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Relationship between texture and pectin composition of two apple cultivars during storage
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Postharvest Biology and Technology 47 (2008) 315–324
Relationship between texture and pectin composition of two apple cultivars during storage Ludivine Billy a,b , Emira Mehinagic a,∗ , Ga¨elle Royer a , Catherine M.G.C. Renard c , Ga¨elle Arvisenet b , Carole Prost b , Fr´ed´erique Jourjon a a
Ecole Sup´erieure d’Agriculture, Groupe de Recherche en Agro-industrie sur les Produits et les Proc´ed´es (GRAPPE), 55 rue Rabelais, B.P. 30 748, 49007 Angers Cedex 01, France b ENITIAA, UMR CNRS GEPEA 6144, Laboratoire de Biochimie, rue de la G´ eraudi`ere, B.P. 82 225, 44322 Nantes Cedex 03, France c UMR A 408 INRA, Universit´ e d’Avignon, SQPOV, S´ecurit´e et Qualit´e des Produits d’Origine V´eg´etale, Domaine St. Paul, 84 914 Avignon Cedex 09, France Received 3 April 2007; accepted 13 July 2007
Abstract The texture of two apple cultivars was characterised by sensory and instrumental methods for five different storage periods. The aim of this study was to explain the changes in apple texture during storage by different physical (penetrometry, compression) and chemical measurements (extraction and analysis of pectin composition). The emphasis was on determining the most relevant biochemical markers in relation to different sensory properties of apple texture. Contrary to ‘Fuji’, ‘Golden Delicious’ fruit softened easily during storage, became mealy and had higher neutral sugar concentrations in their alcohol-insoluble residues (AIR) and more galacturonic acid in the water-soluble pectin extracts (WSP). The most relevant biochemical marker linked to texture change was the galacturonic acid content in the water-soluble pectin extracts. High and positive correlation coefficients were observed between sensory mealiness (R = 0.84) and galacturonic acid content in the WSP while, sensory crunchiness and instrumentally measured firmness were negatively correlated with this component. The total neutral sugar content in the alcohol-insoluble residues and in the water-soluble pectin fractions also changed with apple texture properties. © 2007 Elsevier B.V. All rights reserved. Keywords: Apple; Malus domestica Borkh; Texture; Cell wall; Mechanical properties; Sensory analysis
1. Introduction Apple texture is one of the most important quality properties that influence consumer acceptability of these fruit (Stow, 1995). Thus, postharvest softening of apples during storage and market distribution is a serious commercial problem resulting in quality losses for growers and distributors. Therefore, our research aims to explain the development of apple softening during storage using different physical (penetrometry, compression) and chemical measurements (extraction and analysis of pectin composition). Knowledge of the mechanism of fruit tissue softening, cellwall structure and cell-wall breakdown is very important for understanding and improving the texture and quality of apple
∗
Corresponding author. Tel.: +33 2 41 23 55 55; fax: +33 2 41 23 55 65. E-mail address: [email protected] (E. Mehinagic).
0925-5214/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2007.07.011
fruit. A better understanding of the relationship between fruit texture and biochemical composition could lead to improvements in quality control and process design in the food industry and the marketplace. Many studies have already addressed either the changes in apple texture during storage (Abbott et al., 1984; Grotte et al., 2001; Johnston et al., 2001; Mehinagic et al., 2004; Varela et al., 2007) or changes in pectin composition during softening (Yoshioka et al., 1994; Massiot et al., 1996; Nara et al., 2001), but few studies tried to connect these two phenomena. Moreover, for the majority of these, sensory perception of changes in fruit texture was never carried out. Ben and Gaweda (1985) showed high correlation coefficients between the content of protopectin (non-cellulosic neutral sugar residues) and the firmness of ‘Jonathan’ apples in one storage season only. A study performed on ‘Golden Delicious’ apples attempted to determine the possible contribution of changes in cell-wall composition to fruit softening under various storage conditions (Siddiqui et al., 1996). More recently, Pena and Carpita (2004) reported a study
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of the chronology of the biochemical events associated with loss of firm texture and cell separation, and Lo Scalzo et al. (2005) described the results of research on pectin composition just after reddening and after cold storage of reddened ‘Annurca’ apple fruit in different atmospheres. Loss of firm texture and cell adhesion are processes associated with ripening that affect fruit quality and postharvest storage. The cell walls of fruit have received considerable attention, as changes occurring in their structure and composition are clearly determining in fruit firmness (Knee, 1973a,b; Bartley, 1976; De Vries et al., 1981; Renard et al., 1990b, 1991a,b,c,d; Renard and Thibault, 1993; Massiot et al., 1994). Indeed, it is well known that separation and degradation of water-soluble pectic substances, erosion of the middle lamella and disintegration of the primary cell wall lead to softening of fruit flesh. Pectic substances are the most abundant class of macromolecules within the cell-wall matrix and are the main components of the middle lamellae. They have a role in the adhesion between cells, and in regulation of intercellular adhesion. Consequently, pectic substances have been assigned an important role in changes in texture of fruit tissues (Yoshioka et al., 1992; Grant Reid, 1997) and notably, changes in pectin composition have been implicated in decreased adhesion between cells (Ilker and Szczesniak, 1990). During fruit softening, pectins typically undergo solubilisation and depolymerisation, which are thought to contribute to wall loosening and disintegration. In apple, there is usually an increase in water-soluble pectin (WSP), and a decrease in galactose and arabinose residues (Knee, 1973b), with little depolymerisation occurring in any pectin fraction during ripening (Yoshioka et al., 1992; Fischer and Amado, 1994; Fischer et al., 1994). However, the association of biochemical changes with changes in fruit texture is still unclear; for example, it is still not known if the processes of pectin solubilisation and loss of galactose are causal, coincidental, or a consequence of apple softening (Redgwell et al., 1997). We aimed to study the relationship between texture and pectin composition of stored apples. Two cultivars with different textural characteristics were studied in order to investigate whether the differences in texture between different apple cultivars were due to differences in their pectin composition. ‘Fuji’ is a crunchy and juicy apple that stores well and ‘Golden Delicious’ apples become mealy during storage (Mehinagic et al., 2004). The apples were stored for up to 7 months at 2 ◦ C. Their texture was assessed using two deformation tests (penetrometry and compression) and sensory analysis. At the same time, cell-wall content and pectin extractability of these fruit were characterised in order to try to relate texture modifications and sensory perceptions to different chemical changes occurring in apple cell walls. 2. Materials and methods 2.1. Fruit Two different apple cultivars (Malus domestica Borkh), ‘Fuji’ (FU) and ‘Golden Delicious’ (GO), were studied. One hundred and forty fruit of each cultivar harvested in 2004
at commercial maturity were provided by a fruit-bearing test center (“La Morini`ere”, Saint Epain, France). Their ripening stage was verified by starch regression measurements. These fruit were selected on the basis of uniformity and absence of damage or blemishes. They were all stored in the same cool room at 2 ◦ C and under ambient atmospheric pressure and humidity for five different periods (1, 2, 3, 4 and 7 months). The choice of these storage periods was based on a previous study which showed that they resulted in a large range of apple mechanical properties (Mehinagic et al., 2004). For each storage period, 28 fruit per cultivar were brought up to room temperature 24 h before being analysed. Half of each fruit (n = 28) was analysed by sensory panel and the second half by penetrometry (n = 14) or compression (n = 14). Sensory and mechanical methods were carried out simultaneously and immediately after the apples were cut. For penetrometry and compression tests, apples were manually and carefully peeled by the same operator using the same vegetable knife. Fruit analysed by penetrometry were then sampled (taking care to remove the crushed part before sampling) and freeze-dried prior to chemical analysis. 2.2. Sensory evaluation The sensory panel included 14 permanent trained panellists from the staff of the Ecole Sup´erieure d’Agriculture (ESA, Angers, France). These panellists, selected in 1999, are trained every week to evaluate apple texture according to the recommendations of AFNOR (1995) and of Fortin and Desplancke (1998). Their performances are evaluated once a year to ensure reliability. The sensory attributes studied on apple flesh were crunchiness, juiciness, mealiness, chewiness and fondant. These sensory attributes are defined and described in Table 1. During a sensory session, each panellist peeled and analysed two different half fruit from each cultivar. The washed unpeeled half apples, coded by a random three-digit code, were randomly presented to the panellists, at room temperature and under red light illumination to avoid apple cultivar identification. A 10 cm line scale was used for evaluation. The left end of the scale corresponded to the lowest intensity (value 0) and the right end to the highest (value 10). Each panellist used mineral water as a rinse between sample analyses. Five sessions, corresponding to five storage periods, were organised. In this way, each panellist analysed 2 cultivars × 2 half apples during each session. Thus, Table 1 Texture sensory descriptors used for apples Texture attribute
Definition (Mehinagic et al., 2003)
Crunchiness
Force required for the first bite and the noise resulting from this bite Amount of liquid released on mastication just before swallowing Dry and crumbly texture Time and number of chewing movements needed to grind the sample prior to swallowing Force required to crush a piece of apple between the tongue and palate
Juiciness Mealiness Chewiness Fondant
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Fig. 1. The penetrometric curve obtained on peeled half apples, using the MTS (Synergie 200H) traction machine (cylindrical probe with a 4 mm diameter convex tip; penetration speed of 50 mm min−1 ; depth of 10 mm) (FLC: flesh limit compression force; S: slope of the force-deformation curve; D: deformation associated with flesh limit compression force; WFLC : work associated with FLC; W7 : work required to attain a flesh deformation of 7 mm).
for each cultivar and each storage period, 28 fruit samples were analysed. 2.3. Penetrometry According to Mehinagic et al. (2004), a cylindrical probe with a 4 mm diameter convex tip was used to perforate peeled half apples in an MTS (Synergie 2000H) traction machine. Peeled half apples were placed with the flat side facing down on the plate and punched on their rounded side in an equatorial position. The equipment used detected the sample automatically; penetration speed was set at 50 mm min−1 , and the test was stopped after penetration to 10 mm. This distance was short enough to avoid any influence of the core in the measurement. Fifty points were recorded during a penetrometric test. Penetrometry curves were analysed and five parameters were studied (Fig. 1): flesh limit compression force (FLC), deformation associated with flesh limit compression force (D), slope of the force-deformation curve (S), work associated with FLC (WFLC ) and work required to attain a flesh deformation of 7 mm (W7 ) (Duprat et al., 2000). Definitions of these parameters are given in Table 2. For each cultivar and each storage period, 14 fruit samples were analysed. 2.4. Compression According to Mehinagic et al. (2004), two parallel plates (50 mm of diameter and 7 mm of thickness) were used to compress peeled half apples. These were placed with the flat side facing down on the plate, with the equatorial section of their rounded side targeted in the same MTS (Synergie 2000H) traction machine. The equipment detected the diameter and height of the sample automatically: the height of peeled half apples was
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Fig. 2. Double compression force/time curve obtained on peeled half apples, using an MTS (Synergie 200H) traction machine (2 parallel plates; 50 mm min−1 ; 7 mm) (1: the first compression; 2: the second compression; H: maximal force; WH : surface area under the compression curve; S: slope of the compression).
compressed by 20% and the loading rate of the crosshead was 50 mm min−1 . Two successive compressions were carried out on each sample. Compression curves were analysed and six parameters were studied (Fig. 2): maximal force associated with the first compression, usually described as hardness (H1 ), maximal force associated with the second compression (H2 ), surface area under the first compression curve (WH1 ), surface area under the second compression curve (WH2 ), slope of the first compression (S1 ) and slope of the second compression (S2 ). For each cultivar and each storage period, 14 fruit samples were analysed. 2.5. Apple powder preparation Peeled apple pieces were freeze-dried, then frozen with liquid N2 and immediately homogenised in a commercial blender for 1 min. The apple powders obtained were prepared in triplicate for each batch (2 cultivars × 5 storage durations) and stored in a desiccator under vacuum prior to chemical analysis. 2.6. Alcohol-insoluble residue (AIR) preparation Cell walls were prepared as alcohol-insoluble residues for each sample triplicate. According to Renard (2005), the apple powder (7 g) was blended with 50 mL of 70% ethanol in an empty 75 mL Sep-pack prep column (InterchimTM ) equipped with a sinter, porosity 20 m. The mixture was stirred at room temperature for 20 min then filtered under vacuum. The residue was re-extracted with 70 mL of 70% ethanol eight times, as described above. At the end of these washings, the filtrates were sugar-free (absence of sugars was tested by the phenol-sulphuric
Table 2 Definitions of penetrometric parameters Parameters
Calculation
Definitions (Duprat et al., 2000; Grotte et al., 2001)
FLC (N) D (mm) S (N mm−1 ) WFLC (N mm) W7 (N mm)
Maximal force before the drop in force at the moment of penetration Deformation associated with FLC Gradient on the curve (between 0 and FLC) Area under the curve between 0 and D Area under the curve between 0 and 7 mm
Force representing the limit of flesh elasticity Gradient measuring penetrometric firmness Work required for rupture of the flesh Work required to attain a flesh deformation of 7 mm
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method of Dubois et al., 1956). The alcohol-insoluble residues were dried by solvent exchange with 96% ethanol (three times), and acetone (three times), and finally overnight in an oven at 40 ◦ C. 2.7. Water-soluble pectin extraction From each AIR preparation, water-soluble pectin was extracted by water. For each extraction, approximately 100 mg of cell-wall preparation and 6 mL of water (Renard, 2005) were incubated for 2 h at room temperature in an empty 8 mL Sep-pack prep column (Interchim TM ) equipped with a sinter, porosity 20 m, under slow planetary agitation. After incubation, the extract and cell walls were separated by filtration under vacuum. This extraction was repeated three times for each AIR preparation; the three water extracts were pooled and frozen before analysis. 2.8. Analytical methods Galacturonic acid content (GA) was determined by automated m-hydroxydiphenyl assays on an Alliance Instruments (M´ery sur Oise, France) autoanalyser after sulphuric acid prehydrolysis and hydrolysis (26N sulphuric acid, 1 h, room temperature followed by 2N, 3 h, 100 ◦ C) for the alcoholinsoluble residues (Saeman et al., 1954), or saponification (0.1N NaOH, 30 min, room temperature) for water-soluble pectin extracts (Thibault, 1979). The results are expressed as anhydrogalacturonic acid. Total neutral sugar content (NS) was determined by the orcinol method (Tollier and Robin, 1979) on the same hydrolysates and saponified pectin solutions. Methanol was determined according to Klavons and Bennett (1986). The degree of methylation (DM) was calculated as the molar ratio of methanol to galacturonic acid. The degree of acetylation (DA) was determined by high performance liquid chromatography after saponification according to the method of Voragen et al. (1986). 2.9. Statistical analysis Data acquisition and statistical treatment were performed with Statgraphics Plus 5.1 software (Sigmaplus, Toulouse,
France). The analysis of variance (ANOVA) was carried out independently for each cultivar and for each of the studied variables measured by sensory analysis, penetrometry, compression and pectin analysis. Two-way ANOVA was performed to determine significant differences in sensory quality between apples. The factors studied were the panellist effect and the storage period. An interaction of these two factors was evaluated for each cultivar and each sensory attribute to make sure that these main effects were not confused. One-way ANOVA was carried out on instrumental measurements (penetrometry, compression and pectin analysis) to test the effects of storage duration on each cultivar. For each analysis, a significant level of 5% was used. To compare the effect of 5 storage periods on the different attributes of the two cultivars, Fischer’s least significant difference (LSD) procedures were applied separately to each cultivar. Principal component analysis (PCA) was carried out on the averaged data. Pearson’s correlation coefficients between texture parameters and biochemical data were calculated between the average values. 3. Results and discussion 3.1. Effect of storage duration on sensory attributes and mechanical parameters of fruit texture Variance analysis was carried out independently for each of the five sensory descriptors studied. For all sensory descriptors, no interactions were found between panellist effect and storage period for ‘Fuji’ apples while two significant interactions were found for ‘Golden Delicious’. These two interactions involving mealiness and fondant can be explained by heterogeneity within a fruit batch or differences in sensitivity between panellists. For ‘Golden Delicious’ apples, the storage effect was significant for all descriptors except chewiness. Texture change in ‘Fuji’ was more limited and the storage effect was significant for three descriptors at the 5% level: mealiness, juiciness and fondant. For both cultivars, the intensity of juiciness decreased while the intensity of mealiness increased (Table 3). These results confirmed those of Mehinagic et al. (2004). According to Holt and Schoorl (1984), these changes are related to the deterioration of the mechanical strength of apple tissue during storage.
Table 3 Evolution of sensory quality of 2 apple cultivars during storage Storage (months)/cultivar
Crunchiness
Juiciness
Mealiness
Chewiness
Fondant
1/Fuji 2/Fuji 3/Fuji 4/Fuji 7/Fuji
7.6 7.9 7.6 7.5 7.5
6.9ab
1.8b
6.2c
2.0ab
7.0a 6.5bc 6.2c
1.7b 1.8b 2.3a
5.7 5.8 5.3 5.3 5.3
2.9ab 2.4b 3.1a 3.5a 3.5a
1/Golden 2/Golden 3/Golden 4/Golden 7/Golden
4.5ab 4.6ab 4.2bc 3.7c 4.9a
4.9a 4.3b 4.5ab 3.9b 3.9b
3.9b 3.7b 4.0b 4.8a 4.8a
4.2 4.1 3.8 3.6 4.2
5.5c 5.4c 6.6ab 6.8a 6.1bc
Different letters (a–c) in a column within a cultivar denote values that are significantly different at P < 0.05.
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Fig. 3. Slope of the force-deformation curve (S), obtained by penetrometry (a) and maximal force associated with the second compression (H2 ) (b), of ‘Fuji’ (grey columns) and ‘Golden Delicious’ apples (white columns) after different periods of storage (identical letters on columns for the same cultivar indicate that there is no significant difference with P > 0.05).
It appeared that storage had a significant effect for all penetrometric parameters and for both cultivars. For ‘Golden Delicious’ apples, the effect of storage was also significant for all compression parameters while ‘Fuji’ apples seem more resistant. This could be explained by the better texture conservation of ‘Fuji’ apples. Indeed, Jobling and McGlasson (1995) and Curry (1989) showed that ‘Fuji’ apples maintain their firm texture during storage. As all penetrometric parameters were strongly correlated, only the changes in the slope of the force-deformation curve (S), corresponding to firmness, are represented in Fig. 3a. In this figure, it can be seen that fruit firmness decreased significantly between the second and third month of storage for ‘Fuji’ apples. For ‘Golden Delicious’ fruit, this parameter did not change throughout the 7 months storage period. In Fig. 3b, the maximal force (describing hardness) associated with the second compression is shown for each storage step. The hardness of ‘Fuji’ did not change at all, while ‘Golden Delicious’ apples hardness decreased between the first and second month of storage. Instrumental measurements and sensory analysis showed a good concordance, with a general decrease in fruit firmness during storage and better texture conservation of ‘Fuji’ apples. Moreover, both methods showed that ‘Fuji’ apples were firmer
than ‘Golden Delicious’. These results were in accordance with previous studies. They confirmed (Grotte et al., 2001; Mehinagic et al., 2004) that ‘Fuji’ and ‘Golden Delicious’ apples present mechanical and sensory textural differences. For the temporal aspects, Mehinagic et al. (2004) found a significant change in firmness between apples stored for 3 weeks and those stored for 20 or 30 weeks but they found no significant difference between 20 and 30 weeks of storage. Indeed, according to Johnston et al. (2001), the decrease in firmness for many apple cultivars can be characterised by a curve consisting of three distinct phases. Firstly, fruit soften slowly, then more rapidly, and finally slowly again. 3.2. Effect of storage duration on apple pectin extractability For ‘Fuji’ apples, the storage effect was significant for five biochemical parameters: the total neutral sugar content and the degree of methylation in the AIR, the composition of the WSP extracts (primarily galacturonic acid, less total neutral sugars) and its degree of methylation. For ‘Golden Delicious’ apples, three parameters changed significantly during storage: the galacturonic acid content and the total neutral sugar content in the AIR and the degree of methylation in the WSP fractions.
Table 4 Composition of different apple pectin extracts during storage Alcohol-insoluble residues composition
Water-soluble pectin composition
Storage (months)/cultivar
GA (mg/g)
DM (%)
DA (%)
NS (mg/g)
GA (mg/g)
DM (%)
NS (mg/g)
1/Fuji 2/Fuji 3/Fuji 4/Fuji 7/Fuji
292 270 276 290 286
75a 73a 75a 72ab 69b
5 5 5 5 5
694a 666ab 629b 582c 567c
8b 7b 10b 23a 23a
52b 70a 75a 56b 75a
29a 13b 17b 16b 15b
1/Golden 2/Golden 3/Golden 4/Golden 7/Golden
252b 276a 290a 275a 285a
75 75 74 73 74
6 6 5 6 5
716a 676b 676b 622c 668b
36 35 35 29 31
53c 62b 66b 81a 80a
18 19 16 15 16
Different letters in a column (a–c) within a cultivar denote values that are significantly different at P < 0.05. GA: Galacturonic acid; DM: degree of methylation; DA: degree of acetylation; NS: neutral sugar.
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The values obtained (Table 4) were close to those in the literature. Renard et al. (1990a) and Renard (2005) found that the galacturonic acid content of alcohol-insoluble residues of ‘Golden Delicious’ apples is generally between 250 and 277 mg/g AIR. In the same way, the values of galacturonic acid content of water-soluble pectin extracts agreed with those of Fischer et al. (1994). While the total neutral sugar contents in the WSP fractions were in accordance with those in the literature (Massiot et al., 1996), the total neutral sugar content in the AIR of each cultivar seemed to be higher (Fischer and Amado, 1994). This can be explained by the different methods used: these authors analysed the individual neutral sugar by gas chromatography and we analysed it by the orcinol method, which is known to over-estimate the results, the calibration being done with a hexose. The values for the degree of methylation of the pectic substances of AIR (69–75%) were totally in accordance with those reported by De Vries et al. (1981) (80%) and Renard et al. (1990b) (72%), just as for the values for the degree of acetylation (<10%) (Voragen et al., 1986). In the same way, the degree of methylation of the pectic substances of WSP agreed with previous studies (Renard et al., 1990a; Fischer et al., 1994; Massiot et al., 1996). In the AIR (Table 4), except for a significant increase between the first and second month of storage for ‘Golden Delicious’ apples, no trend of an increase or a decrease in the galacturonic acid content during fruit storage was noticeable for both cultivars. This observation was also valid for the degree of methylation of apples even if a significant decrease in this one was observed at the end of ‘Fuji’ apple storage. Moreover, no changes were observed for the degree of acetylation. All these results were in agreement with those in literature (Fischer and Amado, 1994). However, the total neutral sugar content significantly decreased during apple storage. For both cultivars, this decrease was more marked between the third and fourth month of storage and between the first and second month of storage for ‘Golden Delicious’. These results were in accordance with Fischer and Amado (1994). According to Yoshioka et al. (1994), this decrease is more specifically due to a decline in galactose and arabinose contents. In the WSP fractions (Table 4), for ‘Fuji’ apples, the content of galacturonic acid increased significantly between the third and fourth month of storage (Yoshioka et al., 1994; Massiot et al., 1996; Lo Scalzo et al., 2005). This phenomenon, corresponding to pectin solubilisation, might result from the enzymatic cleavage of linkages between pectin and other cell-wall components rather than from the degradation of pectin chains. Contrary to the literature (Siddiqui et al., 1996; Nara et al., 2001) there was no significant change in ‘Golden Delicious’. The degree of methylation increased along with storage period. This increase was particularly significant between the first and second month of storage for both cultivars, and between the third and fourth month of storage for ‘Golden Delicious’ apples. These results confirm previous observations (Massiot et al., 1996) concerning the biosynthesis of highly methylated pectin during apple storage. The content of total neutral sugars decreased during storage for ‘Fuji’ apples (Massiot et al., 1996; Nara et al., 2001). This change was particularly significant between the first and second
month of storage. As previously shown by Massiot et al. (1996), this decrease more particularly involves arabinose and galactose contents. Yoshioka et al. (1994) suggested that these two types of polyuronide may be lost from the side-chain polysaccharides of the polyuronide molecules and may be involved in the solubilisation of polyuronides. Contrary to ‘Fuji’ apples, this change was not significant for ‘Golden Delicious’, as shown by Nara et al. (2001). To summarise, the total neutral sugar content in the AIR, the composition of the WSP extracts (galacturonic acid and total neutral sugars) and its degree of methylation showed the most obvious changes during apple storage. Moreover, we showed that the total neutral sugar content in the AIR and its degree of acetylation, and the composition of the WSP fractions (galacturonic acid, total neutral sugars) of ‘Golden Delicious’ apples were significantly different from those of ‘Fuji’. Consequently, these five biochemical parameters could explain the mechanical and sensory textural differences observed previously between these two cultivars and storage periods. 3.3. Relationships between apple mechanical properties, texture sensory attributes and biochemical composition of cell-wall pectins A PCA analysis was carried out on sensory, penetrometry, compression and biochemical parameters. The first dimension (PC1) described 66.5% of the variation amongst samples, and the second (PC2) 15% (Fig. 4). Sensory parameters juiciness, crunchiness and chewiness were highly related to the mechanical parameters (firmness, hardness, slope of the force-deformation curve) and negatively linked to mealiness and fondant. These two
Fig. 4. PCA plot of the two cultivars (‘Fuji’ and ‘Golden Delicious’) analysed at five storage periods by five sensory attributes, four parameters measured by penetrometry, six parameters measured by compression and seven biochemical parameters in the plane defined by the first two principal components (GOX : ‘Golden Delicious’ apples stored for X months; FUX : ‘Fuji’ apples stored for X months).
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Table 5 Pearson’s correlation coefficients between instrumental attributes measured by mechanical parameters, sensory descriptors for texture, and biochemical parameters for both cultivars (coefficients inferior to 0.6 were omitted) Biochemical parameters
GA WSP DA AIR
Mechanical parameters
Texture sensory descriptors
S
H1
H2
S1
S2
Crunchiness
Juiciness
Mealiness
Chewiness
Fondant
−0.94 −0.68
−0.85 −0.64
−0.85 −0.63
−0.88
−0.78 −0.63
−0.89 −0.80
−0.86 −0.76
0.84 0.81
−0.90 −0.75
0.89 0.73
GA: Galacturonic acid; WSP: water-soluble pectin; DA: degree of acetylation; AIR: alcohol-insoluble residue.
Fig. 5. Changes in the galacturonic acid content in the water-soluble pectin extracts (—, GA WSP) and the maximal force associated with the second compression (- -, H2 ) (a) or (- -) fondant (b) of ‘Fuji’ apples during storage.
attributes were related to the content of galacturonic acid in the WSP extracts. Highly negative Pearson’s correlation coefficients were found between the content of galacturonic acid in the WSP extracts, mechanical parameters and fruit juiciness, crunchiness and chewiness (Table 5). Indeed, the content of galacturonic acid in water-soluble pectin extracts of ‘Fuji’ apples increased at the same time as the hardness (H2 ) decreased or as the fondant increased (Fig. 5). In the same way, correct Pearson’s correlation
coefficients were obtained between the degrees of acetylation in the AIR and fruit texture parameters. Peasron’s correlation coefficients between mechanical and sensory parameters of texture and the total neutral sugar content in the AIR and in the WSP extracts were not high enough to be significant. However, for both cultivars, the total neutral sugar content in the AIR seemed to follow the same profile as the firmness (Fig. 6a), confirming Ben and Gaweda’s (1985) results.
Fig. 6. Changes in the total neutral sugar content in the alcohol-insoluble residue (—, NS AIR) (a) and in the water-soluble pectin extracts (—, NS WSP) (b) and the slope of the force-deformation curve (- -, S), obtained by penetrometry, of ‘Fuji’ () and ‘Golden Delicious’ (䊉) apples during storage.
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Fig. 7. Changes in the total neutral sugar content in the alcohol-insoluble residue (—, NS AIR) and the (- -) fondant of ‘Fuji’ apples (a) or the (- -) juiciness of ‘Golden Delicious’ apples (b) during storage.
Moreover, this biochemical parameter changed in the same way as juiciness of ‘Golden Delicious’ apples and in the opposite way to fondant of ‘Fuji’ (Fig. 7). As Yoshioka et al. (1994) showed during softening of ‘Red gold’ apples, the firmness of ‘Fuji’ and ‘Golden Delicious’ also changed according to the content of total neutral sugars in their WSP (Fig. 6b). Pena and Carpita (2004) observed that the major biochemical event associated with storage in four M. domestica cultivars is a marked decrease in arabinose content, which always precedes the loss of fruit firmness measured by both breaking strength and compression resistance. Tomato is the model system of choice for studying textural changes during ripening of fleshy fruit. Recently, Van Dijk et al. (2006) showed that polygalacturonase seems a more suitable candidate than -galactosidase to explain the firmness decrease. However, extrapolation of findings in tomato to other fruit species may not be true. Redgwell et al. (1997) showed temporal differences in fruit softening between eight species. ‘Braeburn’ and ‘Cox’s Orange Pippin’ apples softened to a much lesser degree than ‘Billington’ plum. Moreover, cell-walldegrading enzymes responsible for softening are not the same from one fruit species to another. Ripening avocado, tomato and peach fruit possess relatively high levels of polygalacturonase activity while it has been reported to be absent in other species including strawberry, apple and melon (Brummell and Harpster, 2001). Consequently, fruit softening may have to be treated on a case-by-case basis. Moreover, Brummell and Harpster (2001) highlighted that no single cell-wall-modifying enzyme can be identified as being necessary and sufficient for textural changes. Fruit softening is a multi-gene trait with each enzyme activity having its own role to play in textural changes. Goulao et al. (2007) identified different cell-wall-degrading enzymes during growth and ripening of ‘Mondial Gala’ apples. During the ripening of Capsicum annuum fruit, Priya Sethu et al. (1996) found that firmness of bell pepper decreases concomitantly with the increase in polygalacturonase activity and with the decrease in pectin methyl esterase. According to Prabha and Bhagyalakshmi (1998), amylase, pectinases, xylanase and
laminarinase in banana pulp may play a dominant role in the loosening of cell structure and tissue softening during ripening. However, even though several enzyme activities seem to operate during the entire ripening period of papaya fruit, Manrique and Lajolo (2004) observed that the period of greater firmness variation in papaya fruit did not match the periods in which the loss of galactose and the solubilisation of hemicellulose were greater. However, a decreasing value of the neutral sugars/acidic sugars ratio was observed by these authors during solubilisation, giving a key enzymatic role to a polygalacturonase in papaya softening. Ali et al. (2004) suggested that pectin methyl esterase and -galactosidase activity seem relevant and might contribute significantly to the observed variations in softening rates amongst tropical fruit. These previous studies revealed that fruit softening during storage is a complex process; the action of enzymes and proteins modifies cell-wall structure and consequently, fruit texture implying fruit softening. However, without transgenic experiments, it is difficult to determine which cell-wall-modifying enzymes are responsible of fruit softening. According to Owino et al. (2005), the individual roles of these enzymes are still being addressed in part through use of gene silencing techniques (antisense and co-suppression). But another technique is the use of “chimeric” genes to simultaneously silence several target enzymes in order to manipulate entire biochemical pathways. Proteomic strategies will also be useful and an additional approach in identifying genes regulating fruit texture is the use tomato pleiotropic mutations. 4. Conclusion Apple texture was studied by sensory, physical and chemical means in order to research the most relevant biochemical markers in relation to changes in different sensory properties of apple texture. Two cultivars with different textural characteristics were studied for five different storage periods. Fruit softening confirmed previous studies as well as the observed change in pectin composition. Indeed, major changes concern soluble pectin composition. This study showed that the most
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relevant biochemical marker which seemed linked to texture change was the galacturonic acid content analysed in the watersoluble pectin extracts. Indeed, high and positive correlation coefficients were observed between sensory mealiness and this marker while sensory crunchiness and instrumentally measured firmness were negatively correlated to this biochemical component. There were divergences between the two cultivars studied. The physical parameters of the texture of ‘Golden Delicious’ apples were more affected than those of ‘Fuji’ while the chemical composition of cell-wall pectin evaluated in higher extent for ‘Fuji’ than for ‘Golden Delicious’ apples. This means that other factors such as genetic and enzymatic metabolisms should be taken into account in further studies dealing with fruit softening mechanisms. It would be necessary to relate the observed changes in cell-wall pectin to the microscopic aspects (size and shape of the cells, thickness of the cell wall, etc.) of fruit tissues as well as cell turgidity (water content, osmotic pressure, volume of the intercellular space, etc.) in order to explain changes in fruit texture on a macroscopic scale. Acknowledgements This research was supported by the Conseil R´egional des Pays de la Loire. The authors are grateful to Marie-Jeanne Cr´epeau and Matthieu Vicaire for their contribution to this study. They would also like to thank St´ephanie Khaldi and Nad`ege Gibouin for their technical assistance as well as Ronan Symoneaux and the members of the trained sensory panel. We are grateful to the fruit-bearing test center “La Morini`ere” for providing fruit and storage facilities. References Abbott, J.A., Watada, A.E., Massie, D.R., 1984. Sensory and instrument measurement of apple texture. J. Am. Soc. Hort. Sci. 109, 221–228. AFNOR, 1995. Analyse sensorielle-Recherche et s´election de descripteurs pour l’´elaboration d’un profil sensorial, par approche multidimensionnelle. Contrˆole de la qualit´e des produits alimentaires—Analyse sensorielle, 5`eme e´ d. AFNOR, Paris, pp. 276–310. Ali, Z.M., Chin, L.-H., Lazan, H., 2004. A comparative study on wall degrading enzymes, pectin modifications and softening during ripening of selected tropical fruit. Plant Sci. 167, 317–327. Bartley, I.M., 1976. Changes in the glucans of ripening apples. Phytochemistry 15, 625–626. Ben, J., Gaweda, M., 1985. Changes of pectic compounds in Jonathan apples under various storage conditions. Acta Physiol. Plant 7, 45–54. Brummell, D.A., Harpster, M.H., 2001. Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Mol. Biol. 47, 311–340. Curry, E.A., 1989. Effect of harvest date and oxygen level on storability of late season apple cultivars. In: Fellman, J.K. (Ed.), Proceedings of the Fifth International CA Research Conferences. Wanatchee, Wasch, pp. 103–109. De Vries, J.A., Voragen, A.G.J., Rombouts, F.M., Pilnik, W., 1981. Extraction and purification of pectins from alcohol insoluble solids from ripe and unripe apples. Carbohydr. Polym. 1, 117–127. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Duprat, F., Grotte, M., Loonis, D., Pietri, E., 2000. Etude de la possibilit´e de mesurer simultan´ement la fermet´e de la chair et l’´epiderme des pommes. Sci. Alim. 20, 253–265.
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Redgwell, R.J., Fischer, M., Kendal, E., MacRae, E.A., 1997. Galactose loss and fruit ripening, high-molecular-weight arabinogalactans in the pectic polysaccharides of fruit cell walls. Planta 203, 174–181. Renard, C.M.G.C., Thibault, J.F., Voragen, A.G.J., 1990a. Etude chimique et enzymatique de la structure de la paroi de pomme. Cahier Scientifique et Technique, 341–347. Renard, C.M.G.C., Voragen, A.G.J., Thibault, J.F., Pilnik, W., 1990b. Studies on apple protopectin. I: Extraction of insoluble pectin by chemical means. Carbohydr. Polym. 12, 9–25. Renard, C.M.G.C., Searle van Leeuwen, M.J.F., Voragen, A.G.J., Thibault, J.F., Pilnik, W., 1991a. Studies on apple protopectin. II: Apple cell wall degradation by pure polysaccharides and their combinations. Carbohydr. Polym. 14, 295–314. Renard, C.M.G.C., Schols, H.A., Voragen, A.G.J., Thibault, J.F., Pilnik, W., 1991b. Studies on apple protopectin. III: Characterization of the material extracted by pure polysaccharidases from apple cell walls. Carbohydr. Polym. 15, 13–32. Renard, C.M.G.C., Voragen, A.G.J., Thibault, J.F., Pilnik, W., 1991c. Studies on apple protopectin. IV: Apple xyloglucans and influence of pectin extraction treatments on their solubility. Carbohydr. Polym. 15, 387–403. Renard, C.M.G.C., Voragen, A.G.J., Thibault, J.F., Pilnik, W., 1991d. Studies on apple protopectin. V: Structural studies on enzymatically extracted pectins. Carbohydr. Polym. 16, 137–154. Renard, C.M.G.C., Thibault, J.F., 1993. Studies on apple protopectin. VI: Extraction of pectins from apple cell walls with rhamnogalacturonase. Carbohydr. Polym. 22, 203–210. Renard, C.M.G.C., 2005. Variability in cell wall preparations, quantification and comparison of common methods. Carbohydr. Polym. 60, 512–522.
Saeman, J.F., Moore, W.E., Mitchell, R.L., Millett, M.A., 1954. Techniques for the determination of pulp constituents by quantitative paper chromatography. Tappi 37, 336–343. Siddiqui, S., Brackmann, A., Streif, J., Bangerth, F., 1996. Controlled atmosphere storage of apples, cell wall composition and fruit softening. J. Hort. Sci. 71, 613–620. Stow, J., 1995. Quality measurements of apples. Postharvest News Inf. 6, 32–33. Thibault, J.F., 1979. Automatisation du dosage des substances pectiques par la m´ethode au m´eta-hydroxydiph´enyl. Lebensm. -Wiss. Technol. 12, 247–251. Tollier, M.T., Robin, J.P., 1979. Adaptation de la m´ethode a` l’orcinol sulfurique au dosage automatique des glucides neutres totaux, conditions d’application aux extraits d’origine v´eg´etale. Ann. Technol. Agric. 28, 1–15. Varela, P., Salavdor, A., Fiszman, S., 2007. Changes in apple tissue with storage time: rheological, textural and microstructural analyses. J. Food Eng. 78, 622–662. Voragen, A.G.J., Schols, H.A., Pilnik, W., 1986. Determination of the degree of methylation and acetylation of pectins by h.p.l.c. Food Hydrocolloid 1, 65–70. Van Dijk, C., Boeriu, C., Stolle-Smits, T., Tijskens, L.M.M., 2006. The firmness of stored tomatoes (cv Tradiro). 2: Kinetic and Near Infrared models to describe pectin degrading enzymes and firmness loss. J. Food Eng. 77, 585–593. Yoshioka, H., Aoba, K., Kashimura, Y., 1992. Molecular weight and degree of methoxylation in cell wall polyuronide during softening in pear and apple fruit. J. Am. Soc. Hort. Sci. 117, 600–606. Yoshioka, H., Kashimura, Y., Kaneko, K., 1994. Solubilization and distribution of neutral sugar residues derived from polyuronides during the softening in apple fruit. J. Jpn. Soc. Hort. Sci. 63, 173–182.
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Relationship between texture and pectin composition of two apple cultivars during storage Ludivine Billy a,b , Emira Mehinagic a,∗ , Ga¨elle Royer a , Catherine M.G.C. Renard c , Ga¨elle Arvisenet b , Carole Prost b , Fr´ed´erique Jourjon a a
Ecole Sup´erieure d’Agriculture, Groupe de Recherche en Agro-industrie sur les Produits et les Proc´ed´es (GRAPPE), 55 rue Rabelais, B.P. 30 748, 49007 Angers Cedex 01, France b ENITIAA, UMR CNRS GEPEA 6144, Laboratoire de Biochimie, rue de la G´ eraudi`ere, B.P. 82 225, 44322 Nantes Cedex 03, France c UMR A 408 INRA, Universit´ e d’Avignon, SQPOV, S´ecurit´e et Qualit´e des Produits d’Origine V´eg´etale, Domaine St. Paul, 84 914 Avignon Cedex 09, France Received 3 April 2007; accepted 13 July 2007
Abstract The texture of two apple cultivars was characterised by sensory and instrumental methods for five different storage periods. The aim of this study was to explain the changes in apple texture during storage by different physical (penetrometry, compression) and chemical measurements (extraction and analysis of pectin composition). The emphasis was on determining the most relevant biochemical markers in relation to different sensory properties of apple texture. Contrary to ‘Fuji’, ‘Golden Delicious’ fruit softened easily during storage, became mealy and had higher neutral sugar concentrations in their alcohol-insoluble residues (AIR) and more galacturonic acid in the water-soluble pectin extracts (WSP). The most relevant biochemical marker linked to texture change was the galacturonic acid content in the water-soluble pectin extracts. High and positive correlation coefficients were observed between sensory mealiness (R = 0.84) and galacturonic acid content in the WSP while, sensory crunchiness and instrumentally measured firmness were negatively correlated with this component. The total neutral sugar content in the alcohol-insoluble residues and in the water-soluble pectin fractions also changed with apple texture properties. © 2007 Elsevier B.V. All rights reserved. Keywords: Apple; Malus domestica Borkh; Texture; Cell wall; Mechanical properties; Sensory analysis
1. Introduction Apple texture is one of the most important quality properties that influence consumer acceptability of these fruit (Stow, 1995). Thus, postharvest softening of apples during storage and market distribution is a serious commercial problem resulting in quality losses for growers and distributors. Therefore, our research aims to explain the development of apple softening during storage using different physical (penetrometry, compression) and chemical measurements (extraction and analysis of pectin composition). Knowledge of the mechanism of fruit tissue softening, cellwall structure and cell-wall breakdown is very important for understanding and improving the texture and quality of apple
∗
Corresponding author. Tel.: +33 2 41 23 55 55; fax: +33 2 41 23 55 65. E-mail address: [email protected] (E. Mehinagic).
0925-5214/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2007.07.011
fruit. A better understanding of the relationship between fruit texture and biochemical composition could lead to improvements in quality control and process design in the food industry and the marketplace. Many studies have already addressed either the changes in apple texture during storage (Abbott et al., 1984; Grotte et al., 2001; Johnston et al., 2001; Mehinagic et al., 2004; Varela et al., 2007) or changes in pectin composition during softening (Yoshioka et al., 1994; Massiot et al., 1996; Nara et al., 2001), but few studies tried to connect these two phenomena. Moreover, for the majority of these, sensory perception of changes in fruit texture was never carried out. Ben and Gaweda (1985) showed high correlation coefficients between the content of protopectin (non-cellulosic neutral sugar residues) and the firmness of ‘Jonathan’ apples in one storage season only. A study performed on ‘Golden Delicious’ apples attempted to determine the possible contribution of changes in cell-wall composition to fruit softening under various storage conditions (Siddiqui et al., 1996). More recently, Pena and Carpita (2004) reported a study
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of the chronology of the biochemical events associated with loss of firm texture and cell separation, and Lo Scalzo et al. (2005) described the results of research on pectin composition just after reddening and after cold storage of reddened ‘Annurca’ apple fruit in different atmospheres. Loss of firm texture and cell adhesion are processes associated with ripening that affect fruit quality and postharvest storage. The cell walls of fruit have received considerable attention, as changes occurring in their structure and composition are clearly determining in fruit firmness (Knee, 1973a,b; Bartley, 1976; De Vries et al., 1981; Renard et al., 1990b, 1991a,b,c,d; Renard and Thibault, 1993; Massiot et al., 1994). Indeed, it is well known that separation and degradation of water-soluble pectic substances, erosion of the middle lamella and disintegration of the primary cell wall lead to softening of fruit flesh. Pectic substances are the most abundant class of macromolecules within the cell-wall matrix and are the main components of the middle lamellae. They have a role in the adhesion between cells, and in regulation of intercellular adhesion. Consequently, pectic substances have been assigned an important role in changes in texture of fruit tissues (Yoshioka et al., 1992; Grant Reid, 1997) and notably, changes in pectin composition have been implicated in decreased adhesion between cells (Ilker and Szczesniak, 1990). During fruit softening, pectins typically undergo solubilisation and depolymerisation, which are thought to contribute to wall loosening and disintegration. In apple, there is usually an increase in water-soluble pectin (WSP), and a decrease in galactose and arabinose residues (Knee, 1973b), with little depolymerisation occurring in any pectin fraction during ripening (Yoshioka et al., 1992; Fischer and Amado, 1994; Fischer et al., 1994). However, the association of biochemical changes with changes in fruit texture is still unclear; for example, it is still not known if the processes of pectin solubilisation and loss of galactose are causal, coincidental, or a consequence of apple softening (Redgwell et al., 1997). We aimed to study the relationship between texture and pectin composition of stored apples. Two cultivars with different textural characteristics were studied in order to investigate whether the differences in texture between different apple cultivars were due to differences in their pectin composition. ‘Fuji’ is a crunchy and juicy apple that stores well and ‘Golden Delicious’ apples become mealy during storage (Mehinagic et al., 2004). The apples were stored for up to 7 months at 2 ◦ C. Their texture was assessed using two deformation tests (penetrometry and compression) and sensory analysis. At the same time, cell-wall content and pectin extractability of these fruit were characterised in order to try to relate texture modifications and sensory perceptions to different chemical changes occurring in apple cell walls. 2. Materials and methods 2.1. Fruit Two different apple cultivars (Malus domestica Borkh), ‘Fuji’ (FU) and ‘Golden Delicious’ (GO), were studied. One hundred and forty fruit of each cultivar harvested in 2004
at commercial maturity were provided by a fruit-bearing test center (“La Morini`ere”, Saint Epain, France). Their ripening stage was verified by starch regression measurements. These fruit were selected on the basis of uniformity and absence of damage or blemishes. They were all stored in the same cool room at 2 ◦ C and under ambient atmospheric pressure and humidity for five different periods (1, 2, 3, 4 and 7 months). The choice of these storage periods was based on a previous study which showed that they resulted in a large range of apple mechanical properties (Mehinagic et al., 2004). For each storage period, 28 fruit per cultivar were brought up to room temperature 24 h before being analysed. Half of each fruit (n = 28) was analysed by sensory panel and the second half by penetrometry (n = 14) or compression (n = 14). Sensory and mechanical methods were carried out simultaneously and immediately after the apples were cut. For penetrometry and compression tests, apples were manually and carefully peeled by the same operator using the same vegetable knife. Fruit analysed by penetrometry were then sampled (taking care to remove the crushed part before sampling) and freeze-dried prior to chemical analysis. 2.2. Sensory evaluation The sensory panel included 14 permanent trained panellists from the staff of the Ecole Sup´erieure d’Agriculture (ESA, Angers, France). These panellists, selected in 1999, are trained every week to evaluate apple texture according to the recommendations of AFNOR (1995) and of Fortin and Desplancke (1998). Their performances are evaluated once a year to ensure reliability. The sensory attributes studied on apple flesh were crunchiness, juiciness, mealiness, chewiness and fondant. These sensory attributes are defined and described in Table 1. During a sensory session, each panellist peeled and analysed two different half fruit from each cultivar. The washed unpeeled half apples, coded by a random three-digit code, were randomly presented to the panellists, at room temperature and under red light illumination to avoid apple cultivar identification. A 10 cm line scale was used for evaluation. The left end of the scale corresponded to the lowest intensity (value 0) and the right end to the highest (value 10). Each panellist used mineral water as a rinse between sample analyses. Five sessions, corresponding to five storage periods, were organised. In this way, each panellist analysed 2 cultivars × 2 half apples during each session. Thus, Table 1 Texture sensory descriptors used for apples Texture attribute
Definition (Mehinagic et al., 2003)
Crunchiness
Force required for the first bite and the noise resulting from this bite Amount of liquid released on mastication just before swallowing Dry and crumbly texture Time and number of chewing movements needed to grind the sample prior to swallowing Force required to crush a piece of apple between the tongue and palate
Juiciness Mealiness Chewiness Fondant
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Fig. 1. The penetrometric curve obtained on peeled half apples, using the MTS (Synergie 200H) traction machine (cylindrical probe with a 4 mm diameter convex tip; penetration speed of 50 mm min−1 ; depth of 10 mm) (FLC: flesh limit compression force; S: slope of the force-deformation curve; D: deformation associated with flesh limit compression force; WFLC : work associated with FLC; W7 : work required to attain a flesh deformation of 7 mm).
for each cultivar and each storage period, 28 fruit samples were analysed. 2.3. Penetrometry According to Mehinagic et al. (2004), a cylindrical probe with a 4 mm diameter convex tip was used to perforate peeled half apples in an MTS (Synergie 2000H) traction machine. Peeled half apples were placed with the flat side facing down on the plate and punched on their rounded side in an equatorial position. The equipment used detected the sample automatically; penetration speed was set at 50 mm min−1 , and the test was stopped after penetration to 10 mm. This distance was short enough to avoid any influence of the core in the measurement. Fifty points were recorded during a penetrometric test. Penetrometry curves were analysed and five parameters were studied (Fig. 1): flesh limit compression force (FLC), deformation associated with flesh limit compression force (D), slope of the force-deformation curve (S), work associated with FLC (WFLC ) and work required to attain a flesh deformation of 7 mm (W7 ) (Duprat et al., 2000). Definitions of these parameters are given in Table 2. For each cultivar and each storage period, 14 fruit samples were analysed. 2.4. Compression According to Mehinagic et al. (2004), two parallel plates (50 mm of diameter and 7 mm of thickness) were used to compress peeled half apples. These were placed with the flat side facing down on the plate, with the equatorial section of their rounded side targeted in the same MTS (Synergie 2000H) traction machine. The equipment detected the diameter and height of the sample automatically: the height of peeled half apples was
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Fig. 2. Double compression force/time curve obtained on peeled half apples, using an MTS (Synergie 200H) traction machine (2 parallel plates; 50 mm min−1 ; 7 mm) (1: the first compression; 2: the second compression; H: maximal force; WH : surface area under the compression curve; S: slope of the compression).
compressed by 20% and the loading rate of the crosshead was 50 mm min−1 . Two successive compressions were carried out on each sample. Compression curves were analysed and six parameters were studied (Fig. 2): maximal force associated with the first compression, usually described as hardness (H1 ), maximal force associated with the second compression (H2 ), surface area under the first compression curve (WH1 ), surface area under the second compression curve (WH2 ), slope of the first compression (S1 ) and slope of the second compression (S2 ). For each cultivar and each storage period, 14 fruit samples were analysed. 2.5. Apple powder preparation Peeled apple pieces were freeze-dried, then frozen with liquid N2 and immediately homogenised in a commercial blender for 1 min. The apple powders obtained were prepared in triplicate for each batch (2 cultivars × 5 storage durations) and stored in a desiccator under vacuum prior to chemical analysis. 2.6. Alcohol-insoluble residue (AIR) preparation Cell walls were prepared as alcohol-insoluble residues for each sample triplicate. According to Renard (2005), the apple powder (7 g) was blended with 50 mL of 70% ethanol in an empty 75 mL Sep-pack prep column (InterchimTM ) equipped with a sinter, porosity 20 m. The mixture was stirred at room temperature for 20 min then filtered under vacuum. The residue was re-extracted with 70 mL of 70% ethanol eight times, as described above. At the end of these washings, the filtrates were sugar-free (absence of sugars was tested by the phenol-sulphuric
Table 2 Definitions of penetrometric parameters Parameters
Calculation
Definitions (Duprat et al., 2000; Grotte et al., 2001)
FLC (N) D (mm) S (N mm−1 ) WFLC (N mm) W7 (N mm)
Maximal force before the drop in force at the moment of penetration Deformation associated with FLC Gradient on the curve (between 0 and FLC) Area under the curve between 0 and D Area under the curve between 0 and 7 mm
Force representing the limit of flesh elasticity Gradient measuring penetrometric firmness Work required for rupture of the flesh Work required to attain a flesh deformation of 7 mm
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method of Dubois et al., 1956). The alcohol-insoluble residues were dried by solvent exchange with 96% ethanol (three times), and acetone (three times), and finally overnight in an oven at 40 ◦ C. 2.7. Water-soluble pectin extraction From each AIR preparation, water-soluble pectin was extracted by water. For each extraction, approximately 100 mg of cell-wall preparation and 6 mL of water (Renard, 2005) were incubated for 2 h at room temperature in an empty 8 mL Sep-pack prep column (Interchim TM ) equipped with a sinter, porosity 20 m, under slow planetary agitation. After incubation, the extract and cell walls were separated by filtration under vacuum. This extraction was repeated three times for each AIR preparation; the three water extracts were pooled and frozen before analysis. 2.8. Analytical methods Galacturonic acid content (GA) was determined by automated m-hydroxydiphenyl assays on an Alliance Instruments (M´ery sur Oise, France) autoanalyser after sulphuric acid prehydrolysis and hydrolysis (26N sulphuric acid, 1 h, room temperature followed by 2N, 3 h, 100 ◦ C) for the alcoholinsoluble residues (Saeman et al., 1954), or saponification (0.1N NaOH, 30 min, room temperature) for water-soluble pectin extracts (Thibault, 1979). The results are expressed as anhydrogalacturonic acid. Total neutral sugar content (NS) was determined by the orcinol method (Tollier and Robin, 1979) on the same hydrolysates and saponified pectin solutions. Methanol was determined according to Klavons and Bennett (1986). The degree of methylation (DM) was calculated as the molar ratio of methanol to galacturonic acid. The degree of acetylation (DA) was determined by high performance liquid chromatography after saponification according to the method of Voragen et al. (1986). 2.9. Statistical analysis Data acquisition and statistical treatment were performed with Statgraphics Plus 5.1 software (Sigmaplus, Toulouse,
France). The analysis of variance (ANOVA) was carried out independently for each cultivar and for each of the studied variables measured by sensory analysis, penetrometry, compression and pectin analysis. Two-way ANOVA was performed to determine significant differences in sensory quality between apples. The factors studied were the panellist effect and the storage period. An interaction of these two factors was evaluated for each cultivar and each sensory attribute to make sure that these main effects were not confused. One-way ANOVA was carried out on instrumental measurements (penetrometry, compression and pectin analysis) to test the effects of storage duration on each cultivar. For each analysis, a significant level of 5% was used. To compare the effect of 5 storage periods on the different attributes of the two cultivars, Fischer’s least significant difference (LSD) procedures were applied separately to each cultivar. Principal component analysis (PCA) was carried out on the averaged data. Pearson’s correlation coefficients between texture parameters and biochemical data were calculated between the average values. 3. Results and discussion 3.1. Effect of storage duration on sensory attributes and mechanical parameters of fruit texture Variance analysis was carried out independently for each of the five sensory descriptors studied. For all sensory descriptors, no interactions were found between panellist effect and storage period for ‘Fuji’ apples while two significant interactions were found for ‘Golden Delicious’. These two interactions involving mealiness and fondant can be explained by heterogeneity within a fruit batch or differences in sensitivity between panellists. For ‘Golden Delicious’ apples, the storage effect was significant for all descriptors except chewiness. Texture change in ‘Fuji’ was more limited and the storage effect was significant for three descriptors at the 5% level: mealiness, juiciness and fondant. For both cultivars, the intensity of juiciness decreased while the intensity of mealiness increased (Table 3). These results confirmed those of Mehinagic et al. (2004). According to Holt and Schoorl (1984), these changes are related to the deterioration of the mechanical strength of apple tissue during storage.
Table 3 Evolution of sensory quality of 2 apple cultivars during storage Storage (months)/cultivar
Crunchiness
Juiciness
Mealiness
Chewiness
Fondant
1/Fuji 2/Fuji 3/Fuji 4/Fuji 7/Fuji
7.6 7.9 7.6 7.5 7.5
6.9ab
1.8b
6.2c
2.0ab
7.0a 6.5bc 6.2c
1.7b 1.8b 2.3a
5.7 5.8 5.3 5.3 5.3
2.9ab 2.4b 3.1a 3.5a 3.5a
1/Golden 2/Golden 3/Golden 4/Golden 7/Golden
4.5ab 4.6ab 4.2bc 3.7c 4.9a
4.9a 4.3b 4.5ab 3.9b 3.9b
3.9b 3.7b 4.0b 4.8a 4.8a
4.2 4.1 3.8 3.6 4.2
5.5c 5.4c 6.6ab 6.8a 6.1bc
Different letters (a–c) in a column within a cultivar denote values that are significantly different at P < 0.05.
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319
Fig. 3. Slope of the force-deformation curve (S), obtained by penetrometry (a) and maximal force associated with the second compression (H2 ) (b), of ‘Fuji’ (grey columns) and ‘Golden Delicious’ apples (white columns) after different periods of storage (identical letters on columns for the same cultivar indicate that there is no significant difference with P > 0.05).
It appeared that storage had a significant effect for all penetrometric parameters and for both cultivars. For ‘Golden Delicious’ apples, the effect of storage was also significant for all compression parameters while ‘Fuji’ apples seem more resistant. This could be explained by the better texture conservation of ‘Fuji’ apples. Indeed, Jobling and McGlasson (1995) and Curry (1989) showed that ‘Fuji’ apples maintain their firm texture during storage. As all penetrometric parameters were strongly correlated, only the changes in the slope of the force-deformation curve (S), corresponding to firmness, are represented in Fig. 3a. In this figure, it can be seen that fruit firmness decreased significantly between the second and third month of storage for ‘Fuji’ apples. For ‘Golden Delicious’ fruit, this parameter did not change throughout the 7 months storage period. In Fig. 3b, the maximal force (describing hardness) associated with the second compression is shown for each storage step. The hardness of ‘Fuji’ did not change at all, while ‘Golden Delicious’ apples hardness decreased between the first and second month of storage. Instrumental measurements and sensory analysis showed a good concordance, with a general decrease in fruit firmness during storage and better texture conservation of ‘Fuji’ apples. Moreover, both methods showed that ‘Fuji’ apples were firmer
than ‘Golden Delicious’. These results were in accordance with previous studies. They confirmed (Grotte et al., 2001; Mehinagic et al., 2004) that ‘Fuji’ and ‘Golden Delicious’ apples present mechanical and sensory textural differences. For the temporal aspects, Mehinagic et al. (2004) found a significant change in firmness between apples stored for 3 weeks and those stored for 20 or 30 weeks but they found no significant difference between 20 and 30 weeks of storage. Indeed, according to Johnston et al. (2001), the decrease in firmness for many apple cultivars can be characterised by a curve consisting of three distinct phases. Firstly, fruit soften slowly, then more rapidly, and finally slowly again. 3.2. Effect of storage duration on apple pectin extractability For ‘Fuji’ apples, the storage effect was significant for five biochemical parameters: the total neutral sugar content and the degree of methylation in the AIR, the composition of the WSP extracts (primarily galacturonic acid, less total neutral sugars) and its degree of methylation. For ‘Golden Delicious’ apples, three parameters changed significantly during storage: the galacturonic acid content and the total neutral sugar content in the AIR and the degree of methylation in the WSP fractions.
Table 4 Composition of different apple pectin extracts during storage Alcohol-insoluble residues composition
Water-soluble pectin composition
Storage (months)/cultivar
GA (mg/g)
DM (%)
DA (%)
NS (mg/g)
GA (mg/g)
DM (%)
NS (mg/g)
1/Fuji 2/Fuji 3/Fuji 4/Fuji 7/Fuji
292 270 276 290 286
75a 73a 75a 72ab 69b
5 5 5 5 5
694a 666ab 629b 582c 567c
8b 7b 10b 23a 23a
52b 70a 75a 56b 75a
29a 13b 17b 16b 15b
1/Golden 2/Golden 3/Golden 4/Golden 7/Golden
252b 276a 290a 275a 285a
75 75 74 73 74
6 6 5 6 5
716a 676b 676b 622c 668b
36 35 35 29 31
53c 62b 66b 81a 80a
18 19 16 15 16
Different letters in a column (a–c) within a cultivar denote values that are significantly different at P < 0.05. GA: Galacturonic acid; DM: degree of methylation; DA: degree of acetylation; NS: neutral sugar.
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The values obtained (Table 4) were close to those in the literature. Renard et al. (1990a) and Renard (2005) found that the galacturonic acid content of alcohol-insoluble residues of ‘Golden Delicious’ apples is generally between 250 and 277 mg/g AIR. In the same way, the values of galacturonic acid content of water-soluble pectin extracts agreed with those of Fischer et al. (1994). While the total neutral sugar contents in the WSP fractions were in accordance with those in the literature (Massiot et al., 1996), the total neutral sugar content in the AIR of each cultivar seemed to be higher (Fischer and Amado, 1994). This can be explained by the different methods used: these authors analysed the individual neutral sugar by gas chromatography and we analysed it by the orcinol method, which is known to over-estimate the results, the calibration being done with a hexose. The values for the degree of methylation of the pectic substances of AIR (69–75%) were totally in accordance with those reported by De Vries et al. (1981) (80%) and Renard et al. (1990b) (72%), just as for the values for the degree of acetylation (<10%) (Voragen et al., 1986). In the same way, the degree of methylation of the pectic substances of WSP agreed with previous studies (Renard et al., 1990a; Fischer et al., 1994; Massiot et al., 1996). In the AIR (Table 4), except for a significant increase between the first and second month of storage for ‘Golden Delicious’ apples, no trend of an increase or a decrease in the galacturonic acid content during fruit storage was noticeable for both cultivars. This observation was also valid for the degree of methylation of apples even if a significant decrease in this one was observed at the end of ‘Fuji’ apple storage. Moreover, no changes were observed for the degree of acetylation. All these results were in agreement with those in literature (Fischer and Amado, 1994). However, the total neutral sugar content significantly decreased during apple storage. For both cultivars, this decrease was more marked between the third and fourth month of storage and between the first and second month of storage for ‘Golden Delicious’. These results were in accordance with Fischer and Amado (1994). According to Yoshioka et al. (1994), this decrease is more specifically due to a decline in galactose and arabinose contents. In the WSP fractions (Table 4), for ‘Fuji’ apples, the content of galacturonic acid increased significantly between the third and fourth month of storage (Yoshioka et al., 1994; Massiot et al., 1996; Lo Scalzo et al., 2005). This phenomenon, corresponding to pectin solubilisation, might result from the enzymatic cleavage of linkages between pectin and other cell-wall components rather than from the degradation of pectin chains. Contrary to the literature (Siddiqui et al., 1996; Nara et al., 2001) there was no significant change in ‘Golden Delicious’. The degree of methylation increased along with storage period. This increase was particularly significant between the first and second month of storage for both cultivars, and between the third and fourth month of storage for ‘Golden Delicious’ apples. These results confirm previous observations (Massiot et al., 1996) concerning the biosynthesis of highly methylated pectin during apple storage. The content of total neutral sugars decreased during storage for ‘Fuji’ apples (Massiot et al., 1996; Nara et al., 2001). This change was particularly significant between the first and second
month of storage. As previously shown by Massiot et al. (1996), this decrease more particularly involves arabinose and galactose contents. Yoshioka et al. (1994) suggested that these two types of polyuronide may be lost from the side-chain polysaccharides of the polyuronide molecules and may be involved in the solubilisation of polyuronides. Contrary to ‘Fuji’ apples, this change was not significant for ‘Golden Delicious’, as shown by Nara et al. (2001). To summarise, the total neutral sugar content in the AIR, the composition of the WSP extracts (galacturonic acid and total neutral sugars) and its degree of methylation showed the most obvious changes during apple storage. Moreover, we showed that the total neutral sugar content in the AIR and its degree of acetylation, and the composition of the WSP fractions (galacturonic acid, total neutral sugars) of ‘Golden Delicious’ apples were significantly different from those of ‘Fuji’. Consequently, these five biochemical parameters could explain the mechanical and sensory textural differences observed previously between these two cultivars and storage periods. 3.3. Relationships between apple mechanical properties, texture sensory attributes and biochemical composition of cell-wall pectins A PCA analysis was carried out on sensory, penetrometry, compression and biochemical parameters. The first dimension (PC1) described 66.5% of the variation amongst samples, and the second (PC2) 15% (Fig. 4). Sensory parameters juiciness, crunchiness and chewiness were highly related to the mechanical parameters (firmness, hardness, slope of the force-deformation curve) and negatively linked to mealiness and fondant. These two
Fig. 4. PCA plot of the two cultivars (‘Fuji’ and ‘Golden Delicious’) analysed at five storage periods by five sensory attributes, four parameters measured by penetrometry, six parameters measured by compression and seven biochemical parameters in the plane defined by the first two principal components (GOX : ‘Golden Delicious’ apples stored for X months; FUX : ‘Fuji’ apples stored for X months).
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Table 5 Pearson’s correlation coefficients between instrumental attributes measured by mechanical parameters, sensory descriptors for texture, and biochemical parameters for both cultivars (coefficients inferior to 0.6 were omitted) Biochemical parameters
GA WSP DA AIR
Mechanical parameters
Texture sensory descriptors
S
H1
H2
S1
S2
Crunchiness
Juiciness
Mealiness
Chewiness
Fondant
−0.94 −0.68
−0.85 −0.64
−0.85 −0.63
−0.88
−0.78 −0.63
−0.89 −0.80
−0.86 −0.76
0.84 0.81
−0.90 −0.75
0.89 0.73
GA: Galacturonic acid; WSP: water-soluble pectin; DA: degree of acetylation; AIR: alcohol-insoluble residue.
Fig. 5. Changes in the galacturonic acid content in the water-soluble pectin extracts (—, GA WSP) and the maximal force associated with the second compression (- -, H2 ) (a) or (- -) fondant (b) of ‘Fuji’ apples during storage.
attributes were related to the content of galacturonic acid in the WSP extracts. Highly negative Pearson’s correlation coefficients were found between the content of galacturonic acid in the WSP extracts, mechanical parameters and fruit juiciness, crunchiness and chewiness (Table 5). Indeed, the content of galacturonic acid in water-soluble pectin extracts of ‘Fuji’ apples increased at the same time as the hardness (H2 ) decreased or as the fondant increased (Fig. 5). In the same way, correct Pearson’s correlation
coefficients were obtained between the degrees of acetylation in the AIR and fruit texture parameters. Peasron’s correlation coefficients between mechanical and sensory parameters of texture and the total neutral sugar content in the AIR and in the WSP extracts were not high enough to be significant. However, for both cultivars, the total neutral sugar content in the AIR seemed to follow the same profile as the firmness (Fig. 6a), confirming Ben and Gaweda’s (1985) results.
Fig. 6. Changes in the total neutral sugar content in the alcohol-insoluble residue (—, NS AIR) (a) and in the water-soluble pectin extracts (—, NS WSP) (b) and the slope of the force-deformation curve (- -, S), obtained by penetrometry, of ‘Fuji’ () and ‘Golden Delicious’ (䊉) apples during storage.
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Fig. 7. Changes in the total neutral sugar content in the alcohol-insoluble residue (—, NS AIR) and the (- -) fondant of ‘Fuji’ apples (a) or the (- -) juiciness of ‘Golden Delicious’ apples (b) during storage.
Moreover, this biochemical parameter changed in the same way as juiciness of ‘Golden Delicious’ apples and in the opposite way to fondant of ‘Fuji’ (Fig. 7). As Yoshioka et al. (1994) showed during softening of ‘Red gold’ apples, the firmness of ‘Fuji’ and ‘Golden Delicious’ also changed according to the content of total neutral sugars in their WSP (Fig. 6b). Pena and Carpita (2004) observed that the major biochemical event associated with storage in four M. domestica cultivars is a marked decrease in arabinose content, which always precedes the loss of fruit firmness measured by both breaking strength and compression resistance. Tomato is the model system of choice for studying textural changes during ripening of fleshy fruit. Recently, Van Dijk et al. (2006) showed that polygalacturonase seems a more suitable candidate than -galactosidase to explain the firmness decrease. However, extrapolation of findings in tomato to other fruit species may not be true. Redgwell et al. (1997) showed temporal differences in fruit softening between eight species. ‘Braeburn’ and ‘Cox’s Orange Pippin’ apples softened to a much lesser degree than ‘Billington’ plum. Moreover, cell-walldegrading enzymes responsible for softening are not the same from one fruit species to another. Ripening avocado, tomato and peach fruit possess relatively high levels of polygalacturonase activity while it has been reported to be absent in other species including strawberry, apple and melon (Brummell and Harpster, 2001). Consequently, fruit softening may have to be treated on a case-by-case basis. Moreover, Brummell and Harpster (2001) highlighted that no single cell-wall-modifying enzyme can be identified as being necessary and sufficient for textural changes. Fruit softening is a multi-gene trait with each enzyme activity having its own role to play in textural changes. Goulao et al. (2007) identified different cell-wall-degrading enzymes during growth and ripening of ‘Mondial Gala’ apples. During the ripening of Capsicum annuum fruit, Priya Sethu et al. (1996) found that firmness of bell pepper decreases concomitantly with the increase in polygalacturonase activity and with the decrease in pectin methyl esterase. According to Prabha and Bhagyalakshmi (1998), amylase, pectinases, xylanase and
laminarinase in banana pulp may play a dominant role in the loosening of cell structure and tissue softening during ripening. However, even though several enzyme activities seem to operate during the entire ripening period of papaya fruit, Manrique and Lajolo (2004) observed that the period of greater firmness variation in papaya fruit did not match the periods in which the loss of galactose and the solubilisation of hemicellulose were greater. However, a decreasing value of the neutral sugars/acidic sugars ratio was observed by these authors during solubilisation, giving a key enzymatic role to a polygalacturonase in papaya softening. Ali et al. (2004) suggested that pectin methyl esterase and -galactosidase activity seem relevant and might contribute significantly to the observed variations in softening rates amongst tropical fruit. These previous studies revealed that fruit softening during storage is a complex process; the action of enzymes and proteins modifies cell-wall structure and consequently, fruit texture implying fruit softening. However, without transgenic experiments, it is difficult to determine which cell-wall-modifying enzymes are responsible of fruit softening. According to Owino et al. (2005), the individual roles of these enzymes are still being addressed in part through use of gene silencing techniques (antisense and co-suppression). But another technique is the use of “chimeric” genes to simultaneously silence several target enzymes in order to manipulate entire biochemical pathways. Proteomic strategies will also be useful and an additional approach in identifying genes regulating fruit texture is the use tomato pleiotropic mutations. 4. Conclusion Apple texture was studied by sensory, physical and chemical means in order to research the most relevant biochemical markers in relation to changes in different sensory properties of apple texture. Two cultivars with different textural characteristics were studied for five different storage periods. Fruit softening confirmed previous studies as well as the observed change in pectin composition. Indeed, major changes concern soluble pectin composition. This study showed that the most
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relevant biochemical marker which seemed linked to texture change was the galacturonic acid content analysed in the watersoluble pectin extracts. Indeed, high and positive correlation coefficients were observed between sensory mealiness and this marker while sensory crunchiness and instrumentally measured firmness were negatively correlated to this biochemical component. There were divergences between the two cultivars studied. The physical parameters of the texture of ‘Golden Delicious’ apples were more affected than those of ‘Fuji’ while the chemical composition of cell-wall pectin evaluated in higher extent for ‘Fuji’ than for ‘Golden Delicious’ apples. This means that other factors such as genetic and enzymatic metabolisms should be taken into account in further studies dealing with fruit softening mechanisms. It would be necessary to relate the observed changes in cell-wall pectin to the microscopic aspects (size and shape of the cells, thickness of the cell wall, etc.) of fruit tissues as well as cell turgidity (water content, osmotic pressure, volume of the intercellular space, etc.) in order to explain changes in fruit texture on a macroscopic scale. Acknowledgements This research was supported by the Conseil R´egional des Pays de la Loire. The authors are grateful to Marie-Jeanne Cr´epeau and Matthieu Vicaire for their contribution to this study. They would also like to thank St´ephanie Khaldi and Nad`ege Gibouin for their technical assistance as well as Ronan Symoneaux and the members of the trained sensory panel. We are grateful to the fruit-bearing test center “La Morini`ere” for providing fruit and storage facilities. References Abbott, J.A., Watada, A.E., Massie, D.R., 1984. Sensory and instrument measurement of apple texture. J. Am. Soc. Hort. Sci. 109, 221–228. AFNOR, 1995. Analyse sensorielle-Recherche et s´election de descripteurs pour l’´elaboration d’un profil sensorial, par approche multidimensionnelle. Contrˆole de la qualit´e des produits alimentaires—Analyse sensorielle, 5`eme e´ d. AFNOR, Paris, pp. 276–310. Ali, Z.M., Chin, L.-H., Lazan, H., 2004. A comparative study on wall degrading enzymes, pectin modifications and softening during ripening of selected tropical fruit. Plant Sci. 167, 317–327. Bartley, I.M., 1976. Changes in the glucans of ripening apples. Phytochemistry 15, 625–626. Ben, J., Gaweda, M., 1985. Changes of pectic compounds in Jonathan apples under various storage conditions. Acta Physiol. Plant 7, 45–54. Brummell, D.A., Harpster, M.H., 2001. Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Mol. Biol. 47, 311–340. Curry, E.A., 1989. Effect of harvest date and oxygen level on storability of late season apple cultivars. In: Fellman, J.K. (Ed.), Proceedings of the Fifth International CA Research Conferences. Wanatchee, Wasch, pp. 103–109. De Vries, J.A., Voragen, A.G.J., Rombouts, F.M., Pilnik, W., 1981. Extraction and purification of pectins from alcohol insoluble solids from ripe and unripe apples. Carbohydr. Polym. 1, 117–127. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Duprat, F., Grotte, M., Loonis, D., Pietri, E., 2000. Etude de la possibilit´e de mesurer simultan´ement la fermet´e de la chair et l’´epiderme des pommes. Sci. Alim. 20, 253–265.
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