 ± 0.3a * 0.8b * 2.5 ± 0.1a 9.2 ± 0.2a 4.1 ± 0.2a 0.9 ± 0.0a 3.3 ± 0.1a

22 7

43 10

13 47 17 16 11

7

65 86 17 15 45 9

(continued on next page)

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Table 3 (continued) No.A 36 + 37 38 + 39 40 41 42 + 43 44 45 46 47 48 + 49 52 53 My Qu + La Sy Is p-Co Caff

Berry

Juice bc

0.9 ± 0.1 0.9 ± 0.1bc * * 0.6 ± 0.1b 0.7 ± 0.0ab 0.4 ± 0.0c * 0.6 ± 0.0b 0.3 * * 6.0 ± 0.1b 9.5 ± 0.0b 1.8 ± 0.0b 1.1 ± 0.0b 9.4 ± 0.1 2.8 ± 0.0

* 0.5 ± 0.0c * * 0.1 ± 0.1c 0.3 ± 0.1b * * 0.3 ± 0.0b * * * 1.4 ± 0.1bc 1.5 ± 0.1bc 0.3 ± 0.0b * 2.7 ± 0.1 1.0 ± 0.0

Residue I

CRD

a

2.2 ± 0.1 2.0 ± 0.1a * * 1.1 ± 0.0a 1.2 ± 0.4a 0.9 ± 0.0a * 1.1 ± 0.1a 0.9 * * 19 ± 8.2a 25 ± 9.0b 5.1 ± 2.0a 3.9 ± 1.5a 24 4.2

Extract-1

Extract-2

Extract-3

Extract-4

Residue II

1.4 ± 0.3b 1.0 ± 0.3b 0.2 ± 0.1 0.2 ± 0.0 0.7 ± 0.2b 0.8 ± 0.2ab 0.5 ± 0.1b 0.2 0.6 ± 0.1b 0.4 ± 0.1 0.2 0.2 7.4 ± 1.4ab 8.9 ± 1.3b 2.0 ± 0.7ab 1.7 ± 0.6ab 4.0 1.0

0.9 ± 0.2bc 0.6 ± 0.0bc * * 0.5 ± 0.0bc 0.5 ± 0.0b * * 0.6 ± 0.2b 0.7 * * 5.5 ± 1.0b 7.8 ± 1.6b 1.4 ± 0.2b 1.2 ± 0.2b 4.1 ± 0.8 0.8 ± 0.1

0.6 ± 0.2c 0.4 ± 0.0c * * 0.3 ± 0.0c 0.4 ± 0.0b * * 0.4 ± 0.2b 0.6 * * 3.5 ± 0.2b 4.9 ± 0.1b 0.9 ± 0.0b 0.9 ± 0.1b 3.2 ± 0.1 0.6 ± 0.1

0.4 ± 0.0c * * * * * * * 0.6b * * * 2.9 ± 0.7b 5.2 ± 1.3b 0.9 ± 0.3b 0.8 ± 0.2b 4.8 0.6

* * * * * * * * * * * * 0.9c 1.1c * * 6.6 *

A Peak numbers refer to Table 2, * = below the detection limit S/N > 3. Abbreviations indicate hydrolysed aglycones of myricetin (My), quercetin (Qu), laricitrin (La), syringetin (Sy), isorhamnetin (Is), p-coumaric acid (p-Co) and a caffeic acid derivative (Caf). B Contents in each original fraction (Table 1). C Extracts were diluted in water, 25 g/l. Significant differences between samples in each compound based on Tukey’s test (p < 0.05) are marked with superscripts a–d. D Calculated theoretical values of Residue I.

were detected at the end of the chromatogram (Fig. 1B), as well as in the analysis of aglycones after acid hydrolysis of the glycosides (Fig. 1C). Compounds 19, 20, 23 and 24 were not identified and their fragments are shown in Table 2. Peaks 23 and 24 were tentatively identified as quercetin glycosides due to the presence of similar fragments to quercetin galactoside and quercetin glucoside, but the peaks also contained an extra fragment (fragment m/z 68 from the mother ion m/z 533). These four unidentified peaks were most intense in Extract-1 to Extract-4 and were apparently released more efficiently with ethanol than ethyl acetate from Residue I. These compounds might also be formed in the ethanol extraction process. Five other unidentified glycosides were also formed, one for each aglycone, with a mass m/z 266 added to the aglycone (peaks 45–47 and 50 and 51). Peaks 17 and 18 were identified as conjugates of p-coumaric acid according to their UV–Vis spectra. Peak 16 was identified as a caffeic acid derivative. After acid hydrolysis, p-coumaric acid and the caffeic acid derivative were detected based on their UV– Vis spectra. In addition to flavonol glycosides and hydroxycinnamic acid conjugates, traces of catechins, epicatechins and their derivatives were detected from chromatograms, but these compounds were not further identified or quantified.

anins in crowberries are similar to their contents in bilberries and higher than in blackcurrants (Kähkönen et al., 2001; Määttä-Riihinen et al., 2004). In the case of sensory samples, the extracts were diluted in water (25 g/l), and the anthocyanins showed a decreasing trend from Extract-1 to Extract-4. Flavonol glycosides were clearly found in smaller amounts than anthocyanins in the fractions (Table 3). Flavonol glycosides as well as their aglycones showed a continuously decreasing trend according to the repeated extractions. Quercetin and myricetin galactosides were the major glycosides in each fraction as galactosides and xylosides were found at higher levels than glucosides and arabinosides. Four compounds (17, 18, 21 and 22) were found mainly in the extracts. 3.4. Sugars and acids The contents of sugars and organic fruit acids are given in Table 4. Sugars consisted mainly of glucose and fructose with minor amounts of sucrose. The total sugar content of the berry was 70 g/ kg and nearly all of it ended up in the juice after pressing. The sugar

Table 4 Sugars and fruit acids in crowberry in fractionsA and in sensory samplesB. Berry

3.3. Profiles of anthocyanins and other phenolic compounds Most of the anthocyanins and other less abundant phenolic compounds analysed were located in Residue I (Table 1). Four consecutive extractions removed 97% of the total anthocyanins from Residue I. The distribution of the individual anthocyanins and other phenolic compounds in each fraction and samples prepared for sensory analyses is shown in Table 3. The calculated values for each phenolic compound in Residue I are also shown in the table. On average, the calculated values for Residue I were higher than the measured values of the fraction. Galactosides of delphinidin, cyanidin and malvidin were the major anthocyanins in all fractions. These three anthocyanidins have been reported to be the most abundant aglycons in northern crowberry as in the southern crowberry malvidin is the most abundant (Määttä-Riihinen et al., 2004). The total contents of anthocy-

Juice

Residue I

Extract-1

Fractions (g/l kg of berry) Fructose 18 ± 1.1 Glucose 52 ± 2.5 Sucrose 0.8 ± 0.1

17 ± 0.7 54 ± 1.9 1.2 ± 0.0

0.2 ± 0.0 0.7 ± 0.1 –

0.5 ± 0.0 1.6 ± 0.0 –

Malic acid Citric acid Quinic acid

1.9 ± 0.0 0.5 ± 0.0 2.0 ± 0.0

0.04 ± 0.0 0.02 0.02

0.2 ± 0.0 0.1 0.1

Sensory samples (g/100 g) Fructose 1.7 ± 0.2b Glucose 5.2 ± 0.3b Sucrose 0.1 ± 0.0b

2.0 ± 0.1a 6.4 ± 0.2a 0.1 ± 0.0a

0.1 ± 0.0c 0.4 ± 0.0c

0.1 ± 0.0c 0.4 ± 0.0c

0.2 ± 0.0b 0.1 ± 0.0a 0.2 ± 0.0a

0.2 ± 0.0a 0.1 ± 0.0a 0.2 ± 0.0a

0.02 ± 0.0d 0.01 ± 0.0b 0.01 ± 0.0b

0.1 ± 0.0c 0.02b 0.04b

Malic acid Citric acid Quinic acid A

1.8 ± 0.1 0.7 ± 0.1 2.3 ± 0.1

Contents in each original fraction (Table 1). Extract-1 was diluted in water, 25 g/l. Significant differences between samples in each compound based on Tukey’s test (p < 0.05) are marked with superscripts a–d. B

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content of our crowberry juice sample was higher than previously reported (Viljakainen, Visti, & Laakso, 2002). Quinic and malic acids were the main organic acids in each fraction, while citric acid was found in significantly lower amounts. These results are consistent with the previously reported fruit acid content of crowberry (Kallio & Markela, 1982; Viljakainen et al., 2002). Crowberry juice has lower levels of organic fruit acids than other Nordic berries, such as bilberries, lingonberries and blackcurrants (Viljakainen et al., 2002; Sandell et al., 2009). The majority of the organic fruit acids were found in the juice. Practically all the sugars and fruit acids were dissolved during the first ethanol extraction.

These attributes formed different tactile sensations than the reference for astringency (Table 5). The astringent perception can be divided into a wide range of different oral sensations (Bajec & Pickering, 2008). The watery property increased from Extract-1 to Extract-4, indicating a loss of other taste compounds in the extraction process. When analysing Residue II and Residue III with the triangle test, no significant difference was found between samples at the 0.05 level of significance. This showed that the small amount of compounds extracted with SFE had no effect on the sensory properties and that the two residues were practically identical. 3.6. Compounds contributing to orosensory properties

3.5. Orosensory profiles Eight attributes were chosen for the sensory analyses of crowberry (Table 6). The juice was clearly the sweetest of all the samples. It also had some of the strongest total intensities reported, along with some of the extracts. The extracts were perceived as the most bitter and astringent. Two combined extracts in two separate sample sets differed only in their total intensity. The combined extract in sample set A (evaluated with juice and the Residues) was perceived as stronger than in sample set B (evaluated with other Extracts) probably because it was the only extract in sample set A even though the two combined extracts were identical. Extract-1 was the most abundant of the four extracts in most of the attributes, while it was perceived as quite similar to the combined extract. Astringency declined in the four consecutive extracts (Friedman’s test, p = 0.042), with a significant reduction from Residue I to Residue II. Similarly, bitterness, sourness and total intensity also declined significantly during the extraction process. In addition to astringency and the taste attributes, woody, watery and dusty properties were also perceived from the sensory samples. As dusty and woody properties were strongest in the extracts, these attributes could be similar tactile attributes to astringency.

The PLS2-model was applied to the sensory properties and nonvolatile chemical variables of the crowberry fractions, excluding the whole berry, combined extracts and Residue III. The predicted Y-values (sensory properties, n = 8) were computed by applying the model equation to the observed X-variables (chemical variables, n = 45). When the three principal components were taken into account, 96% of the chemical variables explained 85% of the sensory data (Fig. 2). The model again showed strong correlations between Residue I and many phenolic compounds on the righthand side of the plot using PC1, and between juice and sugars at the top of the plot using PC2. Total intensity and sourness did not differ significantly between most of the samples and therefore they were not correlated with any particular sample. Astringency and bitterness correlated with Extract-1 in PC1 along with a dozen chemical variables (compounds 4, 13, 19, 20, 23–25, 40, 41, 46, 52 and 53). The third component (PC3) showed the difference between these 12 compounds correlating with Extract-1 and the rest of the phenolic compounds correlating with Residue I. These compounds as well as some of the other flavonol glycosides also correlated with the dusty and woody attributes. This indicates that flavonol glycosides induce various tactile astringent perceptions.

Table 5 Sensory attributes, descriptions with references and their intensitiesA used in sensory profiling of crowberry fractions.

A

Sensory attribute

Description

Reference

Intensity

Total intensity

–

–

Sweetness Sourness Bitterness Astringency Woody Dusty

Perceived first expression in mouth Sweet taste Sour taste Bitter taste Puckering, drying mouthfeel Woody mouthfeel Dusty, drying mouthfeel

3 4 3 5 5 6

Watery

Watery, flat overall expression

1.0% sucrose (BDH Laboratory Supplies, Poole, UK) 0.10% citric acid monohydrate (J.T. Baker, Deventer, Netherlands) 0.08% caffeine (Yliopiston Apteekki, Helsinki, Finland) 0.10% AlSO4 (Linnan Apteekki, Turku, Finland) Moistured wooden tongue depressors (REF 29000, Medizintechnik KaWe, Asperg, Germany) A piece of filter paper (2  2 cm) placed on the tongue (Whatman no 1, Whatman Laboratory Division, Maidstone, UK) –

–

Scale 0–10.

Table 6 Mean intensitiesA (n = 36) for sensory attributes in crowberry fractions.

Total intensity Sweetness Sourness Bitterness Astringency Woody Dusty Watery

Juice

Residue I

Comb Extract-1

Comb Extract-2

Extract-1

Extract-2

Extract-3

Extract-4

Residue II

Residue III

5.6 ± 1.6a 4.7 ± 1.7a 2.3 ± 1.5a 1.3 ± 1.1de 1.5 ± 1.2ef 1.7 ± 1.2b 1.5 ± 1.3c 2.3 ± 1.8bc

4.2 ± 2.0abc 1.2 ± 0.9b 2.4 ± 1.4a 2.2 ± 1.7bcd 2.1 ± 1.8cde 3.0 ± 1.4a 2.3 ± 1.7bc 3.4 ± 2.4abc

5.0 ± 1.5a 0.5 ± 0.5c 2.3 ± 1.2a 3.9 ± 1.9a 3.8 ± 2.0ab 3.4 ± 2.3a 3.6 ± 1.9ab 4.5 ± 2.4a

3.9 ± 1.9bcd 0.6 ± 0.5bc 1.6 ± 1.2ab 3.0 ± 1.8ab 2.9 ± 1.9abc 3.3 ± 1.8ab 3.4 ± 1.9ab 4.0 ± 2.5ab

4.9 ± 1.7ab 0.6 ± 0.5bc 2.1 ± 1.5a 3.4 ± 2.3ab 3.6 ± 2.2a 4.5 ± 2.3a 3.8 ± 2.4a 4.0 ± 1.6ab

3.7 ± 1.5cd 0.8 ± 0.7bc 1.9 ± 1.4a 2.6 ± 1.4abc 2.9 ± 1.9abcd 2.9 ± 1.8ab 3.1 ± 2.0ab 4.5 ± 2.6a

3.4 ± 1.6cd 0.7 ± 0.6bc 1.5 ± 1.1ab 2.7 ± 1.5abc 2.7 ± 1.7bcde 2.4 ± 1.6ab 3.4 ± 1.9ab 4.4 ± 2.2a

2.7 ± 1.6de 0.9 ± 0.6bc 0.8 ± 0.9bc 1.7 ± 1.3cde 2.4 ± 1.8def 1.6 ± 1.6ab 2.9 ± 2.1abc 5.2 ± 2.7a

1.7 ± 1.2ef 0.7 ± 0.6bc 0.5 ± 0.6c 1.0 ± 1.0e 0.8 ± 1.1f 2.8 ± 1.7ab 2.2 ± 1.9bc 2.5 ± 2.3bc

1.6 ± 1.2f 0.5 ± 0.5c 0.5 ± 0.6c 1.0 ± 1.1e 0.8 ± 1.1f 2.8 ± 1.5ab 2.5 ± 1.9abc 1.9 ± 2.1c

A Scale is from 0 (no sensation) to 10 (very strong sensation). Significant differences between samples based on the Tamhane test (p < 0.05) are marked with a–g. Combined extract was analysed in both sets A and B. Set A contained Comb Extract-1 together with Juice and the Residues. Set B contained Comb Extract-2 with the Extract-1 to -4.

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Correlation Loadings (X and Y) 1.0

PC2

sweetness

citric acid malic acid quinic acid

glucose

Juice fructose sucrose

total intensity sourness

0.5

2

11+12

Residue I 32+33

5 3 14 15 30+31 19+10 6 7+8 22 38+39 27+28 total anthocyanins 21 26 34+35 44 42+43 47 45 36+37 48+49

0

Extract-4 Extract-2 Residue II Extract-3 -0.5 watery

4

20 52 40 25 24 53 Extract-1 astringency 46 23 41 bitterness 19 13 woody

dusty

PC1

-1.0 -1.0

-0.8

X-expl: 49%,24%

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1.0

Y-expl: 34%,34%

Correlation Loadings (X and Y) 1.0

PC3 sucrose quinic acid citric acid malic acid glucose fructose sweetness

Juice 0.5

46 53 52 Extract-1 41 25 40 413 19 23 astringency

total intensity

sourness bitterness 24 dusty 0

Extract-2

woody 32+33 44 42+43 38+39 45 2627+28 21 30+31 34+35 47 22 36+37 48+49 9+10 15 11+12 2 3 51 7+8 14 6 total anthocyanins

Extract-3 Extract-4 Residue II

20

watery

-0.5

Residue I

PC1

-1.0 -1.0

-0.8

X-expl: 49%,23%

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1.0

Y-expl: 34%,17%

Fig. 2. Partial least squares regression (PLS2) plots of the interaction between sensory profiles (n = 8) and chemical variables (n = 45) in crowberry samples (n = 7) with three principal components. The samples are in bold and larger font, and sensory attributes are in italics.

Flavonol glycosides 19 and 20 (unknown flavonol glycosides), 23 and 24 (unknown quercetin glycosides), 25 (myricetin arabinoside), 40 (isorhamnetin arabinoside), 41 (syringetin arabinoside) and 46 (unknown laricitrin glycoside) and free aglycones 52 (syringetin) and 53 (isorhamnetin) contributed significantly to the astringency and bitterness of Extract-1 even in small quantities. It is not clear whether all the compounds identified in the ethanol extracts were in their natural quantities. The possibility that a

proportion of them may have been formed during the extraction process cannot be excluded and further investigations are required. The taste properties of anthocyanins (4 and 13) have not been reported previously. Flavonol glycosides have very low taste thresholds (Scharbert et al., 2004; Schwarz & Hofmann, 2007a) and therefore these might interfere with the perception of anthocyanins. Some of the other phenolic compounds in crowberry fractions that correlated strongly with Residue I might also have astringent

O. Laaksonen et al. / Food Chemistry 124 (2011) 1514–1524

and bitter properties as they were strongly correlated with PC1 as well as Extract-1. As the press residue was significantly less bitter and astringent than the extracts, low extraction of phenolic compounds by human saliva may be possible. These compounds may be bound to the fibre fractions of the skin-rich residue and not be as biologically available in the intestine as they could be after separation. Polyphenols, such as proanthocyanidins and hydrolysable tannins, may cause astringency by precipitating proteins of human saliva (Bajec & Pickering, 2008), but flavonol glycosides do not act in the same way (Schwarz & Hofmann, 2008). Polyphenols binding proteins may cause astringency of certain type, whereas the flavonol glycosides may cause diverse drying or puckering mouthfeel, such as woody, dusty and astringent attributes in our study. Only some of the compounds that might contribute to the sensory attributes perceived from the extracts were identified in this study. The unidentified compounds may include some minor hydroxycinnamic acid conjugates and also proanthocyanidins, which are known to be both astringent and bitter (Hufnagel & Hofmann, 2008; Schwarz & Hofmann, 2007a), as well as some indolyl glycosides that have been reported to be very astringent in red currants (Schwarz & Hofmann, 2007b). 4. Conclusions The locations of the sensory properties and the corresponding chemical compounds in the respective crowberry fractions were investigated. The berry juice was rich in sugars and low in fruit acids and it was perceived as notably sweet since most of the phenolic compounds were in the press residue, which was rather tasteless. On the other hand, phenolic compounds were soluble in ethanol and the ethanol extracts were bitter and astringent after the ethanol was evaporated and the extract was dissolved in water. Ethanol extraction appeared to be a very efficient way to stepwise decrease the bitter and astringent properties of the residue. It is important to understand the interactions between the sensory properties and the chemical composition of crowberry to discover the key compounds and critical factors contributing to the orosensory profile. The simple fractionation of berries applied is a promising technique in the production of natural ingredients, such as juice, press residue, extracts and the fibre components. Especially the ethanol soluble phenolic compounds may be useful ingredients in different food contexts. The balance between healthpromoting and the sensory properties of the novel products is a challenge. By fractionating crowberry, a poorly utilised natural resource may be more easily exploited in the food industry. This study gives new and significant information about crowberry and its fractions. Phenolic compounds may have wanted or unwanted sensory properties, guiding the industrial applications and food development. Acknowledgements M.Sc. Jenni Vaarno is gratefully thanked for her assistance in the sensory analysis. This study was funded by the Finnish Funding Agency for Technology and Innovation, and food companies under the project ”Novel (bio)processing techniques for flavour design in plant-based foods”, Academy of Finland (Sandell 116165), and the Finnish Cultural Foundation, Varsinais-Suomi Regional Fund. References Anttonen, M. J., & Karjalainen, R. O. (2006). High-performance liquid chromatography analysis of blackcurrant (Ribes nigrum L.) fruit phenolics grown either conventionally or organically. Journal of Agricultural and Food Chemistry, 54, 7530–7538.

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Bajec, M. R., & Pickering, G. J. (2008). Astringency: Mechanisms and perception. Critical Reviews in Food Science and Nutrition, 48, 858–875. Boots, A. W., Haenen, G. R. M. M., & Bast, A. (2008). Health effects of quercetin: From antioxidant to nutraceutical. European Journal of Pharmacology, 585, 325–337. Buchert, J., Koponen, J., Suutarinen, M., Mustranta, A., Lille, M., Törrönen, R., et al. (2005). Effect of enzyme-aided pressing on anthocyanin yield and profiles in bilberry and blackcurrant juices. Journal of the Science of Food and Agriculture, 85, 2548–2556. Drewnovski, A., & Gomez-Carneros, C. (2000). Bitter taste, phytonutrients, and the consumer: A review. American Journal of Clinical Nutrition, 72, 1424–1435. Erlund, I., Koli, R., Alfthan, G., Marniemi, J., Puukka, P., Mustonen, P., et al. (2008). Favorable effects of berry consumption on platelet function, blood pressure, and HDL cholesterol. American Journal of Clinical Nutrition, 87, 323–331. Galvano, F., La Fauci, L., Lazzarino, G., Fogliano, V., Ritieni, A., Ciappelano, S., et al. (2004). Cyanidins: Metabolism and biological properties. Journal of Nutritional Biochemistry, 15, 2–11. Hufnagel, J. C., & Hofmann, T. (2008). Orosensory-directed identification of astringent mouthfeel and bitter-tasting compounds in red wine. Journal of Agricultural and Food Chemistry, 56, 1376–1386. Häkkinen, S., Heinonen, M., Kärenlampi, S., Mykkänen, H., Ruuskanen, J., & Törrönen, R. (1999). Screening of selected flavonoids and phenolic acids in 19 berries. Food Research International, 32, 345–353. International Organization for Standardization. (1988a). ISO 8586-1&2. Sensory analysis – General guidance for the selection, training and monitoring of assessors. Switzerland. International Organization for Standardization. (1988b). ISO 8589. Sensory analysis – General guidance for the design of the rooms. Switzerland. International Organization for Standardization. (1992). ISO/DIS 11035. Sensory analysis – Methodology – Identification of descriptors for establishing a sensory profile. Switzerland. International Organization for Standardization. (2004). ISO 4120. Sensory analysis – Methodology –Triangle test. Switzerland. Järvinen, R., Kaimainen, M., & Kallio, H. (2010). Cutin composition of selected northern berries and seeds. Food Chemistry, 122, 137–144. Kallio, H., & Markela, E. (1982). Nonvolatile acids in the juice crowberry, Empetrum nigrum coll. Journal of Food Science, 47(5), 1466–1468. 1477. Kallio, H., Nieminen, R., Tuomasjukka, S., & Hakala, M. (2006). Cutin composition of five Finnish berries. Journal of Agricultural and Food Chemistry, 54, 457–462. Koponen, J. M., Happonen, A. M., Auriola, S., Kontkanen, H., Buchert, J., Poutanen, K. S., et al. (2008). Characterization and fate of blackcurrant and bilberry flavonols in enzyme-aided processing. Journal of Agricultural and Food Chemistry, 56, 3136–3144. Kähkönen, M. P., Hopia, A. I., & Heinonen, M. (2001). Berry phenolics and their antioxidant activity. Journal of Agricultural and Food Chemistry, 49, 4076–4082. Kärppä, J., Kallio, H., Peltonen, I., & Linko, R. (1984). Anthocyanins of crowberry, Empetrum-Nigrum Coll. Journal of Food Science, 49, 634–636. Lätti, A. K., Riihinen, K. R., & Kainulainen, P. S. (2008). Analysis of anthocyanin variation in wild populations of bilberry (Vaccinium myrtillus L.) in Finland. Journal of Agricultural and Food Chemistry, 56, 190–196. Määttä, K., Kamal-Eldin, A., & Törrönen, R. (2001). Phenolic compounds in berries of black, red, green and white currants (Ribes sp.). Antioxidants & Redox Signaling, 3, 981–993. Määttä, K. R., Kamal-Eldin, A., & Törrönen, A. R. (2003). High-performance liquid chromatography (HPLC) analysis of phenolic compounds in berries with diode array detector electrospray ionization mass spectrometer (MS) detection: Ribes species. Journal of Agricultural and Food Chemistry, 52, 6736–6744. Määttä-Riihinen, K. R., Kamal-Eldin, A., Mattila, P. H., Gonzáles-Paramás, A. M., & Törrönen, A. R. (2004). Distribution and contents of phenolic compounds in eighteen Scandinavian berry species. Journal of Agricultural and Food Chemistry, 52, 4477–4485. Ogawa, K., Sakakibara, H., Iwata, R., Ishii, T., Sato, T., Goda, T., et al. (2008). Anthocyanin composition and antioxidant activity of the crowberry (Empetrum nigrum) and other berries. Journal of Agricultural and Food Chemistry, 56, 4457–4462. 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. Riihinen, K., Jaakola, L., Kärenlampi, S., & Hohtola, A. (2008). Organ-specific distribution of phenolic compounds in bilberry (Vaccinium myrtillus) and ‘northblue’ blueberry (Vaccinium corymbosum  V. angustifolium). Food Chemistry, 110, 156–160. Sandell, M., Laaksonen, O., Järvinen, R., Rostiala, N., Pohjanheimo, T., Tiitinen, K., et al. (2009). Orosensory profiles and chemical composition of blackcurrant (Ribes nigrum) juice and fractions of press residue. Journal of Agricultural and Food Chemistry, 57(9), 3718–3728. Scharbert, S., Holzmann, N., & Hofmann, T. (2004). Identification of astringent taste compounds in black tea infusions by combining instrumental analysis and human bioresponse. Journal of Agricultural and Food Chemistry, 52, 2498–2508. Schwarz, B., & Hofmann, T. (2007a). Sensory-guided decomposition of red currant juice (Ribes rubrum) and structure determination of key astringent compounds. Journal of Agricultural and Food Chemistry, 55, 1394–1404. Schwarz, B., & Hofmann, T. (2007b). Isolation, structure determination, and sensory activity of mouth-drying and astringent nitrogen-containing phytochemicals isolated from red currants (Ribes rubrum). Journal of Agricultural and Food Chemistry, 55, 1405–1410.

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O. Laaksonen et al. / Food Chemistry 124 (2011) 1514–1524

Schwarz, B., & Hofmann, T. (2008). Is there a direct relationship between oral astringency and human salivary protein binding? European Food research and Technology, 227, 1693–1698. Tiitinen, K., Hakala, M., & Kallio, H. (2005). Quality components of sea buckthorn (Hippophae rhamnoides) varieties. Journal of Agricultural and Food Chemistry, 53, 1692–1699.

Viljakainen, S., Visti, A., & Laakso, S. (2002). Concentrations of organic acids and soluble sugars in juices from Nordic berries. Acta Agriculturae Scandinavica, Section B, Soil and Plant Science, 52, 101–109. Wu, X., Gu, L., Prior, R. L., & McKay, S. (2004). Characterization of anthocyanins and proanthocyanidins in some cultivars of Ribes, Aronia and Sambucus and their antioxidant capacity. Journal of Agricultural and Food Chemistry, 52, 7846–7856.