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Improving the hardness of thermally processed carrots by selective pretreatments
Food Research International 43 (2010) 1297–1303
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Improving the hardness of thermally processed carrots by selective pretreatments Ans De Roeck, Jan Mols, Daniel Ndaka Sila, Thomas Duvetter, Ann Van Loey, Marc Hendrickx * Laboratory of Food Technology and Leuven Food Science and Nutrition Research Centre (LFoRCe), Department of Microbial and Molecular Systems (M2S), Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium
a r t i c l e
i n f o
Article history: Received 24 August 2009 Accepted 8 March 2010
Keywords: Carrot Texture Thermal processing b-Elimination Degree of pectin methyl-esterification pH Ferulic acid
a b s t r a c t The aim of this study was to improve the texture of thermally processed carrots by selective pretreatments modifying plant-intrinsic properties. Pretreatments were a combination of a thermal or high-pressure (HP) treatment followed by a 1 h soak in a specific solution. Lowering the degree of methylesterification (DM) of the carrot pectin was confirmed to be one strategy to reduce texture degradation. The thermal or HP pretreatment resulted in pectin with a lower DM which is less susceptible to b-eliminative depolymerization. A subsequent Ca2+ soak resulted in an even better texture by enhancing the amount of pectin cross-links within the cell wall. Lowering the pH of the carrots was proven to be another strategy. A thermal or HP pretreatment followed by soaking carrots in solutions of low pH proved to be effective in lowering the internal carrot pH, hereby retarding b-elimination and consequently texture degradation. The composition of the low pH solution was shown to be of importance; soak solutions containing cations and/or Ca2+ complexing agents have to be avoided. Ferulic acid proved to be a good acidifying candidate. In conclusion, for texture improvement of thermally processed carrots, lowering the susceptibility for b-elimination and enhancing cell wall cross-links are two main targets which both can be reached by manipulating different plant-intrinsic properties. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Texture is a major quality characteristic of fruits and vegetables and is remarkably changed during a thermal process. The notion of ‘‘texture engineering” has been introduced in the context of texture optimization of processed fruits and vegetables (Van Buggenhout, Sila, Duvetter, Van Loey, & Hendrickx, 2009). By manipulating the plant-intrinsic properties (via specific pretreatments), the product becomes less susceptible to processing-induced texture changes. Main focus lays on pectin because of its crucial role in cell–cell adhesion and its sensitivity to biochemical and chemical modifications (Van Buren, 1979). Pectin is a very complex cell wall polysaccharide, consisting of alternating rhamnogalacturonan (RG) and homogalacturonan (HG) regions (Vincken et al., 2003). RG is substituted by side chains rich in neutral sugars, e.g. arabinose and galactose. HG is a linear chain of which the galacturonic acid residues can be methyl-esterified. Beta-eliminative depolymerization of pectin is mainly responsible for the extensive softening of (low-acid) fruits and vegetables during a thermal process (Sila, Smout, Elliot, Van Loey, & Hendrickx, 2006). This reaction proceeds on uronic acids which possess a glycosidic linkage on C-4 in the b-position of the carboxyl group at C-5 * Corresponding author. Tel.: +32 16 321572; fax: +32 16 321960. E-mail address: [email protected] (M. Hendrickx). URL: http://www.biw.kuleuven.be/lmt/vdt/ (M. Hendrickx). 0963-9969/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.03.011
(Kiss, 1974). A prerequisite is the presence of a methyl-ester group at C-6, rendering H-5 sufficiently acidic to be removed by an alkali. This results in the formation of unstable, intermediary anions that are stabilized by losing the C–O linkage in the b-position. This depolymerization leads to pectin solubilization and, consequently, to decreased cell–cell adhesion, resulting in tissue softening. It has been established that the reaction rate is strongly dependent on the pH (Fraeye et al., 2007), the degree of pectin methyl-esterification (Sajjaanantakul, Van Buren, & Downing, 1989) and the presence of ions (Keijbets & Pilnik, 1974; Sajjaanantakul, Van Buren, & Downing, 1993). In the context of texture optimization by means of texture engineering, different approaches exist. One strategy is to render the plant tissue less vulnerable to the b-elimination reaction during heating. Next to this, intercellular adhesion can be enhanced by promoting formation of cell wall cross-links. It has already widely been demonstrated that the former can be achieved by reducing the degree of pectin methyl-esterification (Ng & Waldron, 1997; Smout, Sila, Vu, Van Loey, & Hendrickx, 2005). By a thermal pretreatment stimulating pectin methylesterase (PME) activity low-esterified pectin is obtained which is less susceptible to b-elimination and can be cross-linked by calcium ions naturally present in the tissue or exogenously added. More recently, due to the growing interest in HP technology for food preservation, it was found that HP is more beneficial in enhancing PME activity than a thermal pretreatment
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and that a HP pretreatment consequently results in a better texture (Sila, Smout, Vu, & Hendrickx, 2004). The strong pH dependence of the b-elimination reaction (Fraeye et al., 2007) opens up perspectives for counteracting b-elimination of pectin in another way, namely by lowering the pH of the plant tissue. This was the objective of the current study. It was investigated whether lowering the pH of carrot tissue could improve the texture of thermally processed carrots. By including both approaches (i.e. lowering the pectin DM and lowering the pH of the tissue) in the study, the effectiveness of the new approach could be assessed.
2. Materials and methods 2.1. Materials Carrots (Daucus carota var. Nerac, 3–4 cm diameter) were obtained from a local shop in Belgium and stored at 4 °C for a maximum period of 1 week before use. Disks (10 mm height and 12 mm diameter) were excised from the cortex of the carrots before processing and analysis. All chemicals used were of analytical grade. 2.2. Pretreatments 2.2.1. Thermal pretreatment (low temperature blanching – LTB) Carrot disks vacuum packed in a polyethylene bag were heated at 60 °C for 40 min in a temperature controlled water bath and subsequently cooled in an ice water bath. To accelerate heat transfer, carrot disks were spread out into one uniform layer. 2.2.2. High-pressure pretreatment HP pretreatments were carried out in a single vessel (590 mL) HP apparatus (Engineered Pressure Systems International, Belgium). This equipment allows pressures up to 600 MPa in combination with temperatures between 30 °C and 100 °C. The pressure transfer medium was a mixture of 60% DowcalN (The Dow Chemical Company, Switzerland) in demineralized water. Carrot disks vacuum packed in a polyethylene bag were treated at 60 °C and 400 MPa for 15 min and subsequently cooled in an ice water bath. 2.2.3. Soaking After thermal or HP pretreatment, carrot disks were suspended in specific (depending on the experiment) soak solutions at room temperature (22 °C) and atmospheric pressure for 1 h. Afterwards, the solution was drained and the carrots disks were thermally treated. The used soak solutions were demineralized water, 0.1 M sodium acetate–acetic acid (AA) buffer (pH 5.4, 4.7, 4.1), 0.1 M sodium citrate–citric acid (CA) buffer (pH 6.0, 5.3, 4.8, 4.2), an aqueous solution of 0.5% (w/v) CaCl2, or an aqueous solution of 0.1% (w/v) ferulic acid (FA), cinnamic acid (CA), vanillic acid (VA) or vanillin (VA). 2.3. Thermal treatment Carrot disks (10) encapsulated in stainless steel tubes (110 mm long, 13 mm internal diameter, and 1 mm thickness) filled with the respective soak solution (to prevent leaching) were heated at 100 °C for 20 min in a temperature controlled oil bath. The samples were immediately cooled in an ice water bath and the residual texture was determined. Thermal treatment conditions were chosen to imitate a domestic cooking process.
2.4. Texture measurement Texture is a multi-parameter attribute (Szczesniak, 2002). The parameter considered in this study was hardness. The carrot tissue hardness was evaluated by a compression test using a TA-XT2i Texture Analyzer (Stable Micro Systems, UK). The following parameters were used: load cell = 25 kg, probe = 25 mm diameter aluminium cylinder, and test speed = 1 mm/s. The hardness of a carrot disk was defined as the maximum force needed to compress the disk to 70% of its height. Each hardness value reported was the mean of 20 measurements and expressed relatively to the hardness of the fresh material. Texture results were assayed statistically using One-Way ANOVA (SAS Enterprise Guide 3.0, Cary, USA). Significant differences among texture means were analyzed using a Tukey test. The significance level was set at a = 0.05. 2.5. Preparation of alcohol-insoluble residue (AIR) Cell wall material was isolated as AIR following the procedure described by McFeeters and Armstrong (1984). Approximately 10 g of carrot tissue was completely homogenized in 64 mL of 95% ethanol using a mixer (Büchi mixer B-400, Switzerland). The suspension was filtered and the residue was re-homogenized in 32 mL of 95% ethanol and filtered again. The residue was homogenized in 32 mL of acetone before final filtration followed by drying overnight at 40 °C. The AIR was ground using a mortar and pestle and stored in a dessicator until analysis. 2.6. Determination of degree of methyl-esterification of carrot pectin The DM of the pectin was estimated by taking the ratio of moles of methoxyl groups to the moles of anhydrous galacturonic acid in the AIR. To determine the latter, pectin was hydrolyzed with concentrated sulfuric acid according to the method of Ahmed and Labavitch (1977). Subsequently, the galacturonic acid content was determined colorimetrically as described by Blumenkrantz and Asboe-Hansen (1973). The methoxyl content was estimated by hydrolyzing the ester bonds of the pectin with NaOH (Ng & Waldron, 1997) and colorimetric quantification of the released methanol as described by Klavons and Bennett (1986). In both procedures, the respective hydrolyzes were performed in duplicate, and three colorimetrical analyses were carried out for each hydrolysate. 2.7. Determination of ferulic acid content of carrots Cell wall material was extracted with 2 M NaOH for 24 h at 22 °C under nitrogen in the dark, after which the suspension was filtered. The filtrate was acidified to pH < 2 with concentrated HCl and extracted three times with ethyl acetate (three volumes). The ethyl acetate extracts were evaporated to dryness under vacuum at 30 °C (Rotational-Vacuum-Concentrator, Christ, Germany). The residue was re-dissolved in 1 mL 50% (v/v) aqueous methanol prior to analysis by HPLC. Ferulic acid was identified and quantified by reversed-phase HPLC. The system consisted of an Agilent 1200 Series HPLC apparatus equipped with a UV-PAD detector (Agilent Technologies, Belgium). Separation was carried out at 25 °C using a Prevail C18 column (250 4.6 mm, 5 lm particle size, Alltech, USA), protected with a C18 guard column (7.5 4.6 mm, 5 lm particle size). Elution was performed at a flow rate of 1 mL/min using a gradient system which increased the relative amounts of methanol and acetonitrile present in 1 mM aqueous trifluoroacetic acid according to Waldron, Parr, Ng, and Ralph (1996). Identification and quantification of ferulic acid was performed at 325 nm. The extraction was done in duplicate and each extract was analyzed once by HPLC.
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2.8. Determination of calcium content of carrots
Relative hardness (% of raw)
The dry ashing mineralization method as described by Vidal, Pascual-Marti, Salvador, and Llabata (2002) was used to determine the calcium content of the carrots. Four grams of minced carrot tissue was weighed into a porcelain crucible and dried in an oven at 100 °C. The dry sample was calcinated in a muffle furnace at 600 °C. The carbon-free ash residue was dissolved with 3 M HCl (10 mL) and diluted to 100 mL. The calcium content was determined using an atomic absorption spectrophotometer (Unicam Solaar 969, UK) at a wavelength of 422.7 nm. The calcium content was determined once.
120 a 100 80 60
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2.9. Experimental setup Fig. 1 gives a schematic overview of the experimental setup. For each experiment, one batch of carrot disks was made. These disks were either not (raw), either thermally (LTB) or HP pretreated. Subsequently, the pretreated disks were suspended in the different soak solutions (20 disks per solution) and afterwards thermally processed. All (pre)treatments were carried out once. Finally, the residual hardness of the carrots was measured. For specific carrot samples, the Ca2+ content, the ferulic acid content and the pectin DM after pretreatment were determined. 3. Results and discussion Thermal processing of (most) fruits and vegetables is known to result in extensive tissue softening. There is an initial loss of hardness due to membrane damage and the associated turgor pressure loss (Greve, McArdle, Gohlke, & Labavitch, 1994), which is followed by a substantial but more gradual softening. In case of carrots (and other low-acid fruits and vegetables), the latter has been attributed to b-eliminative depolymerization of pectin (Greve et al., 1994; Sila et al., 2006). It was investigated whether by modifying plantintrinsic properties the overall texture loss could be reduced. As the b-elimination reaction is strongly dependent on the pectin DM and the pH, these two factors were targeted. 3.1. Modifying the degree of methyl-esterification of the carrot pectin This well-established method for texture improvement of thermally processed carrots (Sila, Smout, Vu, Van Loey, & Hendrickx, 2005) was involved in the study to be able to compare the effectiveness of different approaches. Carrots were LTB or HP pretreated followed either or not by a Ca2+ soak after which they were thermally processed for 20 min at 100 °C. Fig. 2 shows the residual hardness (relative to the hardness of raw carrots) of the processed carrot disks. When no pretreatment was applied, a major texture
1 batch of carrot disks raw 20
LTB or HP pretreatment
control
LTB
LTB + Ca2+
HP
HP + Ca2+
Fig. 2. Relative hardness (±standard error) of pretreated carrot disks after a thermal treatment of 20 min at 100 °C. Raw: untreated carrot, control: non-pretreated carrot, LTB: low temperature blanched carrot, LTB + Ca2+: LTB followed by Ca2+ soak, HP: HP pretreated carrot, HP + Ca2+: HP followed by Ca2+ soak. Means with a different letter are significantly different.
loss (94%) was observed. By applying pretreatments, this loss could be minimized to about 53%. LTB or HP pretreatment alone resulted already in a significant texture improvement. In combination with a Ca2+ soak, an even larger texture improvement was obtained. The chosen pretreatment conditions have previously been identified as optimal for stimulation of PME activity (Sila et al., 2004; Smout et al., 2005). Consequently, pectin with a lower DM is obtained which is less susceptible to b-elimination and can form cross-links with calcium ions present. Indeed, LTB or HP pretreatment lowered the DM of the carrot pectin (Table 1). Observed from the differences in DM, HP was more effective in stimulating PME activity than temperature. The stimulating effect of pressure on PME activity can be explained by the principle of Le Chatelier which states that any phenomenon accompanied by a decrease in volume is favoured by an increase in pressure (Cheftel, 1995). Solvation of the charged groups created by pectin demethoxylation is accompanied by volume reduction resulting from electrostriction. It is not sure whether the higher residual hardness of the HP pretreated carrots is totally due to the lower DM; other additional mechanisms might play. Macroscopically, it was observed that HP pretreated carrot disks were smaller, more dense which could have partly attributed to the higher compression force. A subsequent Ca2+ soak enhanced the calcium content of the carrots by a factor 10 (Table 1). Presumably, the preceding pretreatment step permeabilized the tissue, hereby facilitating uptake of Ca2+ ions. These Ca2+ ions could form fortifying cross-links with the non-methyl-esterified galacturonic acid residues. The obtained results confirmed the observations made by Sila et al. (2005). It is obvious that pectin engineering by lowering the pectin DM an effective strategy is to improve the texture of thermally processed carrots. Within this context, a HP pretreatment seems to have more potential than a traditional thermal pretreatment.
Ca2+ content
3.2. Influencing the internal pH of carrots
preparation AIR
3.2.1. Effect of adding acid on the texture of thermally processed carrots The strong pH dependence of the b-elimination reaction may lead to another approach to improve the texture of thermally processed carrots. From the foregoing, it is clear that a traditional LTB pretreatment is, besides activating PME, effective in permeabilizing tissue, hereby facilitating uptake of exogenous components. Aiming at lowering the internal carrot pH, carrots were first thermally pretreated and afterwards soaked in 0.1 M sodium
20
soaking 20
20
20
…
20
DM ferulic acid content
thermal treatment 20
20
20
…
20
texture measurement Fig. 1. Schematic overview of the experimental setup.
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Table 1 Degree of pectin methyl-esterification and calcium content of carrots after different pretreatment conditions. Raw: untreated carrot, LTB: low temperature blanched carrot, LTB + Ca2+: LTB followed by Ca2+ soak, HP: HP pretreated carrot, HP + Ca2+: HP followed by Ca2+ soak. Pretreatment
Raw a
Degree of methyl-esterification (%) Calcium content (mg Ca2+/g carrot)
LTB + Ca2+
HP
HP + Ca2+
52.7 ± 1.1 0.097
52.6 ± 2.5 0.755
48.5 ± 1.4 0.086
45.7 ± 0.9 0.918
Standard deviation.
b-elimination. Na+ ions can bind to these carboxyl groups, hereby diminishing the electrostatic repulsion of hydroxyl ions. In addition, Na+ ions can compete with Ca2+ ions for binding to pectin, thereby weakening the pectin network. The observation that ‘‘LTB” and ‘‘LTB + AA pH 4.7” do not result in a significant texture difference although having a distinct different pH can be explained by the presence of Na+ ions in the latter solution counteracting the texture improving effect of a lower pH. The effect of soaking in a low pH solution on the residual hardness of carrot disks after heating is dependent on the composition of the soak solution, as was illustrated by performing the same experiment in citric acid–sodium citrate buffer (Fig. 4). Again, the Na+ concentration in the different buffer solutions was adjusted to the Na+ concentration of the buffer with the highest pH (6.0). In this case, the Na+ concentration amounted 0.266 M, which was significantly higher than in the previous experiment. When comparing the hardness of the carrots soaked in solutions of different pH, the same observation can be made: the lower the pH of the solution, the higher the residual hardness was. However, it is remarkable that in all four cases the residual hardness is lower than after a single LTB, especially in case of ‘‘LTB + CA pH 6.0” where the pH is almost similar. Clearly, there are components in the soak solution (and correspondingly in the carrot) which have an enormous stimulating effect on the texture degradation. The soak solutions consisted of Na+ ions and citrate ions (depending on the pH dissociated or not). As previously indicated Na+ ions have a stimulating effect on the b-elimination (and are here in even higher concentrations present). However, according to Keijbets et al. (1974), citrate ions also stimulate the b-elimination (without having an explanation for this). Moreover, citrate is an ideal complexing agent. So by chelating Ca2+ ions the cross-linked pectin network is weakened resulting in tissue softening (Vu, Smout, Sila, Van Loey, & Hendrickx, 2006). This stresses that not all pH lowering components are suitable candidates for texture improvement.
acetate–acetic acid buffers of different pH values. An acetic acid buffer was chosen because, with a pKa of 4.76, it is an ideal buffering agent in the pH region 3.7–5.6 which is below the carrot pH (which was about 6.2 for the cortex of this carrot batch). Given that the b-elimination is also influenced by ions (Keijbets & Pilnik, 1974; Sajjaanantakul et al., 1993) and that sodium acetate-acetic acid buffers contain a significant amount of Na+ ions, the Na+ concentration in the different buffer solutions was adjusted, by adding NaCl, to the Na+ concentration (0.086 M) of the buffer with the highest pH (5.4) to exclude any Na+ effect. After the 1 h soak of the LTB pretreated carrots, the pH of each soak solution was measured as an indication for the pH differences between the differently soaked carrots. Again, the texture loss due to a thermal treatment (‘‘control”) and the texture improvement due to a preceding LTB pretreatment (‘‘LTB”) were observed (Fig. 3). The acetic acid solutions differing in pH clearly resulted in a different degree of texture loss. The lower the pH, the higher the residual hardness was. At the Na+ concentration present, the pH had to be lower than 4.7 to have an additional effect on a pure LTB pretreatment. Due to the use of buffers, the pH of the soak solutions was only slightly influenced by the soaking of the carrots. The decelerating effect of H+ ions on the texture degradation can be explained by considering the hydroxyl-catalyzed mechanism of the b-elimination reaction (Kiss, 1974). Adding H+ ions lowers the amount of hydroxyl ions available for catalyzing the reaction. That Na+ ions indeed have an influence on the texture can be deduced from Fig. 3 by comparing ‘‘LTB” (carrots soaked in demineralized water) with ‘‘LTB + Na+” (carrots soaked in 0.086 M NaCl) or ‘‘LTB + AA pH 5.4”. Although their pH values are almost identical, the two latter samples have a higher Na+ concentration which is in accordance with a lower hardness. The accelerating effect of Na+ ions on the texture degradation can be explained as follows. Simultaneously with b-elimination chemical demethoxylation of pectin occurs, resulting in many negatively charged carboxyl groups which repel approaching hydroxyl ions and prevent them from catalyzing the
Relative hardness (% of raw)
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a
59.1 ± 1.2 0.088
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2.5 raw
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LTB + Na+ LTB + AA LTB + AA LTB + AA pH 5.4 pH 4.7 pH 4.1
Fig. 3. (Bars): Relative hardness (±standard error) of pretreated (LTB + 1 h soak in acetic acid (AA) buffers of different pH values but with a fixed Na+ concentration (0.086 M)) carrot disks after a thermal treatment of 20 min at 100 °C. Raw: untreated carrot, control: non-pretreated carrot, LTB: LTB carrot soaked in water, LTB + Na+: LTB carrot soaked in 0.086 M NaCl. Means with a different letter are significantly different. ( ): pH of soak solution after 1 h soak of LTB pretreated carrot disks.
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6.5 a
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Relative hardness (% of raw)
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3 raw
control
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LTB + CA LTB + CA LTB + CA LTB + CA pH 6.0 pH 5.3 pH 4.8 pH 4.2
Fig. 4. (Bars): Relative hardness (±standard error) of pretreated (LTB + 1 h soak in citric acid (CA) buffers of different pH values but with a fixed Na+ concentration (0.266 M)) carrot disks after a thermal treatment of 20 min at 100 °C. Raw: untreated carrot, control: non-pretreated carrot, LTB: LTB carrot soaked in water. Means with a different letter are significantly different. ( ): pH of soak solution after 1 h soak of LTB pretreated carrot disks.
3.2.2. Ferulic acid as pH lowering component In search for a suitable pH lowering soak solution, it is clear from previous results that chelators which complex Ca2+ ions and ions which stimulate the b-elimination have to be avoided. An exception to the latter is Ca2+ ions which are desirable. Although they stimulate the b-elimination, their potential to cross-link pectin chains prevails (as can be seen in Fig. 2 where there is clear texture improvement after a Ca2+ soak). Given current consumer demand for high-quality foods based on natural ingredients, it would be interesting to use an acid component naturally present in fruits and vegetables. Such a candidate is ferulic acid. Ferulic acid is a phenolic acid ubiquitous in nature, present in plant cell walls. In monocotyledons, ferulic acid is esterified to the O-5 of arabinose residues in arabinoxylans. In dicotyledons (to which fruits and vegetables belong), ferulic acid is associated with the pectin side chains and is ester linked to either the O-2 of arabinose or the O-6 of galactose residues. By cross-linking cell wall polysaccharides, ferulic acid imparts structural rigidity and strengthens cell wall architecture (Waldron, Smith, Parr, Ng, & Parker, 1997). At the same time, ferulic acid is commercially prepared and used as a functional food ingredient as it exhibits a wide range of therapeutic effects against various diseases, including cancer, diabetes, cardiovascular and neurodegenerative diseases, due to its potent antioxidant capacity (Itagaki et al., 2009). Considering the qualities just listed, it seems a valuable pH lowering agent. Fig. 5 shows the residual hardness of thermally treated carrot disks which were LTB
pretreated followed by a 1 h soak in 0.1% (w/v) ferulic acid. At the same time, three other components structurally related to ferulic acid were tested to be able to attribute the effect to a specific functional group. Soaking in ferulic acid resulted in a significant texture improvement. A similar or even larger texture improvement was observed after soaking in cinnamic acid or vanillic acid whereas soaking in vanillin had no effect. Again, the lower the pH the better the texture retention was. By comparing the chemical structures, it was clear that the texture improvement could be related to the acid functional group, present in ferulic acid, cinnamic acid and vanillic acid but absent in vanillin. The initial pH values of the different soak solutions were 3.5, 3.4, 3.4 and 5.0, respectively for ferulic acid, cinnamic acid, vanillic acid and vanillin. It is clear that these solutions, in contrast to the buffer solutions used earlier, undergo a pH shift during soaking of the carrots. However, the pH is still low enough to induce a pH shift in the carrots and correspondingly to result in a reduced texture loss. A thermal pretreatment proved once more to be an efficient way to permeabilize the tissue and facilitate uptake of exogenous components. Due to its solubility in organic solvents, it was expected that all exogenous ferulic acid would be washed away during preparation of AIR of ‘‘LTB + FA” treated carrots and that ferulic acid found in the AIR would be of endogenous origin. In the three washing solutions for preparation of the AIR, i.e. 64 mL 95% ethanol, 32 mL 95% ethanol and 32 mL acetone, respectively 3.72 (±0.33), 0.47 (±0.04) and 0.05 (±0.01) mg ferulic acid was found
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Relative hardness (% of raw)
120
2.5
0 raw
control
LTB
LTB + FA LTB + CA LTB + VA LTB + VN pH 3.5 pH 3.4 pH 3.4 pH 5.0
Fig. 5. (Bars): Relative hardness (±standard error) of pretreated (LTB + 1 h soak in different solutions) carrot disks after a thermal treatment of 20 min at 100 °C. FA: ferulic acid, CA: cinnamic acid, VA: vanillic acid, VN: vanillin. Raw: untreated carrot, control: non-pretreated carrot, LTB: LTB carrot soaked in water. Means with a different letter are significantly different. ( ): pH of soak solution after 1 h soak of LTB pretreated carrot disks.
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as determined by HPLC, confirming that ferulic acid had been taken up by the tissue. When analyzing the ferulic acid content of the AIR after saponification with 2 M NaOH it was noticed that there was still exogenous ferulic acid present. Raw and LTB pretreated carrots contained respectively 5.25 (±0.09) and 5.82 (±0.26) lg ferulic acid/g AIR whereas LTB pretreated carrots followed by FA soak contained 31.50 (±0.55) lg FA/g AIR which was considerably more. So clearly, a large amount of ferulic acid had been taken up by the tissue. It could be concluded that a thermal pretreatment followed by a ferulic acid soak was effective in lowering the pH of the carrot tissue, and hereby a suitable strategy for texture improvement of thermally processed carrots. Next to lowering the pH, one might suggest that ferulic acid also contributes to a better texture in another way, namely by enhancing cross-links between cell wall polymers. The evidence that ferulic acid cross-links contribute to the thermal stability of for example Chinese water chestnut has stimulated researchers in exploiting this mechanism in other plant tissues (Waldron et al., 1997). Chinese water chestnut is a monocotyledon, having high endogenous ferulic acid content. Most edible fruits and vegetables are dicotyledons and only a few, for example sugar beet and beetroot, contain similar levels of ferulic acid. Any increase in ferulic acid cross-linking will, in most dicotyledons, necessitate an increase in ferulic acid that is attached to polysaccharides at appropriate locations in the cell wall. In addition, enzymes that are able to cross-link ferulic acid will have to be present, including peroxidases, and those involved in the production of hydrogen peroxide. Conca (2003) tested the theory that (HP) infusion of peroxidase (POD) with and without ferulic acid into carrots and beets (having respectively a low and high endogenous ferulic acid content) could promote FA cross-linking resulting in maintenance of texture during thermal processing. POD and POD/FA infused carrot and beet samples had enhanced hardness when compared to the uninfused samples. An increase in cross-linked FA was seen in the POD and POD/FA infused beets but not for the carrots. The increase in cross-linking found in the POD infused beets must be directly related to cross-linking of existing wall bound ferulic acid. However, the POD/FA samples that were infused with additional ferulic acid resulted in further cross-linking with corresponding further firming. Conca (2003) proposed an inter-dimer cross-linking theory to explain the more enhanced hardness of the POD/FA infused over the POD infused beets. For the observed texture enhancement of carrots they found no explanation. Based on our results, a pH lowering effect of the infusion solutions used seems responsible for the enhanced hardness. In addition, the HP treatment might have had a firming effect (e.g. by stimulating PME activity) which was not investigated. Likewise, these two effects might have contributed to the enhanced hardness of beets. That ferulic acid cross-linking did not contribute to the texture improvement of the carrots in this study can easily be seen when comparing Fig. 5 with Fig. 3. When LTB treated carrots were soaked in ferulic, cinnamic or vanillic acid, the pH of the solution was about 4.3 and the residual hardness was about 34%. This corresponded very well with LTB treated carrots soaked in acetic acid buffer pH 4.1 where the pH of the solution was 4.2 and the residual hardness was 36%. 3.2.3. HP pretreatment in combination with ferulic acid soaking From Fig. 2 it is clear that a HP pretreatment is even more effective concerning texture improvement of thermally processed carrots than an LTB pretreatment. Whereas an LTB pretreatment resulted in a residual hardness (after thermal processing) of about 20%, HP pretreated carrots had a residual hardness of about 38%. The effect of a HP pretreatment followed by a ferulic acid soak was therefore investigated. In Fig. 6, it can be seen that a HP pretreatment followed by a ferulic acid soak resulted in a significant texture improvement (residual hardness of 45%) in addition to
120
Relative hardness (% of raw)
1302
a 100 80 b
60 c d
40 20
e
0 raw
control
HP
HP + FA
HP + Ca2+ & FA
Fig. 6. Relative hardness (±standard error) of pretreated carrot disks after a thermal treatment of 20 min at 100 °C. Raw: untreated carrot, control: non-pretreated carrot, HP: HP pretreated carrot, HP + FA: HP followed by ferulic acid soak, HP + Ca2+ and FA: HP followed by combined Ca2+ and ferulic acid soak. Means with a different letter are significantly different.
the texture improvement already obtained due to a HP pretreatment. This texture improvement was in the same order of magnitude as in the case of a HP pretreatment followed by a Ca2+ soak (residual hardness of 47%) (Fig. 2). When applying a combined Ca2+ and ferulic acid soak, an even better texture was obtained due to the combination of lowered b-elimination susceptibility and enhanced pectin cross-linking. By applying these two pretreatment steps, thermally processed carrots could retain about 53% of the hardness of raw carrots in contrast to only 6% when no pretreatment was applied. 4. Conclusion Thermal processing for food preservation results in an undesired high level of softening of carrots, mainly due to b-eliminative depolymerization of pectin which results in decreased cell adhesion. In context of texture improvement of thermally processed carrots, next to the established strategy of an LTB or HP pretreatment followed by a Ca2+ soak, a second strategy proved to be useful: treatment at low pH. By respectively lowering the DM of the carrot pectin or lowering the pH of the carrot tissue, the susceptibility to b-elimination was reduced. The composition of the pH lowering solution appeared important: cations and Ca2+ chelators have to be avoided. Ferulic acid showed to be an effective pH lowering component. By combining a HP pretreatment with a soak in ferulic acid and Ca2+ thermally processed carrots could retain more than 50% of the hardness of unprocessed carrots, due to the accumulative effect of the different modifications. Seen increasing consumer demand for high-quality, natural foods, ferulic acid would be a good acidifying component as it is naturally present in fruits and vegetables and is already used as a functional food ingredient. However, one has to remind that, although it is a very important quality characteristic, texture is not the only quality parameter of concern. Therefore, the influence of the different plant modifications on other quality parameters, for example taste of carrots, has to be evaluated to see whether the final product meets consumer demands. Acknowledgements This study has been carried out with financial support from the Research Fund K.U. Leuven, the Industrial Research Fund K.U. Leuven (KP/08/004) and the Commission of the European Communities, Framework 6, Priority 5 ‘Food Quality and Safety’, Integrated Project NovelQ FP6-CT-2006-015710.
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References Ahmed, A. E. R., & Labavitch, J. M. (1977). A simplified method for accurate determination of cell wall uronide content. Journal of Food Biochemistry, 1, 361–365. Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Analytical Biochemistry, 54(2), 484–489. Cheftel, J. C. (1995). Review: High-pressure, microbial inactivation and food preservation. Food Science and Technology International, 1(2–3), 75–90. Conca, K. R. (2003). Ferulic acid and peroxidase infusion into disparate vegetables and its effect on texture. USA: University of Rhode Island. Fraeye, I., De Roeck, A., Duvetter, T., Verlent, I., Hendrickx, M., & Van Loey, A. (2007). Influence of pectin properties and processing conditions on thermal pectin degradation. Food Chemistry, 105(2), 555–563. Greve, L. C., McArdle, R. N., Gohlke, J. R., & Labavitch, J. M. (1994). Impact of heating on carrot firmness: Changes in cell wall components. Journal of Agricultural and Food Chemistry, 42, 2900–2906. Greve, L. C., Shackel, K. A., Ahmadi, H., McArdle, R. N., Gohlke, J. R., & Labavitch, J. M. (1994). Impact of heating on carrot firmness: Contribution of cellular turgor. Journal of Agricultural and Food Chemistry, 42, 2896–2899. Itagaki, S., Kurokawa, T., Nakata, C., Saito, Y., Oikawa, S., Kobayashi, M., et al. (2009). In vitro and in vivo antioxidant properties of ferulic acid: A comparative study with other natural oxidation inhibitors. Food Chemistry, 114, 466–471. Keijbets, M. J., & Pilnik, W. (1974). Beta-elimination of pectin in presence of anions and cations. Carbohydrate Research, 33(2), 359–362. Kiss, J. (1974). Beta-eliminative degradation of carbohydrates containing uronic acid residues. In R. S. Tipson & D. Horton (Eds.), Advances in carbohydrate chemistry and biochemistry (pp. 229–298). New York and London: Academic Press. Klavons, J. A., & Bennett, R. D. (1986). Determination of methanol using alcohol oxidase and its application to methyl-ester content of pectins. Journal of Agricultural and Food Chemistry, 34(4), 597–599. McFeeters, R. F., & Armstrong, S. A. (1984). Measurement of pectin methylation in plant cell walls. Analytical Biochemistry, 139, 212–217. Ng, A., & Waldron, K. W. (1997). Effect of cooking and pre-cooking on cell-wall chemistry in relation to firmness of carrot tissues. Journal of the Science of Food and Agriculture, 73(4), 503–512. Sajjaanantakul, T., Van Buren, J. P., & Downing, D. L. (1993). Effect of cations on heat degradation of chelator-soluble carrot pectin. Carbohydrate Polymers, 20(3), 207–214.
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Sajjaanantakul, T., Van Buren, J. P., & Downing, D. L. (1989). Effect of methyl-ester content on heat degradation of chelator-soluble carrot pectin. Journal of Food Science, 54(5), 1272–1277. Sila, D. N., Smout, C., Elliot, F., Van Loey, A., & Hendrickx, M. (2006). Non-enzymatic depolymerization of carrot pectin: Toward a better understanding of carrot texture during thermal processing. Journal of Food Science, 71(1), E1–E9. Sila, D. N., Smout, C., Vu, T. S., & Hendrickx, M. E. (2004). Effects of high-pressure pretreatment and calcium soaking on the texture degradation kinetics of carrots during thermal processing. Journal of Food Science, 69(5), E205–E211. Sila, D. N., Smout, C., Vu, T. S., Van Loey, A., & Hendrickx, M. (2005). Influence of pretreatment conditions on the texture and cell wall components of carrots during thermal processing. Journal of Food Science, 70(2), E85–E91. Smout, C., Sila, D. N., Vu, T. S., Van Loey, A. M. L., & Hendrickx, M. E. G. (2005). Effect of preheating and calcium pre-treatment on pectin structure and thermal texture degradation: A case study on carrots. Journal of Food Engineering, 67(4), 419–425. Szczesniak, A. S. (2002). Texture is a sensory property. Food Quality and Preference, 13(4), 215–225. Van Buggenhout, S., Sila, D. N., Duvetter, T., Van Loey, A., & Hendrickx, M. (2009). Pectins in processed fruits and vegetables: Part III – Texture engineering. Comprehensive Reviews in Food Science and Food Safety, 8, 105–117. Van Buren, J. P. (1979). The chemistry of texture in fruits and vegetables. Journal of Texture Studies, 10, 1–23. Vidal, M. T., Pascual-Marti, M. C., Salvador, A., & Llabata, C. (2002). Determination of essential metals in complete diet feed by flow injection and flame atomic absorption spectrometry. Microchemical Journal, 72, 221–228. Vincken, J. P., Schols, H. A., Oomen, R. J., McCann, M. C., Ulvskov, P., Voragen, A. G., et al. (2003). If homogalacturonan were a side chain of rhamnogalacturonan I. Implications for cell wall architecture. Plant Physiology, 132(4), 1781–1789. Vu, T. S., Smout, C., Sila, D. N., Van Loey, A., & Hendrickx, M. (2006). The effect of brine ingredients on carrot texture during thermal processing in relation to pectin depolymerization due to the beta-elimination reaction. Journal of Food Science, 71(9), E370–E375. Waldron, K. W., Parr, A. J., Ng, A., & Ralph, J. (1996). Cell wall esterified phenolic dimers: Identification and quantification by reverse phase high performance liquid chromatography and diode array detection. Phytochemical Analysis, 7, 305–312. Waldron, K. W., Smith, A. C., Parr, A. J., Ng, A., & Parker, M. L. (1997). New approaches to understanding and controlling cell separation in relation to fruit and vegetable texture. Trends in Food Science & Technology, 8, 213–221.
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Food Research International journal homepage: www.elsevier.com/locate/foodres
Improving the hardness of thermally processed carrots by selective pretreatments Ans De Roeck, Jan Mols, Daniel Ndaka Sila, Thomas Duvetter, Ann Van Loey, Marc Hendrickx * Laboratory of Food Technology and Leuven Food Science and Nutrition Research Centre (LFoRCe), Department of Microbial and Molecular Systems (M2S), Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium
a r t i c l e
i n f o
Article history: Received 24 August 2009 Accepted 8 March 2010
Keywords: Carrot Texture Thermal processing b-Elimination Degree of pectin methyl-esterification pH Ferulic acid
a b s t r a c t The aim of this study was to improve the texture of thermally processed carrots by selective pretreatments modifying plant-intrinsic properties. Pretreatments were a combination of a thermal or high-pressure (HP) treatment followed by a 1 h soak in a specific solution. Lowering the degree of methylesterification (DM) of the carrot pectin was confirmed to be one strategy to reduce texture degradation. The thermal or HP pretreatment resulted in pectin with a lower DM which is less susceptible to b-eliminative depolymerization. A subsequent Ca2+ soak resulted in an even better texture by enhancing the amount of pectin cross-links within the cell wall. Lowering the pH of the carrots was proven to be another strategy. A thermal or HP pretreatment followed by soaking carrots in solutions of low pH proved to be effective in lowering the internal carrot pH, hereby retarding b-elimination and consequently texture degradation. The composition of the low pH solution was shown to be of importance; soak solutions containing cations and/or Ca2+ complexing agents have to be avoided. Ferulic acid proved to be a good acidifying candidate. In conclusion, for texture improvement of thermally processed carrots, lowering the susceptibility for b-elimination and enhancing cell wall cross-links are two main targets which both can be reached by manipulating different plant-intrinsic properties. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Texture is a major quality characteristic of fruits and vegetables and is remarkably changed during a thermal process. The notion of ‘‘texture engineering” has been introduced in the context of texture optimization of processed fruits and vegetables (Van Buggenhout, Sila, Duvetter, Van Loey, & Hendrickx, 2009). By manipulating the plant-intrinsic properties (via specific pretreatments), the product becomes less susceptible to processing-induced texture changes. Main focus lays on pectin because of its crucial role in cell–cell adhesion and its sensitivity to biochemical and chemical modifications (Van Buren, 1979). Pectin is a very complex cell wall polysaccharide, consisting of alternating rhamnogalacturonan (RG) and homogalacturonan (HG) regions (Vincken et al., 2003). RG is substituted by side chains rich in neutral sugars, e.g. arabinose and galactose. HG is a linear chain of which the galacturonic acid residues can be methyl-esterified. Beta-eliminative depolymerization of pectin is mainly responsible for the extensive softening of (low-acid) fruits and vegetables during a thermal process (Sila, Smout, Elliot, Van Loey, & Hendrickx, 2006). This reaction proceeds on uronic acids which possess a glycosidic linkage on C-4 in the b-position of the carboxyl group at C-5 * Corresponding author. Tel.: +32 16 321572; fax: +32 16 321960. E-mail address: [email protected] (M. Hendrickx). URL: http://www.biw.kuleuven.be/lmt/vdt/ (M. Hendrickx). 0963-9969/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.03.011
(Kiss, 1974). A prerequisite is the presence of a methyl-ester group at C-6, rendering H-5 sufficiently acidic to be removed by an alkali. This results in the formation of unstable, intermediary anions that are stabilized by losing the C–O linkage in the b-position. This depolymerization leads to pectin solubilization and, consequently, to decreased cell–cell adhesion, resulting in tissue softening. It has been established that the reaction rate is strongly dependent on the pH (Fraeye et al., 2007), the degree of pectin methyl-esterification (Sajjaanantakul, Van Buren, & Downing, 1989) and the presence of ions (Keijbets & Pilnik, 1974; Sajjaanantakul, Van Buren, & Downing, 1993). In the context of texture optimization by means of texture engineering, different approaches exist. One strategy is to render the plant tissue less vulnerable to the b-elimination reaction during heating. Next to this, intercellular adhesion can be enhanced by promoting formation of cell wall cross-links. It has already widely been demonstrated that the former can be achieved by reducing the degree of pectin methyl-esterification (Ng & Waldron, 1997; Smout, Sila, Vu, Van Loey, & Hendrickx, 2005). By a thermal pretreatment stimulating pectin methylesterase (PME) activity low-esterified pectin is obtained which is less susceptible to b-elimination and can be cross-linked by calcium ions naturally present in the tissue or exogenously added. More recently, due to the growing interest in HP technology for food preservation, it was found that HP is more beneficial in enhancing PME activity than a thermal pretreatment
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and that a HP pretreatment consequently results in a better texture (Sila, Smout, Vu, & Hendrickx, 2004). The strong pH dependence of the b-elimination reaction (Fraeye et al., 2007) opens up perspectives for counteracting b-elimination of pectin in another way, namely by lowering the pH of the plant tissue. This was the objective of the current study. It was investigated whether lowering the pH of carrot tissue could improve the texture of thermally processed carrots. By including both approaches (i.e. lowering the pectin DM and lowering the pH of the tissue) in the study, the effectiveness of the new approach could be assessed.
2. Materials and methods 2.1. Materials Carrots (Daucus carota var. Nerac, 3–4 cm diameter) were obtained from a local shop in Belgium and stored at 4 °C for a maximum period of 1 week before use. Disks (10 mm height and 12 mm diameter) were excised from the cortex of the carrots before processing and analysis. All chemicals used were of analytical grade. 2.2. Pretreatments 2.2.1. Thermal pretreatment (low temperature blanching – LTB) Carrot disks vacuum packed in a polyethylene bag were heated at 60 °C for 40 min in a temperature controlled water bath and subsequently cooled in an ice water bath. To accelerate heat transfer, carrot disks were spread out into one uniform layer. 2.2.2. High-pressure pretreatment HP pretreatments were carried out in a single vessel (590 mL) HP apparatus (Engineered Pressure Systems International, Belgium). This equipment allows pressures up to 600 MPa in combination with temperatures between 30 °C and 100 °C. The pressure transfer medium was a mixture of 60% DowcalN (The Dow Chemical Company, Switzerland) in demineralized water. Carrot disks vacuum packed in a polyethylene bag were treated at 60 °C and 400 MPa for 15 min and subsequently cooled in an ice water bath. 2.2.3. Soaking After thermal or HP pretreatment, carrot disks were suspended in specific (depending on the experiment) soak solutions at room temperature (22 °C) and atmospheric pressure for 1 h. Afterwards, the solution was drained and the carrots disks were thermally treated. The used soak solutions were demineralized water, 0.1 M sodium acetate–acetic acid (AA) buffer (pH 5.4, 4.7, 4.1), 0.1 M sodium citrate–citric acid (CA) buffer (pH 6.0, 5.3, 4.8, 4.2), an aqueous solution of 0.5% (w/v) CaCl2, or an aqueous solution of 0.1% (w/v) ferulic acid (FA), cinnamic acid (CA), vanillic acid (VA) or vanillin (VA). 2.3. Thermal treatment Carrot disks (10) encapsulated in stainless steel tubes (110 mm long, 13 mm internal diameter, and 1 mm thickness) filled with the respective soak solution (to prevent leaching) were heated at 100 °C for 20 min in a temperature controlled oil bath. The samples were immediately cooled in an ice water bath and the residual texture was determined. Thermal treatment conditions were chosen to imitate a domestic cooking process.
2.4. Texture measurement Texture is a multi-parameter attribute (Szczesniak, 2002). The parameter considered in this study was hardness. The carrot tissue hardness was evaluated by a compression test using a TA-XT2i Texture Analyzer (Stable Micro Systems, UK). The following parameters were used: load cell = 25 kg, probe = 25 mm diameter aluminium cylinder, and test speed = 1 mm/s. The hardness of a carrot disk was defined as the maximum force needed to compress the disk to 70% of its height. Each hardness value reported was the mean of 20 measurements and expressed relatively to the hardness of the fresh material. Texture results were assayed statistically using One-Way ANOVA (SAS Enterprise Guide 3.0, Cary, USA). Significant differences among texture means were analyzed using a Tukey test. The significance level was set at a = 0.05. 2.5. Preparation of alcohol-insoluble residue (AIR) Cell wall material was isolated as AIR following the procedure described by McFeeters and Armstrong (1984). Approximately 10 g of carrot tissue was completely homogenized in 64 mL of 95% ethanol using a mixer (Büchi mixer B-400, Switzerland). The suspension was filtered and the residue was re-homogenized in 32 mL of 95% ethanol and filtered again. The residue was homogenized in 32 mL of acetone before final filtration followed by drying overnight at 40 °C. The AIR was ground using a mortar and pestle and stored in a dessicator until analysis. 2.6. Determination of degree of methyl-esterification of carrot pectin The DM of the pectin was estimated by taking the ratio of moles of methoxyl groups to the moles of anhydrous galacturonic acid in the AIR. To determine the latter, pectin was hydrolyzed with concentrated sulfuric acid according to the method of Ahmed and Labavitch (1977). Subsequently, the galacturonic acid content was determined colorimetrically as described by Blumenkrantz and Asboe-Hansen (1973). The methoxyl content was estimated by hydrolyzing the ester bonds of the pectin with NaOH (Ng & Waldron, 1997) and colorimetric quantification of the released methanol as described by Klavons and Bennett (1986). In both procedures, the respective hydrolyzes were performed in duplicate, and three colorimetrical analyses were carried out for each hydrolysate. 2.7. Determination of ferulic acid content of carrots Cell wall material was extracted with 2 M NaOH for 24 h at 22 °C under nitrogen in the dark, after which the suspension was filtered. The filtrate was acidified to pH < 2 with concentrated HCl and extracted three times with ethyl acetate (three volumes). The ethyl acetate extracts were evaporated to dryness under vacuum at 30 °C (Rotational-Vacuum-Concentrator, Christ, Germany). The residue was re-dissolved in 1 mL 50% (v/v) aqueous methanol prior to analysis by HPLC. Ferulic acid was identified and quantified by reversed-phase HPLC. The system consisted of an Agilent 1200 Series HPLC apparatus equipped with a UV-PAD detector (Agilent Technologies, Belgium). Separation was carried out at 25 °C using a Prevail C18 column (250 4.6 mm, 5 lm particle size, Alltech, USA), protected with a C18 guard column (7.5 4.6 mm, 5 lm particle size). Elution was performed at a flow rate of 1 mL/min using a gradient system which increased the relative amounts of methanol and acetonitrile present in 1 mM aqueous trifluoroacetic acid according to Waldron, Parr, Ng, and Ralph (1996). Identification and quantification of ferulic acid was performed at 325 nm. The extraction was done in duplicate and each extract was analyzed once by HPLC.
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2.8. Determination of calcium content of carrots
Relative hardness (% of raw)
The dry ashing mineralization method as described by Vidal, Pascual-Marti, Salvador, and Llabata (2002) was used to determine the calcium content of the carrots. Four grams of minced carrot tissue was weighed into a porcelain crucible and dried in an oven at 100 °C. The dry sample was calcinated in a muffle furnace at 600 °C. The carbon-free ash residue was dissolved with 3 M HCl (10 mL) and diluted to 100 mL. The calcium content was determined using an atomic absorption spectrophotometer (Unicam Solaar 969, UK) at a wavelength of 422.7 nm. The calcium content was determined once.
120 a 100 80 60
b c
40
d e
20
f
0 raw
2.9. Experimental setup Fig. 1 gives a schematic overview of the experimental setup. For each experiment, one batch of carrot disks was made. These disks were either not (raw), either thermally (LTB) or HP pretreated. Subsequently, the pretreated disks were suspended in the different soak solutions (20 disks per solution) and afterwards thermally processed. All (pre)treatments were carried out once. Finally, the residual hardness of the carrots was measured. For specific carrot samples, the Ca2+ content, the ferulic acid content and the pectin DM after pretreatment were determined. 3. Results and discussion Thermal processing of (most) fruits and vegetables is known to result in extensive tissue softening. There is an initial loss of hardness due to membrane damage and the associated turgor pressure loss (Greve, McArdle, Gohlke, & Labavitch, 1994), which is followed by a substantial but more gradual softening. In case of carrots (and other low-acid fruits and vegetables), the latter has been attributed to b-eliminative depolymerization of pectin (Greve et al., 1994; Sila et al., 2006). It was investigated whether by modifying plantintrinsic properties the overall texture loss could be reduced. As the b-elimination reaction is strongly dependent on the pectin DM and the pH, these two factors were targeted. 3.1. Modifying the degree of methyl-esterification of the carrot pectin This well-established method for texture improvement of thermally processed carrots (Sila, Smout, Vu, Van Loey, & Hendrickx, 2005) was involved in the study to be able to compare the effectiveness of different approaches. Carrots were LTB or HP pretreated followed either or not by a Ca2+ soak after which they were thermally processed for 20 min at 100 °C. Fig. 2 shows the residual hardness (relative to the hardness of raw carrots) of the processed carrot disks. When no pretreatment was applied, a major texture
1 batch of carrot disks raw 20
LTB or HP pretreatment
control
LTB
LTB + Ca2+
HP
HP + Ca2+
Fig. 2. Relative hardness (±standard error) of pretreated carrot disks after a thermal treatment of 20 min at 100 °C. Raw: untreated carrot, control: non-pretreated carrot, LTB: low temperature blanched carrot, LTB + Ca2+: LTB followed by Ca2+ soak, HP: HP pretreated carrot, HP + Ca2+: HP followed by Ca2+ soak. Means with a different letter are significantly different.
loss (94%) was observed. By applying pretreatments, this loss could be minimized to about 53%. LTB or HP pretreatment alone resulted already in a significant texture improvement. In combination with a Ca2+ soak, an even larger texture improvement was obtained. The chosen pretreatment conditions have previously been identified as optimal for stimulation of PME activity (Sila et al., 2004; Smout et al., 2005). Consequently, pectin with a lower DM is obtained which is less susceptible to b-elimination and can form cross-links with calcium ions present. Indeed, LTB or HP pretreatment lowered the DM of the carrot pectin (Table 1). Observed from the differences in DM, HP was more effective in stimulating PME activity than temperature. The stimulating effect of pressure on PME activity can be explained by the principle of Le Chatelier which states that any phenomenon accompanied by a decrease in volume is favoured by an increase in pressure (Cheftel, 1995). Solvation of the charged groups created by pectin demethoxylation is accompanied by volume reduction resulting from electrostriction. It is not sure whether the higher residual hardness of the HP pretreated carrots is totally due to the lower DM; other additional mechanisms might play. Macroscopically, it was observed that HP pretreated carrot disks were smaller, more dense which could have partly attributed to the higher compression force. A subsequent Ca2+ soak enhanced the calcium content of the carrots by a factor 10 (Table 1). Presumably, the preceding pretreatment step permeabilized the tissue, hereby facilitating uptake of Ca2+ ions. These Ca2+ ions could form fortifying cross-links with the non-methyl-esterified galacturonic acid residues. The obtained results confirmed the observations made by Sila et al. (2005). It is obvious that pectin engineering by lowering the pectin DM an effective strategy is to improve the texture of thermally processed carrots. Within this context, a HP pretreatment seems to have more potential than a traditional thermal pretreatment.
Ca2+ content
3.2. Influencing the internal pH of carrots
preparation AIR
3.2.1. Effect of adding acid on the texture of thermally processed carrots The strong pH dependence of the b-elimination reaction may lead to another approach to improve the texture of thermally processed carrots. From the foregoing, it is clear that a traditional LTB pretreatment is, besides activating PME, effective in permeabilizing tissue, hereby facilitating uptake of exogenous components. Aiming at lowering the internal carrot pH, carrots were first thermally pretreated and afterwards soaked in 0.1 M sodium
20
soaking 20
20
20
…
20
DM ferulic acid content
thermal treatment 20
20
20
…
20
texture measurement Fig. 1. Schematic overview of the experimental setup.
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Table 1 Degree of pectin methyl-esterification and calcium content of carrots after different pretreatment conditions. Raw: untreated carrot, LTB: low temperature blanched carrot, LTB + Ca2+: LTB followed by Ca2+ soak, HP: HP pretreated carrot, HP + Ca2+: HP followed by Ca2+ soak. Pretreatment
Raw a
Degree of methyl-esterification (%) Calcium content (mg Ca2+/g carrot)
LTB + Ca2+
HP
HP + Ca2+
52.7 ± 1.1 0.097
52.6 ± 2.5 0.755
48.5 ± 1.4 0.086
45.7 ± 0.9 0.918
Standard deviation.
b-elimination. Na+ ions can bind to these carboxyl groups, hereby diminishing the electrostatic repulsion of hydroxyl ions. In addition, Na+ ions can compete with Ca2+ ions for binding to pectin, thereby weakening the pectin network. The observation that ‘‘LTB” and ‘‘LTB + AA pH 4.7” do not result in a significant texture difference although having a distinct different pH can be explained by the presence of Na+ ions in the latter solution counteracting the texture improving effect of a lower pH. The effect of soaking in a low pH solution on the residual hardness of carrot disks after heating is dependent on the composition of the soak solution, as was illustrated by performing the same experiment in citric acid–sodium citrate buffer (Fig. 4). Again, the Na+ concentration in the different buffer solutions was adjusted to the Na+ concentration of the buffer with the highest pH (6.0). In this case, the Na+ concentration amounted 0.266 M, which was significantly higher than in the previous experiment. When comparing the hardness of the carrots soaked in solutions of different pH, the same observation can be made: the lower the pH of the solution, the higher the residual hardness was. However, it is remarkable that in all four cases the residual hardness is lower than after a single LTB, especially in case of ‘‘LTB + CA pH 6.0” where the pH is almost similar. Clearly, there are components in the soak solution (and correspondingly in the carrot) which have an enormous stimulating effect on the texture degradation. The soak solutions consisted of Na+ ions and citrate ions (depending on the pH dissociated or not). As previously indicated Na+ ions have a stimulating effect on the b-elimination (and are here in even higher concentrations present). However, according to Keijbets et al. (1974), citrate ions also stimulate the b-elimination (without having an explanation for this). Moreover, citrate is an ideal complexing agent. So by chelating Ca2+ ions the cross-linked pectin network is weakened resulting in tissue softening (Vu, Smout, Sila, Van Loey, & Hendrickx, 2006). This stresses that not all pH lowering components are suitable candidates for texture improvement.
acetate–acetic acid buffers of different pH values. An acetic acid buffer was chosen because, with a pKa of 4.76, it is an ideal buffering agent in the pH region 3.7–5.6 which is below the carrot pH (which was about 6.2 for the cortex of this carrot batch). Given that the b-elimination is also influenced by ions (Keijbets & Pilnik, 1974; Sajjaanantakul et al., 1993) and that sodium acetate-acetic acid buffers contain a significant amount of Na+ ions, the Na+ concentration in the different buffer solutions was adjusted, by adding NaCl, to the Na+ concentration (0.086 M) of the buffer with the highest pH (5.4) to exclude any Na+ effect. After the 1 h soak of the LTB pretreated carrots, the pH of each soak solution was measured as an indication for the pH differences between the differently soaked carrots. Again, the texture loss due to a thermal treatment (‘‘control”) and the texture improvement due to a preceding LTB pretreatment (‘‘LTB”) were observed (Fig. 3). The acetic acid solutions differing in pH clearly resulted in a different degree of texture loss. The lower the pH, the higher the residual hardness was. At the Na+ concentration present, the pH had to be lower than 4.7 to have an additional effect on a pure LTB pretreatment. Due to the use of buffers, the pH of the soak solutions was only slightly influenced by the soaking of the carrots. The decelerating effect of H+ ions on the texture degradation can be explained by considering the hydroxyl-catalyzed mechanism of the b-elimination reaction (Kiss, 1974). Adding H+ ions lowers the amount of hydroxyl ions available for catalyzing the reaction. That Na+ ions indeed have an influence on the texture can be deduced from Fig. 3 by comparing ‘‘LTB” (carrots soaked in demineralized water) with ‘‘LTB + Na+” (carrots soaked in 0.086 M NaCl) or ‘‘LTB + AA pH 5.4”. Although their pH values are almost identical, the two latter samples have a higher Na+ concentration which is in accordance with a lower hardness. The accelerating effect of Na+ ions on the texture degradation can be explained as follows. Simultaneously with b-elimination chemical demethoxylation of pectin occurs, resulting in many negatively charged carboxyl groups which repel approaching hydroxyl ions and prevent them from catalyzing the
Relative hardness (% of raw)
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a
59.1 ± 1.2 0.088
LTB
2.5 raw
control
LTB
LTB + Na+ LTB + AA LTB + AA LTB + AA pH 5.4 pH 4.7 pH 4.1
Fig. 3. (Bars): Relative hardness (±standard error) of pretreated (LTB + 1 h soak in acetic acid (AA) buffers of different pH values but with a fixed Na+ concentration (0.086 M)) carrot disks after a thermal treatment of 20 min at 100 °C. Raw: untreated carrot, control: non-pretreated carrot, LTB: LTB carrot soaked in water, LTB + Na+: LTB carrot soaked in 0.086 M NaCl. Means with a different letter are significantly different. ( ): pH of soak solution after 1 h soak of LTB pretreated carrot disks.
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6.5 a
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60 4.2 40
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Relative hardness (% of raw)
120
3 raw
control
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LTB + CA LTB + CA LTB + CA LTB + CA pH 6.0 pH 5.3 pH 4.8 pH 4.2
Fig. 4. (Bars): Relative hardness (±standard error) of pretreated (LTB + 1 h soak in citric acid (CA) buffers of different pH values but with a fixed Na+ concentration (0.266 M)) carrot disks after a thermal treatment of 20 min at 100 °C. Raw: untreated carrot, control: non-pretreated carrot, LTB: LTB carrot soaked in water. Means with a different letter are significantly different. ( ): pH of soak solution after 1 h soak of LTB pretreated carrot disks.
3.2.2. Ferulic acid as pH lowering component In search for a suitable pH lowering soak solution, it is clear from previous results that chelators which complex Ca2+ ions and ions which stimulate the b-elimination have to be avoided. An exception to the latter is Ca2+ ions which are desirable. Although they stimulate the b-elimination, their potential to cross-link pectin chains prevails (as can be seen in Fig. 2 where there is clear texture improvement after a Ca2+ soak). Given current consumer demand for high-quality foods based on natural ingredients, it would be interesting to use an acid component naturally present in fruits and vegetables. Such a candidate is ferulic acid. Ferulic acid is a phenolic acid ubiquitous in nature, present in plant cell walls. In monocotyledons, ferulic acid is esterified to the O-5 of arabinose residues in arabinoxylans. In dicotyledons (to which fruits and vegetables belong), ferulic acid is associated with the pectin side chains and is ester linked to either the O-2 of arabinose or the O-6 of galactose residues. By cross-linking cell wall polysaccharides, ferulic acid imparts structural rigidity and strengthens cell wall architecture (Waldron, Smith, Parr, Ng, & Parker, 1997). At the same time, ferulic acid is commercially prepared and used as a functional food ingredient as it exhibits a wide range of therapeutic effects against various diseases, including cancer, diabetes, cardiovascular and neurodegenerative diseases, due to its potent antioxidant capacity (Itagaki et al., 2009). Considering the qualities just listed, it seems a valuable pH lowering agent. Fig. 5 shows the residual hardness of thermally treated carrot disks which were LTB
pretreated followed by a 1 h soak in 0.1% (w/v) ferulic acid. At the same time, three other components structurally related to ferulic acid were tested to be able to attribute the effect to a specific functional group. Soaking in ferulic acid resulted in a significant texture improvement. A similar or even larger texture improvement was observed after soaking in cinnamic acid or vanillic acid whereas soaking in vanillin had no effect. Again, the lower the pH the better the texture retention was. By comparing the chemical structures, it was clear that the texture improvement could be related to the acid functional group, present in ferulic acid, cinnamic acid and vanillic acid but absent in vanillin. The initial pH values of the different soak solutions were 3.5, 3.4, 3.4 and 5.0, respectively for ferulic acid, cinnamic acid, vanillic acid and vanillin. It is clear that these solutions, in contrast to the buffer solutions used earlier, undergo a pH shift during soaking of the carrots. However, the pH is still low enough to induce a pH shift in the carrots and correspondingly to result in a reduced texture loss. A thermal pretreatment proved once more to be an efficient way to permeabilize the tissue and facilitate uptake of exogenous components. Due to its solubility in organic solvents, it was expected that all exogenous ferulic acid would be washed away during preparation of AIR of ‘‘LTB + FA” treated carrots and that ferulic acid found in the AIR would be of endogenous origin. In the three washing solutions for preparation of the AIR, i.e. 64 mL 95% ethanol, 32 mL 95% ethanol and 32 mL acetone, respectively 3.72 (±0.33), 0.47 (±0.04) and 0.05 (±0.01) mg ferulic acid was found
6.5 5.8
a
5.8
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6 5.5
80
5 4.4
60 40
c d
20
4.2
4.3
bc
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4.5 4 d
3.5 3
e
pH solution after LTB and soak
Relative hardness (% of raw)
120
2.5
0 raw
control
LTB
LTB + FA LTB + CA LTB + VA LTB + VN pH 3.5 pH 3.4 pH 3.4 pH 5.0
Fig. 5. (Bars): Relative hardness (±standard error) of pretreated (LTB + 1 h soak in different solutions) carrot disks after a thermal treatment of 20 min at 100 °C. FA: ferulic acid, CA: cinnamic acid, VA: vanillic acid, VN: vanillin. Raw: untreated carrot, control: non-pretreated carrot, LTB: LTB carrot soaked in water. Means with a different letter are significantly different. ( ): pH of soak solution after 1 h soak of LTB pretreated carrot disks.
A. De Roeck et al. / Food Research International 43 (2010) 1297–1303
as determined by HPLC, confirming that ferulic acid had been taken up by the tissue. When analyzing the ferulic acid content of the AIR after saponification with 2 M NaOH it was noticed that there was still exogenous ferulic acid present. Raw and LTB pretreated carrots contained respectively 5.25 (±0.09) and 5.82 (±0.26) lg ferulic acid/g AIR whereas LTB pretreated carrots followed by FA soak contained 31.50 (±0.55) lg FA/g AIR which was considerably more. So clearly, a large amount of ferulic acid had been taken up by the tissue. It could be concluded that a thermal pretreatment followed by a ferulic acid soak was effective in lowering the pH of the carrot tissue, and hereby a suitable strategy for texture improvement of thermally processed carrots. Next to lowering the pH, one might suggest that ferulic acid also contributes to a better texture in another way, namely by enhancing cross-links between cell wall polymers. The evidence that ferulic acid cross-links contribute to the thermal stability of for example Chinese water chestnut has stimulated researchers in exploiting this mechanism in other plant tissues (Waldron et al., 1997). Chinese water chestnut is a monocotyledon, having high endogenous ferulic acid content. Most edible fruits and vegetables are dicotyledons and only a few, for example sugar beet and beetroot, contain similar levels of ferulic acid. Any increase in ferulic acid cross-linking will, in most dicotyledons, necessitate an increase in ferulic acid that is attached to polysaccharides at appropriate locations in the cell wall. In addition, enzymes that are able to cross-link ferulic acid will have to be present, including peroxidases, and those involved in the production of hydrogen peroxide. Conca (2003) tested the theory that (HP) infusion of peroxidase (POD) with and without ferulic acid into carrots and beets (having respectively a low and high endogenous ferulic acid content) could promote FA cross-linking resulting in maintenance of texture during thermal processing. POD and POD/FA infused carrot and beet samples had enhanced hardness when compared to the uninfused samples. An increase in cross-linked FA was seen in the POD and POD/FA infused beets but not for the carrots. The increase in cross-linking found in the POD infused beets must be directly related to cross-linking of existing wall bound ferulic acid. However, the POD/FA samples that were infused with additional ferulic acid resulted in further cross-linking with corresponding further firming. Conca (2003) proposed an inter-dimer cross-linking theory to explain the more enhanced hardness of the POD/FA infused over the POD infused beets. For the observed texture enhancement of carrots they found no explanation. Based on our results, a pH lowering effect of the infusion solutions used seems responsible for the enhanced hardness. In addition, the HP treatment might have had a firming effect (e.g. by stimulating PME activity) which was not investigated. Likewise, these two effects might have contributed to the enhanced hardness of beets. That ferulic acid cross-linking did not contribute to the texture improvement of the carrots in this study can easily be seen when comparing Fig. 5 with Fig. 3. When LTB treated carrots were soaked in ferulic, cinnamic or vanillic acid, the pH of the solution was about 4.3 and the residual hardness was about 34%. This corresponded very well with LTB treated carrots soaked in acetic acid buffer pH 4.1 where the pH of the solution was 4.2 and the residual hardness was 36%. 3.2.3. HP pretreatment in combination with ferulic acid soaking From Fig. 2 it is clear that a HP pretreatment is even more effective concerning texture improvement of thermally processed carrots than an LTB pretreatment. Whereas an LTB pretreatment resulted in a residual hardness (after thermal processing) of about 20%, HP pretreated carrots had a residual hardness of about 38%. The effect of a HP pretreatment followed by a ferulic acid soak was therefore investigated. In Fig. 6, it can be seen that a HP pretreatment followed by a ferulic acid soak resulted in a significant texture improvement (residual hardness of 45%) in addition to
120
Relative hardness (% of raw)
1302
a 100 80 b
60 c d
40 20
e
0 raw
control
HP
HP + FA
HP + Ca2+ & FA
Fig. 6. Relative hardness (±standard error) of pretreated carrot disks after a thermal treatment of 20 min at 100 °C. Raw: untreated carrot, control: non-pretreated carrot, HP: HP pretreated carrot, HP + FA: HP followed by ferulic acid soak, HP + Ca2+ and FA: HP followed by combined Ca2+ and ferulic acid soak. Means with a different letter are significantly different.
the texture improvement already obtained due to a HP pretreatment. This texture improvement was in the same order of magnitude as in the case of a HP pretreatment followed by a Ca2+ soak (residual hardness of 47%) (Fig. 2). When applying a combined Ca2+ and ferulic acid soak, an even better texture was obtained due to the combination of lowered b-elimination susceptibility and enhanced pectin cross-linking. By applying these two pretreatment steps, thermally processed carrots could retain about 53% of the hardness of raw carrots in contrast to only 6% when no pretreatment was applied. 4. Conclusion Thermal processing for food preservation results in an undesired high level of softening of carrots, mainly due to b-eliminative depolymerization of pectin which results in decreased cell adhesion. In context of texture improvement of thermally processed carrots, next to the established strategy of an LTB or HP pretreatment followed by a Ca2+ soak, a second strategy proved to be useful: treatment at low pH. By respectively lowering the DM of the carrot pectin or lowering the pH of the carrot tissue, the susceptibility to b-elimination was reduced. The composition of the pH lowering solution appeared important: cations and Ca2+ chelators have to be avoided. Ferulic acid showed to be an effective pH lowering component. By combining a HP pretreatment with a soak in ferulic acid and Ca2+ thermally processed carrots could retain more than 50% of the hardness of unprocessed carrots, due to the accumulative effect of the different modifications. Seen increasing consumer demand for high-quality, natural foods, ferulic acid would be a good acidifying component as it is naturally present in fruits and vegetables and is already used as a functional food ingredient. However, one has to remind that, although it is a very important quality characteristic, texture is not the only quality parameter of concern. Therefore, the influence of the different plant modifications on other quality parameters, for example taste of carrots, has to be evaluated to see whether the final product meets consumer demands. Acknowledgements This study has been carried out with financial support from the Research Fund K.U. Leuven, the Industrial Research Fund K.U. Leuven (KP/08/004) and the Commission of the European Communities, Framework 6, Priority 5 ‘Food Quality and Safety’, Integrated Project NovelQ FP6-CT-2006-015710.
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