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Fruit pectins – A suitable tool for screening gelling properties using infrared spectroscopy
Food Hydrocolloids 24 (2010) 1–7
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Fruit pectins – A suitable tool for screening gelling properties using infrared spectroscopy M.S. Lima a, E.P. Paiva a, S.A.C. Andrade b, J.A. Paixa˜o a, * a b
´ rio, 50670-901 Recife, PE, Brazil ˜o, Universidade Federal de Pernambuco, Av.Prof. Moraes Rego, s/n, Campus Universita Departamento de Nutriça ´rio, 50670-901 Recife, PE, Brazil Departamento de Engenharia Quı´mica, Universidade Federal de Pernambuco, Campus Universita
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 May 2008 Accepted 7 April 2009
Found in higher plants, pectins are natural hydrocolloids whose extraction is controversial as it is condition-dependent. To optimize the methodology for extracting and isolating pectins and verifying the effect on their structural characterization, a factorial 23 assay was planned, using the following independent variables: pH, duration of heating and nature of alcohol in fruits with different kinds of morphologies. The fruits were grouped into those that produce good jams and jellies (group I), those that have a variable chemical composition and contain fiber (group II), and those that contain starch (group III). The results were compared using variance analysis and Duncan test. The degree of methoxylation (DM) of the pectin isolated from the mesocarp of citrus and guava fruits (whole) was associated linearly with all independent variables. However, the pectin yield was influenced only by pH and duration of heating. The interaction between the nature of alcohol and the duration of heating were shown to be significant, a longer heating time and ethanol being better for the two kinds of fruit. In group I, the pectins isolated showed best DM associated with high yield, whilst group II yield was below the limit for producing jams and jellies, despite high DM. Group I fruits displayed characteristics that favor gel formation, whilst group II and group III proved to be deficient in at least one of the dependent variables. This study has validated as analytical tool for isolating and characterizing the structure of pectins, mainly those naturally occurring in tropical fruits. Published by Elsevier Ltd.
Keywords: Pectins Extraction Degree of methoxylation Infrared Gel formation
1. Introduction Pectins are heteropolysaccharides composed of hydrocolloids that occur naturally in higher plants, and are widely used in the food industry, owing to their ability to form gels, to stabilize and emulsify (Winning, Viereck, Nørgaarda, Larsen, & Engelsen, 2007). These molecules are found in the primary cell wall and in the intercellular layers known as the middle lamella, and help the cells to adhere to one another, thereby providing vegetable tissue with consistency and mechanical resistance (McCready, 1970; Mesbahi, Jamalian, & Farahnaky, 2005). Pectins differ in their chemical composition, with a predomination of monosaccharides, acids and other functional groups that undergo alterations during growth, ripening and storage (Bartley & Knee, 1982; Melford & Prakash, 1986). Pectins are associated with hemicellulose and cellulose in the form of protopectin, mainly in the primary cell wall, and are found in large quantities in the middle lamella in the form of pectinic acid and calcium pectate (Willats, Knox, & Mikkelsen, 2006).
* Corresponding author. Tel.: þ55 (81) 21268464; fax: þ55 (81) 21268473. E-mail addresses: [email protected], [email protected] (J.A. Paixa˜o). 0268-005X/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.foodhyd.2009.04.002
Chemically, pectins are a mixture of complex polysaccharides, homogalacturonan being the main component. This is a linear polymer made up of repeated units of alpha-(1-4)-linked D-galacturonic acid, to form a long polygalacturonic chain (Capel, Nicolai, Durand, Boulenguer, & Langendorff, 2006; Guillotin, Van Loey, Boulenguer, Schols, & Voragen, 2007; Mesbahi et al., 2005). In their molecular structure, the carboxylic acids of galacturonic monomers may or may not be esterified with methanol, or even acetic acid, in which case the percentage of esterified groups is expressed in the degree of methoxylation (DM) and degree of acetylation, respectively (Fishman, Chau, Hoagland, & Hotchkiss, 2006; Levigne, Thomas, Ralet, Quemener, & Thibault, 2002; Mesbahi et al., 2005; Yapo, Robert, Etienne, Wathelet, & Paquot, 2007). DM may reach the equivalent of 14% methoxyl, which means esterification of between 50 and 80%. These are known as highgrade methoxyl pectins, whilst those with a maximum of 7%, or a degree of esterification below 50% are regarded as low-grade methoxyl pectins (Pomeranz & Meloan, 2000). The extraction of pectins occurs in three main stages: the acid aqueous extraction of the extracted liquor precipitate, followed by the subsequent isolation and characterization of the pectin (Joye & Luzio, 2000; Liu, Shi, & Langrish, 2006; McCready, 1970).
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McCready (1970) and other contemporary researchers have reported that the extraction conditions have pronounced effects on the yield, characterization and chemical structure of the compounds (Joye & Luzio, 2000; Kalapathy & Proctor, 2001; Micard & Thibault, 1999; Pinheiro et al., 2008; Sahari, Akbarian, & Hamedi, 2003), leading to alterations in the functional properties of pectins, notably in their ability to form gels. These alterations allow for different uses in the food industry (Tabilo-Munizaga & BarbosaCanovas, 2005), the pharmaceutical industry (Yamada, 1996; Yamada, Kiyohara, & Matsumoto, 2003) and for biodegradable packaging (Barbosa-Canovas, Kokini, Ma, & Ibarz, 1996). The ability to form gels does not depend only on the type of pectin (methoxylated, acetylated or amidated) but principally on the concentration of pectin (from 0.1 to 1.5%), pH 2.7–3.7, and total solids (64–71%). Models for studies of the tertiary structure of the gel confirm that calcium pectates form an egg-box type network stabilized by bridges of calcium, whilst the hydrophobic chains of sugars and acids and other pectin components sustain an interface with methanol to form a gel when medium (50–60% DM) – to highgrade methoxyl (superior to 60% DM) pectins are used (Grant, Morris, Ree, Smith, & Thom, 1973). The procedure for extracting pectins has raised doubts as to the appropriateness of the method, owing primarily to the influence of the pH conditions (Joye & Luzio, 2000; McCready, 1970; Mesbahi et al., 2005), but also the nature of the acid (Sahari et al., 2003) and alcohol precipitant (Kalapathy & Proctor, 2001). This study aimed to optimize the extraction conditions by using a more appropriate procedure for isolating and characterizing fruit pectins, considering matrix effects (mesocarp and whole) evaluating include properties gelling burnt directly of tropical fruits. 2. Materials and methods 2.1. Material 2.1.1. Citric fruits (mesocarp) and whole fruits The mesocarp of oranges (Rutaceae sp) and passion fruit (Passiflora edulis) was used to monitor the effects of extraction pH on the pectin yield, degree of methoxylation and the size of isolated pectin fragments. Orange mesocarp was used to characterize pectins for industrial purposes, whilst guava (whole fruit) was used in order to obtain the result of the optimization assay for comparison purposes. The method actually proposed in this study was applied to three distinct classes of whole fruit that yield reasonable quantities of pectin and different properties of gelling (Group I): apple (Malus silvestris), guava (Psidium guajava), strawberry (Fragaria vesca L.), grape (Vitia vinifera L.), jaboticaba (Myrcia califora Berg); and those that contain fibers (Group II): pitanga (Brazil cherry) (Eugenia uniflora L.), acerola (West Indian cherry) (Malpighia glabra), cashew fruit (Anacardium orcidentale), mango (Mangifera indica L.), sapoti (naseberry) (Achras sapota L.); and those which contain starch (Group III): graviola (soursop) (Annona muricato L.), pinha (sugar apple) (Annona sp.), banana (Musa paradisiaca). The fruits were thus classified in order to verify the impact of different chemical compositions on the extraction performance and sufficient characterization of pectins. All the fruits selected were determined taken as better stage of maturation on the basis of their characteristic color and consistency. The fruits were peeled, pulped and diluted for exhaustive extraction in ratios previously established. 2.1.2. Standard pectins Standard galacturonic acids were obtained from Merck, DM ¼ 10.59% and from Sigma, DM ¼ 1.26%. Pectins obtained from
Citrus Colloids containing DM ¼ 37.62% and DM ¼ 70.56% and from CP Kelco, DM ¼ 38.15% and DM ¼ 61.05%; Fluka, DM ¼ 71.70%. DM values (%) for all the standards were confirmed using titrimetric methods as described for McCready (1970). 2.2. Methods 2.2.1. Extraction of pectins from mesocarp citrus under different pH One hundred grams of each sample of orange and passion fruit mesocarp were selected from at least one kilogram of peeled fruit. 800 mL of de-ionized water (1:8 m/v) was added and the mixture shredded in a food processor to obtain small fragments. The pH was set at 2.2 using a 10% solution of citric acid and the mixture was heated to boiling for 30 min, cooled and strained using polyester fabric. This filtrate was used as the mother fraction A. 10% HCl was added to one third of fraction A to set the pH at 1.0. It was then boiled for 30 min and cooled, and the pH neutralized to pH 7.0, to form fraction A. 10% NaOH was added to another one third of fraction A to bring the pH up to 12.0. This was maintained for 60 min at 20 C and the pH was neutralized to pH 7.0 to form fraction Aþ. 2.2.2. Isolating pectin directly extracted of tropical fruits The pectins were isolated by full precipitation using a quantum satis volume of 95% ethanol, and the portions obtained after precipitation yielding fractions A, A and Aþ, which were filtered and washed first in 99% ethanol, and then in 99% acetone to remove sugars and pigments respectively. The final precipitate was kept at room temperature for 24 h and then lyophilized for the preparation of KBr pastille. 2.2.3. Experiment design of suitability tool extraction of pectins The experiment was carried out using factorial 23 planning with the following independent variables: pH, extraction time (minutes) and nature of alcohol, which produced 8 assays, each repeated twice. The conditions found are shown in Table 1 (coded and noncoded levels). The response variables were yield (%) and degree of methoxylation (%). 2.2.4. Procedure for optimizing the extraction of pectins from tropical fruits 800 mL of de-ionized water was added to 100 g of material (citric fruit mesocarp) to the ratio of 1:8 m/v or 400 mL, when the whole fruit was used. The pH of the liquor was adjusted to 2.2 or alternatively to pH 3.0. The mixture was then heated to boiling for 15 or alternatively 30 min, then cooled and filtered using polyester fabric. The pectin (from fraction A) was isolated by the addition of alcohol: 95% ethanol or methanol quantum satis. The final precipitate was kept at room temperature for 24 h and then lyophilized for the preparation of KBr pastille. 2.2.5. Measuring the degree of methoxylation and yield of pectins Standard pectins, lyophilized fractions of pectins and KBr were desiccated in a vacuum. The samples were homogenized with KBr Table 1 Coded levels of independent variables. Assay
pH
R-OH
t (min)
1 2 3 4 5 6 7 8
1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1
Legend: 1 and þ1 correspond to 150 and 300 for the time variable; ethanol and methanol for alcohol type and 2.2 and 3.0 for extraction pH respectively.
M.S. Lima et al. / Food Hydrocolloids 24 (2010) 1–7 Table 2 Effect of different initial pH on natural pectin yield from citrus fruits. Yield (%)
0,30
Orange A A Aþ
2.2 8.81 0.03Ab 13.42 0.83Aa 6.83 0.18Ac
Fraction A
0,35
Passion fruit 3.0 2.57 0.04Bab 2.79 0.07BCa 2.10 0.32Bb
2.2 2.47 0.04Cab 3.44 0.62Ba 1.76 0.11BCb
3.0 1.19 0.01 Da 1.34 0.23Ca 1.15 0.24Ca
Column means with different lower-case letters differ statistically by 5% Row means with different upper-case letters differ statistically by 5%.
0,25
Absorbance
Fractions
3
1750 0,20
1650
0,15 0,10
(9:1 salt/sample) and a compressor (Beckman 00-25) used to produce a pellet, which was sent for analysis using an infrared spectrometer (Bruker IFS66). The FT-IR spectrum was collected for absorbance in the 400–4000 cm1 band, with a resolution of 4 cm1. After analysis of each spectrum using FT-IR through identification of the main 1650 and 1750 cm1 bands, the DM was obtained using the equation: [A1750 cm1/(A1650 cm1 þ A1750 cm1)] in accordance with Manrique and Lajolo (2002). The yield pectin (%) was determined after step of lyophilization by weighing residue. All analyses were performed in duplicate.
0,05 4000
3500
3000
2500
2000
1500
Absorbance
1650
0,5
1750 0,4
0,3
2.2.7. Measuring the aperture of the pores of the polyester fabric and the pectin fragments isolated A sample of a single layer of fabric as recommended for filtering was analyzed by sweeping electronic microscopy (Shimadzu) (Voltage: 20 kV; Spot size: 10). The aperture of the pores and the size of the lyophilized pectin fractions were compared using Image J Morphometry software.
0,2 4000
3500
3000
2500
2000
The pectins extracted under different pH conditions showed significant differences in yield profile (Table 2) and degree of methoxylation (Table 3). As confirmed by the spectra presented in Fig. 1, the variation in the degree of methoxylation was associated with the pH. Conditions of extreme acidity (pH 1.0) therefore caused deesterification and de-polymerization of the pectin chains (Pinheiro
Table 3 Effect of different pH on degree of methoxylation of pectin yield from citrus fruits. Fractions
A A Aþ
Degree of methoxylation (%) Orange
Passion fruit
69.50 1.02a 52.15 0.00b 14.02 0.10c
69.22 0.28a 48.64 2.82b 20.44 0.90c
Columns means with different lower-case letters differ statistically by 5%.
1000
500
Fraction A 0,50
+
1650
0,45 0,40 0,35
Absorbance
3.1. Characterization of pectin fractions extracted under different pH
1500
Wave number cm-1
2.2.8. Statistical analyses Variance analysis was then carried out, comparisons being made using the Duncan 5% significance test on the Statistic 6.1 program (Statsoft, 1997). 3. Results and discussion
500
Fraction A-
0,7
0,6
2.2.6. Linear regression The degree of methoxylation of the standard pectins was measured using titrimetric (in quintuplicate) as proposed by McCready (1970). The curve was plotted with the help of the Origin5.0 program, and the results correlated using titrimetric versus infrared spectroscopy. For a regression curve plotted with six points R2 ¼ 93.35; a ¼ 0.19663 and b ¼ 0.00508.
1000
Wave number cm-1
0,30
1750
0,25 0,20 0,15 0,10 0,05 4000
3500
3000
2500
2000
1500
1000
500
Wave number cm-1 Fig. 1. Infrared spectrum of pectins extracted from orange mesocarp at different pH.
et al., 2008). The same occurred at high alkali conditions (pH 12.0), which resulted in saponification of methyl ester groupings mainly these groupings are easily dissolved in this medium. In addition, the reduction of the polygalacturonic chain by b-elimination produced different sized pectins. Both treatments led to a reduction in the degree of methoxylation to levels below 50%, particularly from of the mesocarp of citric fruits, as shown in Table 3.
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Fig. 2. Analysis of polyester fabric used for isolating main fractions.
The size of fragments isolated from orange mesocarp, such as fraction A (pH 2.2; 4478.8 mm), fraction A (pH 1.0; 2842.2 mm) and fraction Aþ (pH 12.0; 2523.1 mm) differed significantly when A was compared with A and Aþ fractions. The reduced size of the lyophilized pectin fragments may reduce the firmness of the gel from the isolation stage onwards. Firmness was also associated with the quantity of pectin present and primarily the degree of methoxylation (a minimum of 50%). Some reports suggest that it is possible to obtain firm gels at levels as low as 0.1% (Kerr & Wicker, 2000). However, the effect of initial pH on extraction of pectins with variable degrees of methoxylation and probable size molecular can be obtained using different analytical tool in accordance with the degree of firmness of the gel (Kerr & Wicker, 2000) and other factors (Winning et al., 2007). Therefore the extraction time also appears to influence the size of particles. Yapo et al. (2007) have confirmed that when extracting pectins from beetroot at pH 1.5, 90 C, varying the duration from 1 to 4 h, caused a marked reduction in the size molecular when the pectin beet was analyzed using molecular exclusion chromatography. The separation of these molecules takes place according to their size. The larger molecules move more easily (eluted with 9 mL), whilst the smaller ones are retained (eluted with 12 mL) when appropriate columns was used. 3.2. Characterization of the polyester fabric used to filter and isolate the pectin The average aperture in size of the pores in the polyester fabric analyzed was 1072.35 mm (Fig. 2), this being the adequate aperture for isolation of the principal fractions such A, A and Aþ. The nature of the fabric or mesh recommended for retaining the fractions, which is especially neglected in the literature, is derived primarily from McCready’s (1970) original methodology, which recommends the use of cheesecloth. The polyester fabric used in order to improve the isolation stage showed itself to be effective in retaining pectin fractions, given that,
when retained, the pectins are at their maximum levels of coalescence, allowing only molecules with a size of less than 2523.1 mm to pass through the fabric. Other researchers have used centrifugation (2700 g) to isolate pectin (Kalapathy & Proctor, 2001) or ultra-filtration with selected 3–11 mm membranes (Yapo et al., 2007) in substitution to polyester fabric. 3.3. Optimization assay for the extraction of pectins from fruit The degree of methoxylation of pectins extracted from orange mesocarp increased linearly with the nature of alcohol and extraction time, while it decreased with increasing pH, as shown in Table 4 and Fig. 3. The yield for the extraction of pectin from orange mesocarp increased linearly with extraction time and decreased with increasing pH (Table 4 and Fig. 4). The highest yield was obtained when the treatment used a longer extraction time and a lower initial pH, although yield did not seem to be influenced by the nature of alcohol. Only increasing pH decreased the degree of methoxylation of pectin obtained from guava, the extraction carried out at pH 2.2 rendering a higher degree of methoxylation, as illustrated in Fig. 5 and Table 4. Table 4 shows that the yield for extraction of pectin from guava displayed the same behavior observed in the case of orange mesocarp in terms of pH and extraction time: the longer the extraction time and the lower the pH, the higher the yield (Fig. 6). The yield for guava was moderately affected by the nature of alcohol, suggesting that ethanol is the most appropriate alcohol for
Table 4 Principal effects of the tropical fruit pectin extraction optimization study. Factors
pH (1) Nature of alcohol (2) Extraction time (3) 1&2 1&3 2&3 1,2 & 3 NS: not significant.
Orange
Guava
Yield (%)
DM (%)
Yield (%)
DM (%)
6.59 NS 6.94 NS 1.79 1.09 NS
3.88 3.61 2.46 NS NS NS NS
0.30 0.07 0.29 0.06 0.17 NS NS
5.37 NS NS NS NS 3.96 4.02 Fig. 3. Degree of methoxylation (%) of orange mesocarp pectin.
M.S. Lima et al. / Food Hydrocolloids 24 (2010) 1–7
5
Fig. 4. Yield (%) obtained in the extraction of orange mesocarp pectin.
Fig. 6. Yield (%) obtained in the extraction of guava pectin.
the precipitation of pectin, corroborating the claim made by McCready in 1970. Recently used by Kalapathy and Proctor (2001), isopropanol may prove to be an interesting alternative for precipitates of smaller diameter, even for modified pectins. In correlation studies, Levigne et al. (2002) reported that the degree of methoxylation tends to rise in proportion to the initial pH of the extraction, when comparing the effect for pH 1.0 and 3.0. Yapo et al. (2007) observed a similar trend on analyzing pectin extracted at pH 1.5 and 2.0, using 96% ethanol as the precipitation agent. In these studies, the strongly acidic pH (1.0 and 1.5) caused de-esterification of the polygalacturonic chain, which also reflects the reduction simultaneous in the degree of methoxylation, as observed in these results and presented in Table 3 and Fig. 1. In addition to the pH, an increase in the concentration of acid from 0.05 N to 0.2 N of HCl reduces the yield from 28% to 21% in the extraction of pectins from soybean pods, when precipitated using isopropanol (Kalapathy & Proctor, 2001). McCready’s (1970) proposal suggests adjusting the pH using citric acid, thereby minimizing the impact of the acid on the pectin and producing reliable results for the original representation of the chemical composition model found in vegetable matter, especially fruit. However, it should be noted that extreme conditions during extraction (pH below 2.2) and extraction time (longer than 30 min) may accelerate the degradation of this heteropolysaccharide (Cho & Hwang, 2000).
There is no evidence of a direct correlation between extraction time and degree of methoxylation, as extraction may, after more than 30 minutes (and at a temperature of over 100 C), lead to the formation of Maillard’s reaction products, such as aldosamines and furfural. These indirectly confirm that de-esterification and depolymerization of the pectin has occurred (Garna, Mabon, Nott, Wathelet, & Paquot, 2006). According to some researchers (Micard & Thibault, 1999; Mesbahi et al., 2005; Yapo et al., 2007), pH and extraction time are parameters that have greater impact on the yield resulting suitable conditions for extraction of pectins, and that temperature may have a slight effect if analyzed separately. Both time and temperature should be taken into consideration as high temperatures for long periods of extraction may result in full hydrolysis of the pectin chain (Mesbahi et al., 2005).
3.4. Evaluation of the degree of methoxylation of fruit pectins as gel-forming capacity DM and pectin yield are important factors in determining the firmness of the gel and, subsequently, the value and possible use of different kinds of fruits as raw material in the food industry. Extraction that aims to obtain a higher yield and better characterization of pectins in terms of DM is a useful tool in selecting Table 5 Characterization of different tropical fruits grouped by differences in chemical composition and gelling properties. pH Group I (produce good jellies) Guava 3.85 Strawberry 3.78 Grape 3.91 Apple 4.0 Jaboticaba 3.0
Fig. 5. Degree of methoxylation (%) of guava pectin.
0.06b 0.13b 0.77b 0.03a 0.28c
Yield (%)
DM (%)
1.74 1.08 0.03 1.72 0.89
71.55 71.65 81.30 75.32 71.00
1.77c 0.07c 0.28a 0.11b 0.0c
0.28c 0.07c 0.56d 0.49a 0.28b
0.08a 0.0b 0.01d 0.03a 0.09c
Group II (contain mainly fiber) Brazil cherry 2.98 0.17c West Indian cherry 4.5 0.42b Cashew 4.6 0.42b Mango 4.55 0.49b Naseberry 5.70 0.00a
0.05 0.00b 0.045 0.01bc 0.024 0.01 cd 1.19 0.01a 0.02 0.01d
53.80 54.05 47.40 68.65 59.70
Group III (contain mainly starch) Soursop 3.9 0.00b Sugar apple 5.47 0.39a Banana 3.4 0.00b
2.15 0.07a 0.41 0.00c 1.05 0.04b
49.40 0.14c 64.25 0.92a 59.80 0.28b
Equal row letters do not differ statistically by 5%.
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M.S. Lima et al. / Food Hydrocolloids 24 (2010) 1–7
different kinds of fruits for technological purposes, mainly for the production of single-fruit and blended jams and jellies. The basic ingredients necessary for gel formation to occur may be present in the fruit itself. However, a deficiency in any one of the parameters that affect the quality of the pectin may also be corrected in the formulas prior to establish onset gel formation. These formulas generally suggest a limit of 65% sugar (sucrose), acidity of around pH 3.0, and high-grade methoxyl pectins (>50%), although it is not known what the ideal concentration of pectin in, traditional formulas suggesting a minimum of 1%. Whilst more recent data obtained using different standard pectins claim that the required consistency for a firm gel can be observed at levels as low as 0.1% (Kerr & Wicker, 2000). Generally speaking, pectin gels are formed when regions of the molecule form cross-links, through a three-dimensional network, where water molecules and co-solutes are retained. Especially in the case of high-grade methoxyl pectins, gel formation occurs when the junction zones are formed by the cross-links of galacturonic acid by hydrogen bridges and hydrophobic forces between the methoxyl groups present, as caused by the high concentration of sugar and low pH (Willats et al., 2006). In lowmethoxyl pectins, gel formation involves simultaneous linkages between calcium ions from free carboxyl groups, according to the egg-box model (Capel et al., 2006; Cardoso, Coimbra, & Lopes da Silva, 2003; Grosso, Bobbio, & Airoldi, 2000; Lo¨fgren & Hermansson, 2007). In fruits evaluated in this experiment and treated as group I (Table 5), the pectins isolated showed high levels of methoxylation (>70.0%); a high percentage yield, with the exception of grape, which showed lower values, although this was compensated for by the high degree of methoxylation (>80.0%); and a pH near to the ideal for gel formation. On analysis of the three parameters presented in Table 5 (pH, pectin yield and degree of methoxylation), it can be seen that in the case of groups II and III there was a deficiency in one of the values established for gel formation, thereby requiring further correction of soluble solids and acidity. Concerning the fruits in group II, despite the degree of methoxylation being high (>50%), with the exception of cashew fruit, which proved to be unsatisfactory in all three parameters, the yield was lower than the minimum established in the literature. The pH of the fruits was high (>3.0) with the exception of pitanga (Brazil cherry). In this group, adjustments need to be made, both to the pH and also the addition of higher-grade methoxyl pectins, or alternatively, blends should be produced as a way of achieving complementary and synergic effects. Of the group III fruits, graviola (soursop) showed a degree of methoxylation below 50%, although the yield and the pH satisfactory. Pinha (sugar apple) displayed a very high pH, although its yield and degree of methoxylation approached adequate levels. Banana, in contrast, behaved like a group I fruit, despite the expected high levels of starch. 4. Conclusions The main results obtained under the experimental conditions reported above allow us to conclude that the yield and DM of pectins are modified by an initial extraction pH that causes simultaneous de-methoxylation and defragmentation, thereby allowing different pectins to be produced for different industrial applications. The size of lyophilized pectin fragments tends to decrease proportionally with the degree of methoxylation in both alkaline and acid media. The polyester fabric analyzed proved to be effective in isolating pectins and is a more easily accessible technique than those usually employed. The procedure for extracting fruit pectin
was optimized in terms of pH, extraction time and nature of alcohol, thus allowing the most efficient and suitable, in terms of yield and DM, to be chosen. Finally, the group I fruits lent themselves easily to the formation of gels, confirming their status as fruits that produce good jellies and jams. In contrast, group II and group III fruits did not display similar behavior on comparison of the principal characteristics evaluated in this experiment. Acknowledgments The authors are grateful to CAPES and The Federal University of Pernambuco for financial and human resource support. References Barbosa-Canovas, G. V., Kokini, J. L., Ma, L., & Ibarz, A. (1996). The rheology of semi liquid foods. Advances in Food and Nutrition Research, 39, 1–69. Bartley, I. M., & Knee, M. (1982). The chemistry of textural changes in fruit during storage. Food Chemistry, 9(1–2), 47–58. Capel, F., Nicolai, T., Durand, D., Boulenguer, P., & Langendorff, V. (2006). Calcium and acid induced gelation of (amidated) low methoxyl pectin. Food Hydrocolloids, 20(6), 901–907. Cardoso, S., Coimbra, M., & Lopes da Silva, J. A. (2003). Temperature dependence of the formation and melting of pectin–Ca2þ networks: a rheological study. Food Hydrocolloids, 17(6), 801–807. Cho, Y.-J., & Hwang, J.-K. (2000). Modeling the yield and intrinsic viscosity of pectin in acidic solubilization of apple pomace. Journal of Food Engineering, 44(2), 85–89. Fishman, M. L., Chau, H. K., Hoagland, P. D., & Hotchkiss, A. T. (2006). Microwaveassisted extraction of lime pectin. Food Hydrocolloids, 20(8), 1170–1177. Garna, H., Mabon, N., Nott, K., Wathelet, B., & Paquot, M. (2006). Kinetic of the hydrolysis of pectin galacturonic acid chains and quantification by ionic chromatography. Food Chemistry, 96(3), 477–484. Grant, G. T., Morris, E. R., Ree, D. A., Smith, P. J. C., & Thom, D. (1973). Biological interactions between polysaccharides and divalent cations: the egg-box model. Federation of European Biochemical Societies Letters, 32(1), 195–198. Grosso, C. R. F., Bobbio, P. A., & Airoldi, C. (2000). Effect of sugar and sorbitol on the formation of low methoxyl pectin gels. Carbohydrate Polymers, 41(4), 421–424. Guillotin, S. E., Van Loey, A., Boulenguer, P., Schols, H. A., & Voragen, A. G. J. (2007). Rapid HPLC method to screen pectins for heterogeneity in methyl-esterification and amidation. Food Hydrocolloids, 21(1), 85–91. Joye, D. D., & Luzio, G. A. (2000). Process for selective extraction of pectins from plant material by differential pH. Carbohydrate Polymers, 43(4), 337–342. Kalapathy, U., & Proctor, A. (2001). Effect of acid extraction and alcohol precipitation conditions on the yield and purity of soy hull pectin. Food Chemistry, 73(4), 393–396. Kerr, W. L., & Wicker, L. (2000). NMR proton relaxation measurements of water associated with high methoxyl and low methoxyl pectins. Carbohydrate Polymers, 42(2), 133–141. Levigne, S., Thomas, M., Ralet, M. C., Quemener, B., & Thibault, J. F. (2002). Determination of the degrees of methylation and acetylation of pectins using a C18 column and internal standards. Food Hydrocolloids, 16(6), 547–550. Liu, Y., Shi, J., & Langrish, T. A. G. (2006). Water-based extraction of pectin from flavedo and albedo of orange peels. Chemical Engineering Journal, 120(3), 203–209. Lo¨fgren, C., & Hermansson, A.-M. (2007). Synergistic rheological behaviour of mixed HM/LM pectin gels. Food Hydrocolloids, 21(3), 480–486. McCready, R. M. (1970). Pectin. In M. A. Joslyn (Ed.), Methods in food analysis: Physical, chemical, and instrumental (pp. 565–599). New York: Academic Press. Manrique, G. D., & Lajolo, F. M. (2002). FT-IR spectroscopy as a tool for measuring degree of methyl esterification in pectins isolated from ripening papaya fruit. Postharverst Biology and Technology, 25(1), 99–107. Melford, A. J., & Prakash, M. D. (1986). Postharvest changes in fruit cell wall. Advances Food Research, 30, 139–193. Mesbahi, G., Jamalian, J., & Farahnaky, A. (2005). A comparative study on functional properties of beet and citrus pectins in foods systems. Food Hydrocolloids, 19(4), 731–738. Micard, V., & Thibault, J. F. (1999). Oxidative gelation of sugar-beet pectins: use of laccases and hydration properties of the cross-linked pectins. Carbohydrate Polymers, 39(3), 265–273. Pinheiro, E. R., Silva, I. M. D. A., Gonzaga, L. V., Amante, E. R., Teo´filo, R. F., Ferreira, M. M. C., et al. (2008). Optimization of extraction of high-ester pectin from passion fruit peel (Passiflora edulis flavicarpa) with citric acid by using response surface methodology. Bioresource Technology, 99, 5561–5566. Pomeranz, Y., & Meloan, C. (2000). Food analysis: Theory and practice. New York: Chapman & Hall. Sahari, A. L., Akbarian, A., & Hamedi, M. (2003). Effect of variety and acid washing method on extraction yield and quality of sunflower head pectin. Food Chemistry, 83(1), 43–47.
M.S. Lima et al. / Food Hydrocolloids 24 (2010) 1–7 Statsoft, Inc. (1997). Statistica for windows 6.0 [Computer program manual]. Tulsa: StatSoft. Tabilo-Munizaga, G., & Barbosa-Canovas, G. V. (2005). Rheology for the food industry. Journal of Food Engineering, 67(1–2), 147–156. Willats, W. G. T., Knox, J. P., & Mikkelsen, J. D. (2006). Pectin: new insights into an old polymer are starting to gel. Trends in Food Science & Tecnology, 17(3), 97–104. Winning, H., Viereck, N., Nørgaard, L., Larsen, J., & Engelsen, S. B. (2007). Quantification of the degree of blockiness in pectins using 1H NMR spectroscopy and chemometrics. Food Hydrocolloids, 21(2), 256–266.
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Yamada, H. (1996). Contribution of pectins on health care. In J. Visser, & A. G. J. Voragen (Eds.), Pectins and pectinases (pp. 173–190). Amsterdam: Elsevier. Yamada, H., Kiyohara, H., & Matsumoto, T. (2003). Recent studies on possible functions of bioactive pectins and pectic polysaccharides from medicinal herbs. In F. Voragen, H. Schols, & R. Visser (Eds.), Advances in pectin and pectinase research (pp. 481–490). Dordrecht: Kluwer Academic Publishers. Yapo, B. M., Robert, C., Etienne, I., Wathelet, B., & Paquot, M. (2007). Effect of extraction conditions on the yield, purity and surface properties of sugar beet pulp pectin extracts. Food Chemistry, 100(4), 1359–1364.
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Fruit pectins – A suitable tool for screening gelling properties using infrared spectroscopy M.S. Lima a, E.P. Paiva a, S.A.C. Andrade b, J.A. Paixa˜o a, * a b
´ rio, 50670-901 Recife, PE, Brazil ˜o, Universidade Federal de Pernambuco, Av.Prof. Moraes Rego, s/n, Campus Universita Departamento de Nutriça ´rio, 50670-901 Recife, PE, Brazil Departamento de Engenharia Quı´mica, Universidade Federal de Pernambuco, Campus Universita
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 May 2008 Accepted 7 April 2009
Found in higher plants, pectins are natural hydrocolloids whose extraction is controversial as it is condition-dependent. To optimize the methodology for extracting and isolating pectins and verifying the effect on their structural characterization, a factorial 23 assay was planned, using the following independent variables: pH, duration of heating and nature of alcohol in fruits with different kinds of morphologies. The fruits were grouped into those that produce good jams and jellies (group I), those that have a variable chemical composition and contain fiber (group II), and those that contain starch (group III). The results were compared using variance analysis and Duncan test. The degree of methoxylation (DM) of the pectin isolated from the mesocarp of citrus and guava fruits (whole) was associated linearly with all independent variables. However, the pectin yield was influenced only by pH and duration of heating. The interaction between the nature of alcohol and the duration of heating were shown to be significant, a longer heating time and ethanol being better for the two kinds of fruit. In group I, the pectins isolated showed best DM associated with high yield, whilst group II yield was below the limit for producing jams and jellies, despite high DM. Group I fruits displayed characteristics that favor gel formation, whilst group II and group III proved to be deficient in at least one of the dependent variables. This study has validated as analytical tool for isolating and characterizing the structure of pectins, mainly those naturally occurring in tropical fruits. Published by Elsevier Ltd.
Keywords: Pectins Extraction Degree of methoxylation Infrared Gel formation
1. Introduction Pectins are heteropolysaccharides composed of hydrocolloids that occur naturally in higher plants, and are widely used in the food industry, owing to their ability to form gels, to stabilize and emulsify (Winning, Viereck, Nørgaarda, Larsen, & Engelsen, 2007). These molecules are found in the primary cell wall and in the intercellular layers known as the middle lamella, and help the cells to adhere to one another, thereby providing vegetable tissue with consistency and mechanical resistance (McCready, 1970; Mesbahi, Jamalian, & Farahnaky, 2005). Pectins differ in their chemical composition, with a predomination of monosaccharides, acids and other functional groups that undergo alterations during growth, ripening and storage (Bartley & Knee, 1982; Melford & Prakash, 1986). Pectins are associated with hemicellulose and cellulose in the form of protopectin, mainly in the primary cell wall, and are found in large quantities in the middle lamella in the form of pectinic acid and calcium pectate (Willats, Knox, & Mikkelsen, 2006).
* Corresponding author. Tel.: þ55 (81) 21268464; fax: þ55 (81) 21268473. E-mail addresses: [email protected], [email protected] (J.A. Paixa˜o). 0268-005X/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.foodhyd.2009.04.002
Chemically, pectins are a mixture of complex polysaccharides, homogalacturonan being the main component. This is a linear polymer made up of repeated units of alpha-(1-4)-linked D-galacturonic acid, to form a long polygalacturonic chain (Capel, Nicolai, Durand, Boulenguer, & Langendorff, 2006; Guillotin, Van Loey, Boulenguer, Schols, & Voragen, 2007; Mesbahi et al., 2005). In their molecular structure, the carboxylic acids of galacturonic monomers may or may not be esterified with methanol, or even acetic acid, in which case the percentage of esterified groups is expressed in the degree of methoxylation (DM) and degree of acetylation, respectively (Fishman, Chau, Hoagland, & Hotchkiss, 2006; Levigne, Thomas, Ralet, Quemener, & Thibault, 2002; Mesbahi et al., 2005; Yapo, Robert, Etienne, Wathelet, & Paquot, 2007). DM may reach the equivalent of 14% methoxyl, which means esterification of between 50 and 80%. These are known as highgrade methoxyl pectins, whilst those with a maximum of 7%, or a degree of esterification below 50% are regarded as low-grade methoxyl pectins (Pomeranz & Meloan, 2000). The extraction of pectins occurs in three main stages: the acid aqueous extraction of the extracted liquor precipitate, followed by the subsequent isolation and characterization of the pectin (Joye & Luzio, 2000; Liu, Shi, & Langrish, 2006; McCready, 1970).
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M.S. Lima et al. / Food Hydrocolloids 24 (2010) 1–7
McCready (1970) and other contemporary researchers have reported that the extraction conditions have pronounced effects on the yield, characterization and chemical structure of the compounds (Joye & Luzio, 2000; Kalapathy & Proctor, 2001; Micard & Thibault, 1999; Pinheiro et al., 2008; Sahari, Akbarian, & Hamedi, 2003), leading to alterations in the functional properties of pectins, notably in their ability to form gels. These alterations allow for different uses in the food industry (Tabilo-Munizaga & BarbosaCanovas, 2005), the pharmaceutical industry (Yamada, 1996; Yamada, Kiyohara, & Matsumoto, 2003) and for biodegradable packaging (Barbosa-Canovas, Kokini, Ma, & Ibarz, 1996). The ability to form gels does not depend only on the type of pectin (methoxylated, acetylated or amidated) but principally on the concentration of pectin (from 0.1 to 1.5%), pH 2.7–3.7, and total solids (64–71%). Models for studies of the tertiary structure of the gel confirm that calcium pectates form an egg-box type network stabilized by bridges of calcium, whilst the hydrophobic chains of sugars and acids and other pectin components sustain an interface with methanol to form a gel when medium (50–60% DM) – to highgrade methoxyl (superior to 60% DM) pectins are used (Grant, Morris, Ree, Smith, & Thom, 1973). The procedure for extracting pectins has raised doubts as to the appropriateness of the method, owing primarily to the influence of the pH conditions (Joye & Luzio, 2000; McCready, 1970; Mesbahi et al., 2005), but also the nature of the acid (Sahari et al., 2003) and alcohol precipitant (Kalapathy & Proctor, 2001). This study aimed to optimize the extraction conditions by using a more appropriate procedure for isolating and characterizing fruit pectins, considering matrix effects (mesocarp and whole) evaluating include properties gelling burnt directly of tropical fruits. 2. Materials and methods 2.1. Material 2.1.1. Citric fruits (mesocarp) and whole fruits The mesocarp of oranges (Rutaceae sp) and passion fruit (Passiflora edulis) was used to monitor the effects of extraction pH on the pectin yield, degree of methoxylation and the size of isolated pectin fragments. Orange mesocarp was used to characterize pectins for industrial purposes, whilst guava (whole fruit) was used in order to obtain the result of the optimization assay for comparison purposes. The method actually proposed in this study was applied to three distinct classes of whole fruit that yield reasonable quantities of pectin and different properties of gelling (Group I): apple (Malus silvestris), guava (Psidium guajava), strawberry (Fragaria vesca L.), grape (Vitia vinifera L.), jaboticaba (Myrcia califora Berg); and those that contain fibers (Group II): pitanga (Brazil cherry) (Eugenia uniflora L.), acerola (West Indian cherry) (Malpighia glabra), cashew fruit (Anacardium orcidentale), mango (Mangifera indica L.), sapoti (naseberry) (Achras sapota L.); and those which contain starch (Group III): graviola (soursop) (Annona muricato L.), pinha (sugar apple) (Annona sp.), banana (Musa paradisiaca). The fruits were thus classified in order to verify the impact of different chemical compositions on the extraction performance and sufficient characterization of pectins. All the fruits selected were determined taken as better stage of maturation on the basis of their characteristic color and consistency. The fruits were peeled, pulped and diluted for exhaustive extraction in ratios previously established. 2.1.2. Standard pectins Standard galacturonic acids were obtained from Merck, DM ¼ 10.59% and from Sigma, DM ¼ 1.26%. Pectins obtained from
Citrus Colloids containing DM ¼ 37.62% and DM ¼ 70.56% and from CP Kelco, DM ¼ 38.15% and DM ¼ 61.05%; Fluka, DM ¼ 71.70%. DM values (%) for all the standards were confirmed using titrimetric methods as described for McCready (1970). 2.2. Methods 2.2.1. Extraction of pectins from mesocarp citrus under different pH One hundred grams of each sample of orange and passion fruit mesocarp were selected from at least one kilogram of peeled fruit. 800 mL of de-ionized water (1:8 m/v) was added and the mixture shredded in a food processor to obtain small fragments. The pH was set at 2.2 using a 10% solution of citric acid and the mixture was heated to boiling for 30 min, cooled and strained using polyester fabric. This filtrate was used as the mother fraction A. 10% HCl was added to one third of fraction A to set the pH at 1.0. It was then boiled for 30 min and cooled, and the pH neutralized to pH 7.0, to form fraction A. 10% NaOH was added to another one third of fraction A to bring the pH up to 12.0. This was maintained for 60 min at 20 C and the pH was neutralized to pH 7.0 to form fraction Aþ. 2.2.2. Isolating pectin directly extracted of tropical fruits The pectins were isolated by full precipitation using a quantum satis volume of 95% ethanol, and the portions obtained after precipitation yielding fractions A, A and Aþ, which were filtered and washed first in 99% ethanol, and then in 99% acetone to remove sugars and pigments respectively. The final precipitate was kept at room temperature for 24 h and then lyophilized for the preparation of KBr pastille. 2.2.3. Experiment design of suitability tool extraction of pectins The experiment was carried out using factorial 23 planning with the following independent variables: pH, extraction time (minutes) and nature of alcohol, which produced 8 assays, each repeated twice. The conditions found are shown in Table 1 (coded and noncoded levels). The response variables were yield (%) and degree of methoxylation (%). 2.2.4. Procedure for optimizing the extraction of pectins from tropical fruits 800 mL of de-ionized water was added to 100 g of material (citric fruit mesocarp) to the ratio of 1:8 m/v or 400 mL, when the whole fruit was used. The pH of the liquor was adjusted to 2.2 or alternatively to pH 3.0. The mixture was then heated to boiling for 15 or alternatively 30 min, then cooled and filtered using polyester fabric. The pectin (from fraction A) was isolated by the addition of alcohol: 95% ethanol or methanol quantum satis. The final precipitate was kept at room temperature for 24 h and then lyophilized for the preparation of KBr pastille. 2.2.5. Measuring the degree of methoxylation and yield of pectins Standard pectins, lyophilized fractions of pectins and KBr were desiccated in a vacuum. The samples were homogenized with KBr Table 1 Coded levels of independent variables. Assay
pH
R-OH
t (min)
1 2 3 4 5 6 7 8
1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1
Legend: 1 and þ1 correspond to 150 and 300 for the time variable; ethanol and methanol for alcohol type and 2.2 and 3.0 for extraction pH respectively.
M.S. Lima et al. / Food Hydrocolloids 24 (2010) 1–7 Table 2 Effect of different initial pH on natural pectin yield from citrus fruits. Yield (%)
0,30
Orange A A Aþ
2.2 8.81 0.03Ab 13.42 0.83Aa 6.83 0.18Ac
Fraction A
0,35
Passion fruit 3.0 2.57 0.04Bab 2.79 0.07BCa 2.10 0.32Bb
2.2 2.47 0.04Cab 3.44 0.62Ba 1.76 0.11BCb
3.0 1.19 0.01 Da 1.34 0.23Ca 1.15 0.24Ca
Column means with different lower-case letters differ statistically by 5% Row means with different upper-case letters differ statistically by 5%.
0,25
Absorbance
Fractions
3
1750 0,20
1650
0,15 0,10
(9:1 salt/sample) and a compressor (Beckman 00-25) used to produce a pellet, which was sent for analysis using an infrared spectrometer (Bruker IFS66). The FT-IR spectrum was collected for absorbance in the 400–4000 cm1 band, with a resolution of 4 cm1. After analysis of each spectrum using FT-IR through identification of the main 1650 and 1750 cm1 bands, the DM was obtained using the equation: [A1750 cm1/(A1650 cm1 þ A1750 cm1)] in accordance with Manrique and Lajolo (2002). The yield pectin (%) was determined after step of lyophilization by weighing residue. All analyses were performed in duplicate.
0,05 4000
3500
3000
2500
2000
1500
Absorbance
1650
0,5
1750 0,4
0,3
2.2.7. Measuring the aperture of the pores of the polyester fabric and the pectin fragments isolated A sample of a single layer of fabric as recommended for filtering was analyzed by sweeping electronic microscopy (Shimadzu) (Voltage: 20 kV; Spot size: 10). The aperture of the pores and the size of the lyophilized pectin fractions were compared using Image J Morphometry software.
0,2 4000
3500
3000
2500
2000
The pectins extracted under different pH conditions showed significant differences in yield profile (Table 2) and degree of methoxylation (Table 3). As confirmed by the spectra presented in Fig. 1, the variation in the degree of methoxylation was associated with the pH. Conditions of extreme acidity (pH 1.0) therefore caused deesterification and de-polymerization of the pectin chains (Pinheiro
Table 3 Effect of different pH on degree of methoxylation of pectin yield from citrus fruits. Fractions
A A Aþ
Degree of methoxylation (%) Orange
Passion fruit
69.50 1.02a 52.15 0.00b 14.02 0.10c
69.22 0.28a 48.64 2.82b 20.44 0.90c
Columns means with different lower-case letters differ statistically by 5%.
1000
500
Fraction A 0,50
+
1650
0,45 0,40 0,35
Absorbance
3.1. Characterization of pectin fractions extracted under different pH
1500
Wave number cm-1
2.2.8. Statistical analyses Variance analysis was then carried out, comparisons being made using the Duncan 5% significance test on the Statistic 6.1 program (Statsoft, 1997). 3. Results and discussion
500
Fraction A-
0,7
0,6
2.2.6. Linear regression The degree of methoxylation of the standard pectins was measured using titrimetric (in quintuplicate) as proposed by McCready (1970). The curve was plotted with the help of the Origin5.0 program, and the results correlated using titrimetric versus infrared spectroscopy. For a regression curve plotted with six points R2 ¼ 93.35; a ¼ 0.19663 and b ¼ 0.00508.
1000
Wave number cm-1
0,30
1750
0,25 0,20 0,15 0,10 0,05 4000
3500
3000
2500
2000
1500
1000
500
Wave number cm-1 Fig. 1. Infrared spectrum of pectins extracted from orange mesocarp at different pH.
et al., 2008). The same occurred at high alkali conditions (pH 12.0), which resulted in saponification of methyl ester groupings mainly these groupings are easily dissolved in this medium. In addition, the reduction of the polygalacturonic chain by b-elimination produced different sized pectins. Both treatments led to a reduction in the degree of methoxylation to levels below 50%, particularly from of the mesocarp of citric fruits, as shown in Table 3.
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M.S. Lima et al. / Food Hydrocolloids 24 (2010) 1–7
Fig. 2. Analysis of polyester fabric used for isolating main fractions.
The size of fragments isolated from orange mesocarp, such as fraction A (pH 2.2; 4478.8 mm), fraction A (pH 1.0; 2842.2 mm) and fraction Aþ (pH 12.0; 2523.1 mm) differed significantly when A was compared with A and Aþ fractions. The reduced size of the lyophilized pectin fragments may reduce the firmness of the gel from the isolation stage onwards. Firmness was also associated with the quantity of pectin present and primarily the degree of methoxylation (a minimum of 50%). Some reports suggest that it is possible to obtain firm gels at levels as low as 0.1% (Kerr & Wicker, 2000). However, the effect of initial pH on extraction of pectins with variable degrees of methoxylation and probable size molecular can be obtained using different analytical tool in accordance with the degree of firmness of the gel (Kerr & Wicker, 2000) and other factors (Winning et al., 2007). Therefore the extraction time also appears to influence the size of particles. Yapo et al. (2007) have confirmed that when extracting pectins from beetroot at pH 1.5, 90 C, varying the duration from 1 to 4 h, caused a marked reduction in the size molecular when the pectin beet was analyzed using molecular exclusion chromatography. The separation of these molecules takes place according to their size. The larger molecules move more easily (eluted with 9 mL), whilst the smaller ones are retained (eluted with 12 mL) when appropriate columns was used. 3.2. Characterization of the polyester fabric used to filter and isolate the pectin The average aperture in size of the pores in the polyester fabric analyzed was 1072.35 mm (Fig. 2), this being the adequate aperture for isolation of the principal fractions such A, A and Aþ. The nature of the fabric or mesh recommended for retaining the fractions, which is especially neglected in the literature, is derived primarily from McCready’s (1970) original methodology, which recommends the use of cheesecloth. The polyester fabric used in order to improve the isolation stage showed itself to be effective in retaining pectin fractions, given that,
when retained, the pectins are at their maximum levels of coalescence, allowing only molecules with a size of less than 2523.1 mm to pass through the fabric. Other researchers have used centrifugation (2700 g) to isolate pectin (Kalapathy & Proctor, 2001) or ultra-filtration with selected 3–11 mm membranes (Yapo et al., 2007) in substitution to polyester fabric. 3.3. Optimization assay for the extraction of pectins from fruit The degree of methoxylation of pectins extracted from orange mesocarp increased linearly with the nature of alcohol and extraction time, while it decreased with increasing pH, as shown in Table 4 and Fig. 3. The yield for the extraction of pectin from orange mesocarp increased linearly with extraction time and decreased with increasing pH (Table 4 and Fig. 4). The highest yield was obtained when the treatment used a longer extraction time and a lower initial pH, although yield did not seem to be influenced by the nature of alcohol. Only increasing pH decreased the degree of methoxylation of pectin obtained from guava, the extraction carried out at pH 2.2 rendering a higher degree of methoxylation, as illustrated in Fig. 5 and Table 4. Table 4 shows that the yield for extraction of pectin from guava displayed the same behavior observed in the case of orange mesocarp in terms of pH and extraction time: the longer the extraction time and the lower the pH, the higher the yield (Fig. 6). The yield for guava was moderately affected by the nature of alcohol, suggesting that ethanol is the most appropriate alcohol for
Table 4 Principal effects of the tropical fruit pectin extraction optimization study. Factors
pH (1) Nature of alcohol (2) Extraction time (3) 1&2 1&3 2&3 1,2 & 3 NS: not significant.
Orange
Guava
Yield (%)
DM (%)
Yield (%)
DM (%)
6.59 NS 6.94 NS 1.79 1.09 NS
3.88 3.61 2.46 NS NS NS NS
0.30 0.07 0.29 0.06 0.17 NS NS
5.37 NS NS NS NS 3.96 4.02 Fig. 3. Degree of methoxylation (%) of orange mesocarp pectin.
M.S. Lima et al. / Food Hydrocolloids 24 (2010) 1–7
5
Fig. 4. Yield (%) obtained in the extraction of orange mesocarp pectin.
Fig. 6. Yield (%) obtained in the extraction of guava pectin.
the precipitation of pectin, corroborating the claim made by McCready in 1970. Recently used by Kalapathy and Proctor (2001), isopropanol may prove to be an interesting alternative for precipitates of smaller diameter, even for modified pectins. In correlation studies, Levigne et al. (2002) reported that the degree of methoxylation tends to rise in proportion to the initial pH of the extraction, when comparing the effect for pH 1.0 and 3.0. Yapo et al. (2007) observed a similar trend on analyzing pectin extracted at pH 1.5 and 2.0, using 96% ethanol as the precipitation agent. In these studies, the strongly acidic pH (1.0 and 1.5) caused de-esterification of the polygalacturonic chain, which also reflects the reduction simultaneous in the degree of methoxylation, as observed in these results and presented in Table 3 and Fig. 1. In addition to the pH, an increase in the concentration of acid from 0.05 N to 0.2 N of HCl reduces the yield from 28% to 21% in the extraction of pectins from soybean pods, when precipitated using isopropanol (Kalapathy & Proctor, 2001). McCready’s (1970) proposal suggests adjusting the pH using citric acid, thereby minimizing the impact of the acid on the pectin and producing reliable results for the original representation of the chemical composition model found in vegetable matter, especially fruit. However, it should be noted that extreme conditions during extraction (pH below 2.2) and extraction time (longer than 30 min) may accelerate the degradation of this heteropolysaccharide (Cho & Hwang, 2000).
There is no evidence of a direct correlation between extraction time and degree of methoxylation, as extraction may, after more than 30 minutes (and at a temperature of over 100 C), lead to the formation of Maillard’s reaction products, such as aldosamines and furfural. These indirectly confirm that de-esterification and depolymerization of the pectin has occurred (Garna, Mabon, Nott, Wathelet, & Paquot, 2006). According to some researchers (Micard & Thibault, 1999; Mesbahi et al., 2005; Yapo et al., 2007), pH and extraction time are parameters that have greater impact on the yield resulting suitable conditions for extraction of pectins, and that temperature may have a slight effect if analyzed separately. Both time and temperature should be taken into consideration as high temperatures for long periods of extraction may result in full hydrolysis of the pectin chain (Mesbahi et al., 2005).
3.4. Evaluation of the degree of methoxylation of fruit pectins as gel-forming capacity DM and pectin yield are important factors in determining the firmness of the gel and, subsequently, the value and possible use of different kinds of fruits as raw material in the food industry. Extraction that aims to obtain a higher yield and better characterization of pectins in terms of DM is a useful tool in selecting Table 5 Characterization of different tropical fruits grouped by differences in chemical composition and gelling properties. pH Group I (produce good jellies) Guava 3.85 Strawberry 3.78 Grape 3.91 Apple 4.0 Jaboticaba 3.0
Fig. 5. Degree of methoxylation (%) of guava pectin.
0.06b 0.13b 0.77b 0.03a 0.28c
Yield (%)
DM (%)
1.74 1.08 0.03 1.72 0.89
71.55 71.65 81.30 75.32 71.00
1.77c 0.07c 0.28a 0.11b 0.0c
0.28c 0.07c 0.56d 0.49a 0.28b
0.08a 0.0b 0.01d 0.03a 0.09c
Group II (contain mainly fiber) Brazil cherry 2.98 0.17c West Indian cherry 4.5 0.42b Cashew 4.6 0.42b Mango 4.55 0.49b Naseberry 5.70 0.00a
0.05 0.00b 0.045 0.01bc 0.024 0.01 cd 1.19 0.01a 0.02 0.01d
53.80 54.05 47.40 68.65 59.70
Group III (contain mainly starch) Soursop 3.9 0.00b Sugar apple 5.47 0.39a Banana 3.4 0.00b
2.15 0.07a 0.41 0.00c 1.05 0.04b
49.40 0.14c 64.25 0.92a 59.80 0.28b
Equal row letters do not differ statistically by 5%.
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M.S. Lima et al. / Food Hydrocolloids 24 (2010) 1–7
different kinds of fruits for technological purposes, mainly for the production of single-fruit and blended jams and jellies. The basic ingredients necessary for gel formation to occur may be present in the fruit itself. However, a deficiency in any one of the parameters that affect the quality of the pectin may also be corrected in the formulas prior to establish onset gel formation. These formulas generally suggest a limit of 65% sugar (sucrose), acidity of around pH 3.0, and high-grade methoxyl pectins (>50%), although it is not known what the ideal concentration of pectin in, traditional formulas suggesting a minimum of 1%. Whilst more recent data obtained using different standard pectins claim that the required consistency for a firm gel can be observed at levels as low as 0.1% (Kerr & Wicker, 2000). Generally speaking, pectin gels are formed when regions of the molecule form cross-links, through a three-dimensional network, where water molecules and co-solutes are retained. Especially in the case of high-grade methoxyl pectins, gel formation occurs when the junction zones are formed by the cross-links of galacturonic acid by hydrogen bridges and hydrophobic forces between the methoxyl groups present, as caused by the high concentration of sugar and low pH (Willats et al., 2006). In lowmethoxyl pectins, gel formation involves simultaneous linkages between calcium ions from free carboxyl groups, according to the egg-box model (Capel et al., 2006; Cardoso, Coimbra, & Lopes da Silva, 2003; Grosso, Bobbio, & Airoldi, 2000; Lo¨fgren & Hermansson, 2007). In fruits evaluated in this experiment and treated as group I (Table 5), the pectins isolated showed high levels of methoxylation (>70.0%); a high percentage yield, with the exception of grape, which showed lower values, although this was compensated for by the high degree of methoxylation (>80.0%); and a pH near to the ideal for gel formation. On analysis of the three parameters presented in Table 5 (pH, pectin yield and degree of methoxylation), it can be seen that in the case of groups II and III there was a deficiency in one of the values established for gel formation, thereby requiring further correction of soluble solids and acidity. Concerning the fruits in group II, despite the degree of methoxylation being high (>50%), with the exception of cashew fruit, which proved to be unsatisfactory in all three parameters, the yield was lower than the minimum established in the literature. The pH of the fruits was high (>3.0) with the exception of pitanga (Brazil cherry). In this group, adjustments need to be made, both to the pH and also the addition of higher-grade methoxyl pectins, or alternatively, blends should be produced as a way of achieving complementary and synergic effects. Of the group III fruits, graviola (soursop) showed a degree of methoxylation below 50%, although the yield and the pH satisfactory. Pinha (sugar apple) displayed a very high pH, although its yield and degree of methoxylation approached adequate levels. Banana, in contrast, behaved like a group I fruit, despite the expected high levels of starch. 4. Conclusions The main results obtained under the experimental conditions reported above allow us to conclude that the yield and DM of pectins are modified by an initial extraction pH that causes simultaneous de-methoxylation and defragmentation, thereby allowing different pectins to be produced for different industrial applications. The size of lyophilized pectin fragments tends to decrease proportionally with the degree of methoxylation in both alkaline and acid media. The polyester fabric analyzed proved to be effective in isolating pectins and is a more easily accessible technique than those usually employed. The procedure for extracting fruit pectin
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