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Extraction of pectin from passion fruit peel (Passiflora edulis f. flavicarpa) by microwave-induced heating
Food Hydrocolloids 38 (2014) 186e192
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Extraction of pectin from passion fruit peel (Passiflora edulis f. flavicarpa) by microwave-induced heating Fernanda L. Seixas a, *, Deise L. Fukuda a, Franciele R.B. Turbiani a, Patrícia S. Garcia a, Carmen L. de O. Petkowicz b, Sheeja Jagadevan c, Marcelino L. Gimenes a a b c
Departamento de Engenharia Química, Universidade Estadual de Maringá, Av. Colombo, 5790, Bloco D90, 87020-900 Maringá, PR, Brazil Departamento de Engenharia Química, Universidade Federal do Paraná, R. XV de Novembro, 1229, 80060-000 Curitiba, PR, Brazil Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 November 2012 Accepted 2 December 2013
Pectin is a heteropolysaccharide obtained from primary cell walls of terrestrial plants, which is a very important raw material for food and pharmaceutical products. Extraction of pectin from the peels of yellow passion fruit (Passiflora edulis f. flavicarpa) under microwave-induced heating was investigated in the present study. Three types of acids (tartaric, acetic and nitric acid) were employed as extracting agents. The effect of extraction time and microwave-power on yield of pectin has been studied using the response surface methodology. The results indicate that exposure time and microwave-power significantly affects the yield of pectin extraction with both nitric and tartaric acids. However, the extractions using acetic acid were significantly affected only by the exposure time. For all scenarios, the highest yields were obtained when the highest levels of power and time were used (628 W and 9 min). Under these conditions, the yield of pectin obtained with nitric and acetic acids were 13 and 12.9% respectively. Tartaric acid emerged as the best extracting agent in terms of yield (18.2%), however, the obtained pectin exhibited low purity and low degree of esterification. Pectin extracted from passion fruit by employing acetic and nitric acid presented better properties: high molar mass (4.625 105 for acetic acid and 4.966 105 for nitric acid), degree of esterification (64.56% for acetic acid and 64.15% for nitric acid) and content of uronic acids (62.5% for acetic acid and 82.3% for nitric acid). Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Microwave Passion fruit peel Pectin extraction Degree of esterification
1. Introduction Passion fruit (Passiflora edulis f. flavicarpa) being a good source of vitamin C is commonly employed in juice processing. The annual production of this juice in Brazil alone is estimated to be more than 920,158 tons in 2010 (IBGE, 2013). Such a wide-scale use of this fruit, inevitably leads to the generation of vast quantities of the fruit peels, which constitutes about half of the fruit mass. These fruit peels are discarded as a major waste, causing a substantial burden on the environment. It is therefore imperative to find adequate disposal of these peels or means to convert the peels into useful products (Liu, Shi, & Langrish, 2006; Pinheiro et al., 2008). Recent studies have shown that the pericarp (integral part of the peel) of passion fruit, even though processed and stored, could be used as raw material for obtaining co-products in food industry, such as dietary fiber and other bioactive compounds (Yapo, 2009a; Yapo & Koffi, 2006).
* Corresponding author. Tel.: þ55 44 32624860. E-mail address: [email protected] (F.L. Seixas). 0268-005X/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodhyd.2013.12.001
Pectin is a polysaccharide found ubiquitously in cell walls of all plants (Carpita & McCann, 2000). This polysaccharide is mainly composed of two moieties (i) homogalacturonan made up of (1-4) linked a-D-galacturonic acid and (ii) rhamnogalacturonan I consisting of (1-2) repeating linked, a-L-rhamnose-(1-4) and a-D-galacturonic acid disaccharide. Rhamnogalacturonan I usually contains neutral side chains of arabinan, galactan or arabinogalactan. The ratio of esterified carboxylic acid units to total carboxylic acid units in pectin is termed the degree of esterification (DE), which has a major influence on gel properties of pectin. Depending on DE, pectin is commercially divided into two major groups: highester pectin, with DE higher than 50%, and low-ester pectin, with DE lower than 50% (Thakur, Singh, & Handa, 1997). Pectin represents a high-value functional food ingredient widely used as gelling agent and stabilizer, particularly in jams and jellies (Willats, Knox, & Mikkelsen, 2006). Pectin extraction is a multiplestage physicochemical process which involves hydrolysis and extraction of pectin macromolecules from plant tissue, purification of the liquid extract and isolation of the extracted pectin from the liquid. These processes are influenced by various factors, mainly temperature, pH, and time (Pagán, Ibarz, Llorca, Pagán, & Barbosa-
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Cánovas, 2001). Usually the traditional technique requires longer extraction time thus running the severe risk of thermal degradation for the thermolabile materials. Conventional techniques for the extraction of constituents are time and solvent consuming and thermally unsafe. Keeping in pace with such requirements recent times has witnessed the use and growth of new extraction techniques with shortened extraction time, reduced solvent consumption, increased pollution prevention concern and with special care for thermolabile constituents (Luque de Castro & Garcia-Ayuso, 1998; Mandal, Mohan, & Hemalatha, 2007). Although it has been shown that the pericarp of passion fruit can be used as raw material for pectin isolation (Kliemann et al., 2009; Pinheiro et al., 2008; Yapo, 2009a, 2009b; Yapo & Koffi, 2006), the extraction under microwave heating was not investigated. The use of microwave for extraction of constituents from plant material has shown tremendous research interest and potential. There are two types of microwave systems currently used: closed extraction vessels and focused microwave ovens. The latter, used in this work, is also named as solvent extraction, in which only a part of the extraction vessel containing the sample is irradiated with microwave, this system operates in atmospheric pressure (openvessel). The use of atmospheric pressure substantial provides advantages over pressurized vessels such as: open-vessel operation is more suitable with thermolabile species as it uses low temperatures relative to closed vessel systems; the low cost of the equipment required; the absence of any requirement for cooling down or depressurization; the ability to add reagents at any time during the treatment, among others (Mandal et al., 2007). In addition, conventionally, pectin is extracted from citrus peels and apple pomace by employing strong mineral acids (May, 1990; Rolin, 1993). The use of such strong acids in the extraction step leads to corrosion of equipment and has deleterious effects on the environment. The need for replacement of these extracting agents with milder weak organic acids could be a potential means to minimize aforementioned adverse effects. As the yield and composition of pectins depends on the conditions used during pectin isolation and purification (Levigne, Ralet, & Thibault, 2002; Rolin, 1993), in this work pectins from yellow passion fruit peels were extracted using different acids under microwave heating. The main aim of this study was to employ two weak organic acids, such as tartaric and acetic, and the nitric acid for extraction of pectin from peels of yellow passion fruit under microwave heating. The factors influencing the yield of pectin such as intensity of electromagnetic field and exposure time were also examined. The extracted pectin should be of high DE, purity and molecular weight and the yield should be as high as possible. 2. Materials and methods Samples of yellow passion fruit peel (P. edulis f. flavicarpa) were obtained from the pulps producing industry located in Paraná, Brazil. These fruit peel represents a waste generated during the fruit processing operations. These peels were composed of an intact pericarp. The commercial citrus pectin, employed for comparative analysis was procured from Vetec P.A., Brazil. All chemical reagents employed in this study were of analytical grade.
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at 50 C until a constant weight was obtained. The dried peels were then milled to 60 mesh size powdered passion fruit peel and the resulting product, referred to as “passion fruit peel flour (PFF)”. The passion fruit peel flour was used as raw material for pectin extraction. The passion fruit peel flour was packaged in a polyethylene bag and stored in a freezer (15 2 C) until required. 2.2. Pectin extraction Pectin was extracted using a modified version of the method presented elsewhere by Canteri-Schemin, Fertonani, Waszczynskyj, and Wosiacki (2005). The extraction process was carried out in 600 mL beakers, followed by heating in a microwave oven (CCE model M e 304). 4 g of passion fruit peel flour was added to 100 mL of distilled water in a beaker. This was followed by addition of 100 mL of an acidic solution, with concentration adjusted so as to maintain a final pH of 2 for the solutions. The beaker was partially covered with a glass lid and then subjected to microwave heating for predetermined time intervals (3, 6 and 9 min). As the extraction system remained partially open, gradual evaporation of the solvent was observed. In order to maintain a constant suspension concentration, the amount of evaporated water was replenished to the system at regular intervals. The suspension, still warm, was vacuum filtered in synthetic fabrics; the retentate was discarded and the filtrate (containing the soluble pectin) was cooled to 4 C. To isolate the soluble pectins from the filtrate, the extracted liquid was slowly added under magnetic stirring to two volumes of absolute ethyl alcohol, both maintained at 4 C. This mixture was stirred for 10 min, after which it was allowed to rest for 30 min to facilitate the flotation of the pectin. The pectin thus obtained, was separated by vacuum filtration on a filter paper. The extracted pectin in a gel form was immersed in absolute ethyl alcohol for about 12 h and then was partially dehydrated by immersion in acetone for a few minutes. This was followed by drying pectin in an air-circulated oven at 40 C until constant weight was obtained (approximately five hours). The resulting material was milled to dry powdered pectin. The extraction process of pectin is schematized in Fig. 1. 2.3. Experimental design Response surface methodology was used to determine the optimum condition for pectin extraction from passion fruit peel flour. In order to evaluate the effect that the variables extraction time (tEx) and microwave power (P) present on the yield of pectin extracted (response) a complete factorial experimental design 22 was elaborated with the insertion of a central point. Experiments in the centre of the design were performed in order to make the estimation of pure error possible. The variable extraction time was evaluated at the levels 3, 6 and 9 min and the microwave power was evaluated at levels 356, 450 and 627.9 W. The pH of the solution was maintained constant (2.0) for the three kinds of acids studied. The complete design consisted of 4 experiments for each type of acid examined and three replicates in center point (Table 1). All the tests were conducted in duplicate at random. The t-Student test was performed with a significance level of 5%. For data analysis was used the software Statistica 10.
2.1. Preparation of passion fruit peel flour (PFF) 2.4. Degree of esterification The fruit peels were thoroughly washed in running water and subjected to a blanching treatment, which results in enzymatic inactivation. For this, the peels were immersed in water at a temperature of 97 C for 3 min, then, the peels were transferred to another water bath at room temperature and maintained for 15 min. Subsequently, the peels were dried in an air-circulated oven
The degree of esterification (DE) of pectin samples were determined by potentiometric titration, as described by Bochek, Zabivalova, and Petropavlovskii (2001). The percentage of gravimetric yield of pectin was calculated from the ratio of the mass of dried pectin extracted and the mass of flour used as raw material.
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solution, with respect to changes in solute concentration) was determined using five concentrations in the 1e0.2 mg ml1 range using a Waters 2410 differential refractometer. The average molar mass (Mw) and polydispersity index were determined from HPSECMALLS data using Wyatt Technology ASTRA software.
Passion fruit peel flour (PFF) Distilled water Suspension Acid solution (100 mL)
(4 g PFF + 100 mL distilled water, Ratio solid: liquid of 1:25 (g/mL))
(acetic, tartaric or nitric acid)
pH = 2 Microwave heating (Controlled variables: time and power extraction)
Insoluble residue
Filtration Pectin solubilized Flotation Pectin in alcohol (Ratio pectin solution:alcohol 1:2 mL/mL)
Rinsing with ethanol
Filtration Pectin gel Drying at 40 °C
Dry pectin Fig. 1. Flow diagram for the extraction of pectin.
The data were statistically analyzed by applying the Tukey test with a significance level of 5% using the software Statistica 10.
The extracted pectin samples were analyzed by high performance size exclusion chromatography (HPSEC) coupled to a refractive index (RI) and a Wyatt Technology Dawn-F multi-angle laser light scattering (MALLS) detector. The RI detector provides data proportional to the concentration of pectin, whereas the MALLS furnishes data on the molar mass. Four ultrahydrogel columns e 120, 250, 500 and 2000 were used with exclusion limits of 5 103, 8 104, 4 105 and 7 106, respectively. A 0.1 M NaNO2 solution containing 200 ppm NaN3 was employed as an eluent. The samples were dissolved at a concentration of 1.5 w/v in the eluent. Before analysis all samples were filtered through cellulose acetate membranes with pore size of 0.22 mm. The analyses were carried out at 25 C. For the average molar mass (Mw) calculation, the dn/dc value (the refractive index increment of the solventesolute
Table 1 Yield of pectins extracted passion fruit peels under microwave heating using nitric acid, acetic acid and tartaric acid. Variable (coded level)
a
Power (W)
Time (min)
Nitric acid
Acetic acid
Tartaric acid
356 356 628 628 450 450 450
3 9 3 9 6 6 6
9.1 9.9 9.2 13.0 10.3 10.9 11.0
9.4 12.7 11.4 12.9 12.0 11.8 12.4
15.3 18.2 16.0 30.3 17.0 16.8 16.4
a
(1) (1) (1) (1) (0) (0) (0)
Yield (%)
Based on passion fruit peel flour.
2.6.1. Acidic monosaccharides content The determination of uronic acid was performed by the method described by Blumenkrantz and Asboe-Hansen (1973), with galacturonic acid as a standard solution, taken at concentrations of 10e100 mg/mL and the absorbance read at 520 nm. Analyses were performed in quadruplicates. 2.6.2. Neutral monosaccharides The samples were hydrolyzed with 2 M trifluoroacetic acid for 5 h at 100 C (Biermann, 1989). The monosaccharides were reduced with sodium borohydride for 16 h at 4 C (Wolfrom & Thompson, 1963b). Subsequently, samples were treated with strong acid cation resin and methanol as previously described (Vriesmann, Teófilo, & Petkowicz, 2011). The alditol formed were acetylated with pyridineeacetic anhydride (1:1, v/v, 16 h, at 25 C) (Wolfrom & Thompson, 1963a). The alditol acetates formed were extracted with chloroform and submitted to successive treatments with copper sulphate and 5% distilled water for elimination of pyridine. The alditol acetates were analyzed by gaseliquid chromatography (GLC) using a (Trace GC Ultra, Thermo Electron Corporation) equipped with a DB-225 capillary column (0.25 mm 30 m). The temperatures of injector and flame ionization detector (FID) were maintained at 250 C and 300 C, respectively. The oven temperature was programmed from 100 C to 215 C at a heating rate of 40 C/ min. Helium was used as carrier gas at a flow rate of 1.0 mL/min. 2.7. Morphology
2.5. High performance size exclusion chromatography
(1) (1) (1) (1) (0) (0) (0)
2.6. Monosaccharide composition
In order to visualize the morphology of the samples of passion fruit peel flour, scanning electron microscope (Shimadzu SS - 550, Superscan, software Superscan SS-550) image were taken before and after the extraction process. The samples were adhered to a support with the use of a double-sided conductive carbon tape and then metallized with gold to ensure electrical conductivity to the observed surface. 3. Results and discussion 3.1. Extraction The experimental yields of pectin obtained for extraction using three different acids (nitric, acetic and tartaric) are presented in Table 1. Was adjusted a factorial model which analyzed the influences of factors tEx, P and the interaction tEx P. Multiple regression coefficients are shown in Table 2. The results showed that for extractions carried out with nitric acid and tartaric, both variables (tEx and P) as well as their interactions (tEx P) was significant by t-Student test at a significance level of 0.05. For the case of extractions performed with acetic acid results suggest that only the extraction time significantly affects the extraction yield. In this case, the power of the microwave is not very important. Thus, the extraction process using acetic acid may be conducted at the lowest level of radiation (356 W) with no damage as the yields obtained. Figs. 2e4 show the response surfaces coded levels obtained from the fitted model for the three acids analyzed. The quality of the model can be observed by the high R2 values obtained
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Table 2 Coded regression coefficients, standard error and p values for the model built. Factor
Interc. P (W) tEx (min) P tEx Adj. R2
Acetic acid
Nitric acid
Coeff
Std. Err.
p
11.80* 1.10 2.40* 0.90 0.93
0.163 0.432 0.432 0.432
9.0 8.4 1.2 1.3
E-6 E-2 E-2 E-1
Tartaric acid
Coeff
Std. Err.
p
10.49* 1.60* 2.30* 1.50* 0.94
0.170 0.450 0.450 0.450
9.0 3.8 1.5 4.5
E-6 E-2 E-2 E-2
Coeff
Std. Err.
p
17.78* 9.15* 5.85* 8.45* 0.96
0.535 1.414 1.414 1.414
6.0 7.5 2.6 9.4
E-5 E-3 E-2 E-3
*Significative at significance level of 0.05.
(see Table 2). Beyond what we can assume that normality, independence and randomness of the residuals were satisfied. From these results it is possible to observe that for all acids analyzed the highest yields were obtained when the highest field strength (628 W) and exposure time (9 min) were used (Figs. 2e4). Tartaric acid was the best extracting agent (Table 1), with yields between 15.32 and 30.29%. For acetic acid, yields remained between 9.43 and 12.91% and for nitric acid between 9.5 and 13.1%. The results showed that not only concentration, but also acid type influenced the extracted pectin yields (Yapo, 2009a, 2009b) and that the use of tartaric acid in place of strong mineral acids is an efficient alternative as the former performed better in terms of extraction yield. These results are in accordance to study conducted by Yapo (2009a, 2009b), wherein, 3e14% yields were obtained for extractions carried out by using citric, nitric and sulfuric acids at different concentrations (10 mM and 30 mM) and pH (1.8 and 2.5). Similar pectin yields (8e13.1%) were obtained by Fishman, Chau, Hoagland, and Hotchkiss (2006), while studying the effect of microwaveassisted extraction under pressure on lime flavedo, albedo and pulp. The extractions were carried out at 630 W of power in a solution of HCl (pH 2.0) with heating times ranged from 1 to 10 min. However, although the tartaric acid has afforded the highest yields, pectin extracted with this agent had some undesirable qualities, such as: low molar mass and lower uronic acid content. On the other hand, pectin extracted with acetic acid showed a high molar mass and high uronic acid content. Therefore, as they are
both mild acids further studies are needed to explain what are the mechanisms of action, during the extraction process, of such acids and thus to understand the differences in the properties of pectin obtained. Preliminary extraction studies performed on a condensing thermostatic bath (conventional process), conducted at pH 2.0, 90 C and 40 min, resulted in 19.40%, 10.20% and 12.94% yields for extractions carried out with tartaric acid, acetic acid and nitric acid respectively (results not shown). Comparing these results with those from Table 1, an increased yield of pectin was observed for extractions conducted under microwave heating, especially while employing organic acid as an extraction medium. This effect may be due to the changes occurring in plant tissues when subjected to microwaves. The electromagnetic field energy is converted to heat, especially in polar substances, causing formation of intense vapors inside the capillary porous structure of the plant material, thereby leading to modification in their physical properties (Kratchanova, Pavlova, & Panchev, 2004). In the present study, an increase in temperature from microwaves may have caused rupture of the parenchymal cells of the plant material. The damage to the plant tissue increased with increasing intensity of the microwave field, which is explained by an increase in intracellular spaces. The images of scanning electron microscopy for the passion fruit peel flour, before (a) and after (b) undergoing the extraction process can be seen in Fig. 5. It can be clearly seen that extraction under microwave heating promotes an increase in capillary porosity of the passion fruit peel, so the cell will split and the pectin and other inclusion can release itself.
Fig. 2. Response surface showing the effect of extraction time (coded values) and power (coded values) on yield of pectin for extractions carried out with acetic acid.
Fig. 3. Response surface showing the effect of extraction time (coded values) and power (coded values) on yield of pectin for extractions carried out with nitric acid.
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F.L. Seixas et al. / Food Hydrocolloids 38 (2014) 186e192 Table 3 Monosaccharide composition and DE of pectins extracted from passion fruit peels using acetic acid, tartaric acid and nitric acid compared with the commercial citrus pectin. b
Monosaccharide (%)
Acetic acid
Tartaric acid
Nitric acid
Commercial
Rhamnose Fucose Arabinose Xylose Mannose Galactose Glucose Uronic acid
4.0 0.5 9.5 2.5 1.8 6.8 12.4 62.5
5.9 0.6 8.1 2.5 1.7 9.2 13.5 58.5
2.6 0.3 4.1 1.6 0.7 3.6 4.8 82.3
3.1 0.1 6.8 0.5 0.9 12.7 0.0 75.8
DEa
64.56 (1.90)
50.00 (1.48)
64.15 (1.65)
70.0 (0.55)
a
Mean (standard deviation) (3 repetitions). Neutral monosaccharides were determined by GLC and uronic acid content was obtained by colorimetric method. b
Fig. 4. Response surface showing the effect of extraction time (coded values) and power (coded values) on yield of pectin for extractions carried out with tartaric acid.
Zhongdong, Guohua, Yunchang, and Kennedy (2006) extracted pectin from orange skin and also observed that microwave radiation has the stronger destructive effect on the organization structure of orange skin, and the swelling effect of the microwave event forces of the cells to split. Thus, if one compares the extraction process under microwave heating with the conventional extraction process it is observed that in addition higher yields it is possible to get significant energy savings, since the conventional process lasts for about an hour, while microwave process has duration of only a few minutes. 3.2. Characterization of pectin Pectin extracted under the conditions that generated the highest yield (9 min and 628 W), for the three types of acid, were characterized and compared with commercially available citrus pectin (Vetec) for its degree of esterification, chemical composition and molar mass distribution. 3.2.1. Degree of esterification Table 3 enlists the values of the degree of esterification (DE) of pectins isolated from passion fruit peels which were in the range of 50.00e64.56%. The DE values were influenced by the kind of acid used in the extraction. The highest DE were observed for pectins
obtained with acetic acid (64.56%) and nitric acid (64.15%) while the extraction with tartaric acid afforded pectins with the lowest DE (50.00). The DE values for pectins extracted with acetic acid and nitric acid were close to that found for commercially available citrus pectin (70%). The pectins obtained in the present work had a medium to high degree of esterification (above 50%) and hence can be considered to be of great economic interest, as per the conventional classifications (Thakur et al., 1997). It is of interest to note that unlike conventional methods of extraction, microwave extraction, produces high DE pectin (Fishman et al., 2006). Pinheiro et al. (2008) extracted pectins from passion fruit peel using citric acid at different concentrations and extraction times. They obtained pectins with DE ranging from 27.52% to 78.59%. Using a surface response methodology these authors found the satisfactory condition for extraction of high DE pectin with citric acid (0.086% w/v citric acid for 60 min). The extraction with acetic acid for 9 min under microwave heating resulted in a pectic fraction with DE 64.56. Yapo (2009b) used pure lemon juice and citric acid to extract pectins from passion fruit peel at 80 C for 90 min and obtained polysaccharides with DE 52e73%. In the present work, high DE pectins with DE w65% were isolated from passion fruit peel using lower times of extraction (9 min), showing the advantage of this method of extraction when compared with the traditional ones. 3.2.2. Monosaccharide composition The monosaccharide composition of pectins extracted from passion fruit peels with different acids, compared with the commercial citrus pectin is shown in Table 3. Since the commercial
Fig. 5. Scanning electron microscopy for the passion fruit peel flour before (a) and after (b) extraction under microwave heating (magnification 200).
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pectin is standardized using glucose, the glucose content of the sample (w26%) was not considered in the monomeric composition of the polymer. The pectins contain uronic acid as the main component (58.5%e82.3%). Among the neutral monosaccharides, glucose, galactose, arabinose and rhamnose were found in higher amounts. Galactose, arabinose and rhamnose arise from rhamnogalacturonan region. The commercial sample showed a greater level of galactose (12.7%) compared to samples obtained experimentally (3.6%e9.2%). Pectins extracted with nitric acid and acetic acid which had the highest DE also displayed the highest uronic acid contents, 82.3% and 62.5%, respectively. These levels of uronic acid are higher than those found by Yapo (2009b) for pectins extracted from passion fruit peel using pure lemon juice and citric acid which ranged from 63.8% to 78.3%. However, lower uronic acid content (58.5%) was observed for pectins extracted with tartaric acid. As established by FAO and EU, a minimum of 65% of galacturonic acid is required to be considered as pectin (Willats et al., 2006). Thus, the polysaccharides extracted with nitric and acetic acid from passion fruit peel and commercial citrus pectin (Vetec) can be classified as pectin. Value below the required standard was found for pectin extracted from passion fruit peel with tartaric acid. The ratio of uronic acid to rhamnose, an indicator of the proportion of homogalacturonan and rhamnogalacturonan I in the polymer, remained between 20 and 32 during extractions with acetic and nitric acid, respectively. The results suggest that these pectin samples consist mainly of linear regions of galacturonic acid. These results are in accordance to the data recently published for pectins extracted from yellow passion fruit peel where galacturonic acid to rhamnose ratios ranged from 14 to 35 (Yapo, 2009a). However, for pectin extracted with tartaric acid, the uronic acid to rhamnose ratio was 10. 3.2.3. Molar mass The pectins were analyzed by high-performance size-exclusion chromatography (HPSEC) equipped with multi-angle laser light scattering (MALLS) and refractive index (RI) detectors. The results of dn/dc, average molar mass (Mw) and polydispersity index (Mw/ MN) calculated from HPSEC-MALLS are given in Table 4. The highest Mw values were found for pectins extracted with nitric acid and acetic acid, 4.966 105 g/mol and 4.625 105, respectively. The pectin extracted with tartaric acid had the lowest Mw, 2.298 105 g/mol, which was lower than that calculated for the commercial sample (3.073 105 g/mol). These results are similar to those found by Fishman et al. (2006) for pectin extracted from lime under 3 min of microwave heating (3.1e5.15 105 g/mol). The polydispersity index (Mw/MN) of the polymers ranged from 2.492 for pectin extracted with acetic acid to 1.250 for pectin extracted with nitric acid. The highest Mw/MN for pectin extracted with acetic acid indicating a broader range of polymer size distribution in this sample. The differential molar mass distribution for pectins obtained with different acids under microwave heating is shown in Fig. 6. It is possible to observe that the samples shown a uniform distribution of sizes. The curves show how much material (differential Table 4 Results from HPSEC-MALLS for pectins extracted from passion fruit peels under microwave heating using nitric acid, acetic acid and tartaric acid, compared with the commercial citrus pectin. Pectin sample
dn/dc
Mw (g/mol)
Acetic acid Nitric acid Tartaric acid Commercial
0.127 0.124 0.128 0.142
4.625 4.966 2.298 3.073
105 105 105 105
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Fig. 6. Curves of differential weight fraction as a function of molecular weight of pectins extracted from passion fruit peels under microwave heating using nitric acid, acetic acid and tartaric acid, compared with the commercial citrus pectin.
weight fraction) is contained in any molar mass interval. It is possible to observe that the pectins obtained by extracting with nitric acid present a sharp peak, with the narrowest distribution, in accordance with its lower Mw/MN. By the other side, pectin extracted with acetic acid showed the wider distribution of molecules, between 2 104 g/mol and 2 106 g/mol. Comparing the organic acids used in this work, it is noteworthy that the curve for the pectin extracted with tartaric acid was shifted to lower molar mass values. This is due to the fact that tartaric acid has two terminal carboxyl groups, thus, its higher hydrolytical power was greater than that of acetic acid which has only one terminal carboxylic. Therefore, despite the use of tartaric acid provide higher yields, your higher hydrolytical power degrades the pectin extracted. 4. Conclusions The greatest extraction yields in pectin were obtained when highest field strength and exposure time was employed. The type of acid used also affected the yield of extraction and the composition and molar mass of pectin obtained. The hydrolytic power of tartaric acid makes it more active for extraction and also more active for degrading pectin than the other acids investigated. By using the response surface analysis, we can conclude that extraction time significantly affects the yield of pectin obtained using all acids. The microwave power significantly affects the yield only in the case of extractions carried out with nitric and tartaric acids. The pectin extracted from the peels of passion fruit exhibited medium to high degree of esterification (50.00%e64.56%). Pectins obtained by the use of acetic acid and nitric had a yield of approximately 13.0% and showed the highest DE, molar mass and uronic acid contents. Highest yields were obtained the use of tartaric acid (30%), however, the extracted pectin had some undesirable qualities, such as: low molar mass and lower uronic acid content (58.5%) and DE (50%). Acknowledgment The authors gratefully acknowledge the Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico (CNPq) of Brazil for the financial support.
Mw/MN 2.492 1.250 1.638 1.414
0.026 0.010 0.021 0.016
References Biermann, C. J. (1989). Hydrolysis and the other cleavage of glycosidic linkages. In C. J. Biermann, & G. D. McGinnis (Eds.), Analysis of carbohydrates by GLC and MS (pp. 27e41). Florida: CRC Press.
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Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Analytical Biochemistry, 54, 484e489. Bochek, A. M., Zabivalova, N. M., & Petropavlovskii, G. A. (2001). Determination of the esterification degree of polygalacturonic acid. Russian Journal of Applied Chemistry, 75, 796e799. Canteri-Schemin, M. H., Fertonani, H. C. R., Waszczynskyj, N., & Wosiacki, G. (2005). Extraction of pectin from Apple pomace. Brazilian Archives of Biology and Technology, 48, 259e266. Carpita, N., & McCann, M. C. (2000). The cell wall. In B. Buchanan (Ed.), Biochemistry and the molecular biology of plants (pp. 52e108). Fishman, M. L., Chau, H. K., Hoagland, P. D., & Hotchkiss, A. T. (2006). Microwaveassisted extraction of lime pectin. Food Hydrocolloids, 20, 1170e1177. IBGE, Instituto Brasileiro de Geografia e estatística. Available in. Accessed 01.05.13. Kliemann, E., Simas, K. N., Amante, E. R., Prudêncio, E. S., Teófilo, R. F., Ferreira, M. M. C., et al. (2009). Optimisation of pectin acid extraction from passion fruit peel (Passiflora edulis flavicarpa) using response surface methodology. International Journal of Food Science & Technology, 44, 476e483. Kratchanova, M., Pavlova, E., & Panchev, I. (2004). The effect of microwave heating of fresh orange peels on the fruit tissue and quality of extracted pectin. Carbohydrate Polymers, 56, 181e185. Levigne, S., Ralet, M.-C., & Thibault, J.-F. (2002). Characterization of pectins extracted from fresh sugar beet under different conditions using an experimental design. Carbohydrate Polymers, 49, 145e153. 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, 203e209. Luque de Castro, M. D., & Garcia-Ayuso, L. E. (1998). Soxhlet extraction of solid matrices: an outdated technique with a promising innovative future. Analytica Chimica Acta, 369, 1e10. Mandal, V., Mohan, Y., & Hemalatha, S. (2007). Microwave assisted extraction e an innovative and promising extraction tool for medicinal plant research. Pharmacognosy Reviews, 1, 7e18.
May, C. D. (1990). Industrial pectins: sources, production and applications. Carbohydrate Polymers, 12, 79e99. Pagán, J., Ibarz, A., Llorca, M., Pagán, A., & Barbosa-Cánovas, G. V. (2001). Extraction and characterization of pectin from stored peach pomace. Food Research International, 34, 605e612. Pinheiro, E. R., Silva, I. M. D. A., Gonzaga, L. V., Amante, E. R., Teófilo, 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, 5561e5566. Rolin, C., & Pectin. (1993). In R. L. Whistler, & J. N. BeMiller (Eds.), Industrial gums (3rd ed.) (pp. 145e180). Academic Press: San Diego. Thakur, B. R., Singh, R. K., & Handa, A. K. (1997). Chemistry and uses of pectin e a reviews. Critical Reviews in Food Science, 37, 47e73. Vriesmann, L. C., Teófilo, R. F., & Petkowicz, C. L. D. O. (2011). Optimization of nitric acid-mediated extraction of pectin from cacao pod husks (Theobroma cacao L.) using response surface methodology. Carbohydrate Polymers, 84, 1230e1236. Willats, W. G. T., Knox, J. P., & Mikkelsen, J. D. (2006). Pectin: new insights into an old polymer are starting to gel. Food Science Technology, 17, 97e104. Wolfrom, M. L., & Thompson, A. (1963a). Acetylation. Methods in Carbohydrate Chemistry, 2, 211e215. Wolfrom, M. L., & Thompson, A. (1963b). Reduction with sodium borohydride. Methods in Carbohydrate Chemistry, 2, 65e68. Yapo, B. M. (2009a). Biochemical characteristics and gelling capacity of pectin from yellow passion fruit rind as affected by acid extractant nature. Journal of Agricultural and Food Chemistry, 57, 1572e1578. Yapo, B. M. (2009b). Lemon juice improves the extractability and quality characteristics of pectin from yellow passion fruit by-product as compared with commercial citric acid extractant. Bioresource Technology, 100, 3147e3151. Yapo, B. M., & Koffi, K. L. (2006). Yellow passion fruit rind e a potential source of low-methoxyl pectin. Journal of Agricultural and Food Chemistry, 54, 2738e2744. Zhongdong, L., Guohua, W., Yunchang, G., & Kennedy, J. F. (2006). Image study of pectin extraction from orange skin assisted by microwave. Carbohydrate Polymers, 64, 548e552.
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Extraction of pectin from passion fruit peel (Passiflora edulis f. flavicarpa) by microwave-induced heating Fernanda L. Seixas a, *, Deise L. Fukuda a, Franciele R.B. Turbiani a, Patrícia S. Garcia a, Carmen L. de O. Petkowicz b, Sheeja Jagadevan c, Marcelino L. Gimenes a a b c
Departamento de Engenharia Química, Universidade Estadual de Maringá, Av. Colombo, 5790, Bloco D90, 87020-900 Maringá, PR, Brazil Departamento de Engenharia Química, Universidade Federal do Paraná, R. XV de Novembro, 1229, 80060-000 Curitiba, PR, Brazil Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 November 2012 Accepted 2 December 2013
Pectin is a heteropolysaccharide obtained from primary cell walls of terrestrial plants, which is a very important raw material for food and pharmaceutical products. Extraction of pectin from the peels of yellow passion fruit (Passiflora edulis f. flavicarpa) under microwave-induced heating was investigated in the present study. Three types of acids (tartaric, acetic and nitric acid) were employed as extracting agents. The effect of extraction time and microwave-power on yield of pectin has been studied using the response surface methodology. The results indicate that exposure time and microwave-power significantly affects the yield of pectin extraction with both nitric and tartaric acids. However, the extractions using acetic acid were significantly affected only by the exposure time. For all scenarios, the highest yields were obtained when the highest levels of power and time were used (628 W and 9 min). Under these conditions, the yield of pectin obtained with nitric and acetic acids were 13 and 12.9% respectively. Tartaric acid emerged as the best extracting agent in terms of yield (18.2%), however, the obtained pectin exhibited low purity and low degree of esterification. Pectin extracted from passion fruit by employing acetic and nitric acid presented better properties: high molar mass (4.625 105 for acetic acid and 4.966 105 for nitric acid), degree of esterification (64.56% for acetic acid and 64.15% for nitric acid) and content of uronic acids (62.5% for acetic acid and 82.3% for nitric acid). Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Microwave Passion fruit peel Pectin extraction Degree of esterification
1. Introduction Passion fruit (Passiflora edulis f. flavicarpa) being a good source of vitamin C is commonly employed in juice processing. The annual production of this juice in Brazil alone is estimated to be more than 920,158 tons in 2010 (IBGE, 2013). Such a wide-scale use of this fruit, inevitably leads to the generation of vast quantities of the fruit peels, which constitutes about half of the fruit mass. These fruit peels are discarded as a major waste, causing a substantial burden on the environment. It is therefore imperative to find adequate disposal of these peels or means to convert the peels into useful products (Liu, Shi, & Langrish, 2006; Pinheiro et al., 2008). Recent studies have shown that the pericarp (integral part of the peel) of passion fruit, even though processed and stored, could be used as raw material for obtaining co-products in food industry, such as dietary fiber and other bioactive compounds (Yapo, 2009a; Yapo & Koffi, 2006).
* Corresponding author. Tel.: þ55 44 32624860. E-mail address: [email protected] (F.L. Seixas). 0268-005X/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodhyd.2013.12.001
Pectin is a polysaccharide found ubiquitously in cell walls of all plants (Carpita & McCann, 2000). This polysaccharide is mainly composed of two moieties (i) homogalacturonan made up of (1-4) linked a-D-galacturonic acid and (ii) rhamnogalacturonan I consisting of (1-2) repeating linked, a-L-rhamnose-(1-4) and a-D-galacturonic acid disaccharide. Rhamnogalacturonan I usually contains neutral side chains of arabinan, galactan or arabinogalactan. The ratio of esterified carboxylic acid units to total carboxylic acid units in pectin is termed the degree of esterification (DE), which has a major influence on gel properties of pectin. Depending on DE, pectin is commercially divided into two major groups: highester pectin, with DE higher than 50%, and low-ester pectin, with DE lower than 50% (Thakur, Singh, & Handa, 1997). Pectin represents a high-value functional food ingredient widely used as gelling agent and stabilizer, particularly in jams and jellies (Willats, Knox, & Mikkelsen, 2006). Pectin extraction is a multiplestage physicochemical process which involves hydrolysis and extraction of pectin macromolecules from plant tissue, purification of the liquid extract and isolation of the extracted pectin from the liquid. These processes are influenced by various factors, mainly temperature, pH, and time (Pagán, Ibarz, Llorca, Pagán, & Barbosa-
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Cánovas, 2001). Usually the traditional technique requires longer extraction time thus running the severe risk of thermal degradation for the thermolabile materials. Conventional techniques for the extraction of constituents are time and solvent consuming and thermally unsafe. Keeping in pace with such requirements recent times has witnessed the use and growth of new extraction techniques with shortened extraction time, reduced solvent consumption, increased pollution prevention concern and with special care for thermolabile constituents (Luque de Castro & Garcia-Ayuso, 1998; Mandal, Mohan, & Hemalatha, 2007). Although it has been shown that the pericarp of passion fruit can be used as raw material for pectin isolation (Kliemann et al., 2009; Pinheiro et al., 2008; Yapo, 2009a, 2009b; Yapo & Koffi, 2006), the extraction under microwave heating was not investigated. The use of microwave for extraction of constituents from plant material has shown tremendous research interest and potential. There are two types of microwave systems currently used: closed extraction vessels and focused microwave ovens. The latter, used in this work, is also named as solvent extraction, in which only a part of the extraction vessel containing the sample is irradiated with microwave, this system operates in atmospheric pressure (openvessel). The use of atmospheric pressure substantial provides advantages over pressurized vessels such as: open-vessel operation is more suitable with thermolabile species as it uses low temperatures relative to closed vessel systems; the low cost of the equipment required; the absence of any requirement for cooling down or depressurization; the ability to add reagents at any time during the treatment, among others (Mandal et al., 2007). In addition, conventionally, pectin is extracted from citrus peels and apple pomace by employing strong mineral acids (May, 1990; Rolin, 1993). The use of such strong acids in the extraction step leads to corrosion of equipment and has deleterious effects on the environment. The need for replacement of these extracting agents with milder weak organic acids could be a potential means to minimize aforementioned adverse effects. As the yield and composition of pectins depends on the conditions used during pectin isolation and purification (Levigne, Ralet, & Thibault, 2002; Rolin, 1993), in this work pectins from yellow passion fruit peels were extracted using different acids under microwave heating. The main aim of this study was to employ two weak organic acids, such as tartaric and acetic, and the nitric acid for extraction of pectin from peels of yellow passion fruit under microwave heating. The factors influencing the yield of pectin such as intensity of electromagnetic field and exposure time were also examined. The extracted pectin should be of high DE, purity and molecular weight and the yield should be as high as possible. 2. Materials and methods Samples of yellow passion fruit peel (P. edulis f. flavicarpa) were obtained from the pulps producing industry located in Paraná, Brazil. These fruit peel represents a waste generated during the fruit processing operations. These peels were composed of an intact pericarp. The commercial citrus pectin, employed for comparative analysis was procured from Vetec P.A., Brazil. All chemical reagents employed in this study were of analytical grade.
187
at 50 C until a constant weight was obtained. The dried peels were then milled to 60 mesh size powdered passion fruit peel and the resulting product, referred to as “passion fruit peel flour (PFF)”. The passion fruit peel flour was used as raw material for pectin extraction. The passion fruit peel flour was packaged in a polyethylene bag and stored in a freezer (15 2 C) until required. 2.2. Pectin extraction Pectin was extracted using a modified version of the method presented elsewhere by Canteri-Schemin, Fertonani, Waszczynskyj, and Wosiacki (2005). The extraction process was carried out in 600 mL beakers, followed by heating in a microwave oven (CCE model M e 304). 4 g of passion fruit peel flour was added to 100 mL of distilled water in a beaker. This was followed by addition of 100 mL of an acidic solution, with concentration adjusted so as to maintain a final pH of 2 for the solutions. The beaker was partially covered with a glass lid and then subjected to microwave heating for predetermined time intervals (3, 6 and 9 min). As the extraction system remained partially open, gradual evaporation of the solvent was observed. In order to maintain a constant suspension concentration, the amount of evaporated water was replenished to the system at regular intervals. The suspension, still warm, was vacuum filtered in synthetic fabrics; the retentate was discarded and the filtrate (containing the soluble pectin) was cooled to 4 C. To isolate the soluble pectins from the filtrate, the extracted liquid was slowly added under magnetic stirring to two volumes of absolute ethyl alcohol, both maintained at 4 C. This mixture was stirred for 10 min, after which it was allowed to rest for 30 min to facilitate the flotation of the pectin. The pectin thus obtained, was separated by vacuum filtration on a filter paper. The extracted pectin in a gel form was immersed in absolute ethyl alcohol for about 12 h and then was partially dehydrated by immersion in acetone for a few minutes. This was followed by drying pectin in an air-circulated oven at 40 C until constant weight was obtained (approximately five hours). The resulting material was milled to dry powdered pectin. The extraction process of pectin is schematized in Fig. 1. 2.3. Experimental design Response surface methodology was used to determine the optimum condition for pectin extraction from passion fruit peel flour. In order to evaluate the effect that the variables extraction time (tEx) and microwave power (P) present on the yield of pectin extracted (response) a complete factorial experimental design 22 was elaborated with the insertion of a central point. Experiments in the centre of the design were performed in order to make the estimation of pure error possible. The variable extraction time was evaluated at the levels 3, 6 and 9 min and the microwave power was evaluated at levels 356, 450 and 627.9 W. The pH of the solution was maintained constant (2.0) for the three kinds of acids studied. The complete design consisted of 4 experiments for each type of acid examined and three replicates in center point (Table 1). All the tests were conducted in duplicate at random. The t-Student test was performed with a significance level of 5%. For data analysis was used the software Statistica 10.
2.1. Preparation of passion fruit peel flour (PFF) 2.4. Degree of esterification The fruit peels were thoroughly washed in running water and subjected to a blanching treatment, which results in enzymatic inactivation. For this, the peels were immersed in water at a temperature of 97 C for 3 min, then, the peels were transferred to another water bath at room temperature and maintained for 15 min. Subsequently, the peels were dried in an air-circulated oven
The degree of esterification (DE) of pectin samples were determined by potentiometric titration, as described by Bochek, Zabivalova, and Petropavlovskii (2001). The percentage of gravimetric yield of pectin was calculated from the ratio of the mass of dried pectin extracted and the mass of flour used as raw material.
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solution, with respect to changes in solute concentration) was determined using five concentrations in the 1e0.2 mg ml1 range using a Waters 2410 differential refractometer. The average molar mass (Mw) and polydispersity index were determined from HPSECMALLS data using Wyatt Technology ASTRA software.
Passion fruit peel flour (PFF) Distilled water Suspension Acid solution (100 mL)
(4 g PFF + 100 mL distilled water, Ratio solid: liquid of 1:25 (g/mL))
(acetic, tartaric or nitric acid)
pH = 2 Microwave heating (Controlled variables: time and power extraction)
Insoluble residue
Filtration Pectin solubilized Flotation Pectin in alcohol (Ratio pectin solution:alcohol 1:2 mL/mL)
Rinsing with ethanol
Filtration Pectin gel Drying at 40 °C
Dry pectin Fig. 1. Flow diagram for the extraction of pectin.
The data were statistically analyzed by applying the Tukey test with a significance level of 5% using the software Statistica 10.
The extracted pectin samples were analyzed by high performance size exclusion chromatography (HPSEC) coupled to a refractive index (RI) and a Wyatt Technology Dawn-F multi-angle laser light scattering (MALLS) detector. The RI detector provides data proportional to the concentration of pectin, whereas the MALLS furnishes data on the molar mass. Four ultrahydrogel columns e 120, 250, 500 and 2000 were used with exclusion limits of 5 103, 8 104, 4 105 and 7 106, respectively. A 0.1 M NaNO2 solution containing 200 ppm NaN3 was employed as an eluent. The samples were dissolved at a concentration of 1.5 w/v in the eluent. Before analysis all samples were filtered through cellulose acetate membranes with pore size of 0.22 mm. The analyses were carried out at 25 C. For the average molar mass (Mw) calculation, the dn/dc value (the refractive index increment of the solventesolute
Table 1 Yield of pectins extracted passion fruit peels under microwave heating using nitric acid, acetic acid and tartaric acid. Variable (coded level)
a
Power (W)
Time (min)
Nitric acid
Acetic acid
Tartaric acid
356 356 628 628 450 450 450
3 9 3 9 6 6 6
9.1 9.9 9.2 13.0 10.3 10.9 11.0
9.4 12.7 11.4 12.9 12.0 11.8 12.4
15.3 18.2 16.0 30.3 17.0 16.8 16.4
a
(1) (1) (1) (1) (0) (0) (0)
Yield (%)
Based on passion fruit peel flour.
2.6.1. Acidic monosaccharides content The determination of uronic acid was performed by the method described by Blumenkrantz and Asboe-Hansen (1973), with galacturonic acid as a standard solution, taken at concentrations of 10e100 mg/mL and the absorbance read at 520 nm. Analyses were performed in quadruplicates. 2.6.2. Neutral monosaccharides The samples were hydrolyzed with 2 M trifluoroacetic acid for 5 h at 100 C (Biermann, 1989). The monosaccharides were reduced with sodium borohydride for 16 h at 4 C (Wolfrom & Thompson, 1963b). Subsequently, samples were treated with strong acid cation resin and methanol as previously described (Vriesmann, Teófilo, & Petkowicz, 2011). The alditol formed were acetylated with pyridineeacetic anhydride (1:1, v/v, 16 h, at 25 C) (Wolfrom & Thompson, 1963a). The alditol acetates formed were extracted with chloroform and submitted to successive treatments with copper sulphate and 5% distilled water for elimination of pyridine. The alditol acetates were analyzed by gaseliquid chromatography (GLC) using a (Trace GC Ultra, Thermo Electron Corporation) equipped with a DB-225 capillary column (0.25 mm 30 m). The temperatures of injector and flame ionization detector (FID) were maintained at 250 C and 300 C, respectively. The oven temperature was programmed from 100 C to 215 C at a heating rate of 40 C/ min. Helium was used as carrier gas at a flow rate of 1.0 mL/min. 2.7. Morphology
2.5. High performance size exclusion chromatography
(1) (1) (1) (1) (0) (0) (0)
2.6. Monosaccharide composition
In order to visualize the morphology of the samples of passion fruit peel flour, scanning electron microscope (Shimadzu SS - 550, Superscan, software Superscan SS-550) image were taken before and after the extraction process. The samples were adhered to a support with the use of a double-sided conductive carbon tape and then metallized with gold to ensure electrical conductivity to the observed surface. 3. Results and discussion 3.1. Extraction The experimental yields of pectin obtained for extraction using three different acids (nitric, acetic and tartaric) are presented in Table 1. Was adjusted a factorial model which analyzed the influences of factors tEx, P and the interaction tEx P. Multiple regression coefficients are shown in Table 2. The results showed that for extractions carried out with nitric acid and tartaric, both variables (tEx and P) as well as their interactions (tEx P) was significant by t-Student test at a significance level of 0.05. For the case of extractions performed with acetic acid results suggest that only the extraction time significantly affects the extraction yield. In this case, the power of the microwave is not very important. Thus, the extraction process using acetic acid may be conducted at the lowest level of radiation (356 W) with no damage as the yields obtained. Figs. 2e4 show the response surfaces coded levels obtained from the fitted model for the three acids analyzed. The quality of the model can be observed by the high R2 values obtained
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Table 2 Coded regression coefficients, standard error and p values for the model built. Factor
Interc. P (W) tEx (min) P tEx Adj. R2
Acetic acid
Nitric acid
Coeff
Std. Err.
p
11.80* 1.10 2.40* 0.90 0.93
0.163 0.432 0.432 0.432
9.0 8.4 1.2 1.3
E-6 E-2 E-2 E-1
Tartaric acid
Coeff
Std. Err.
p
10.49* 1.60* 2.30* 1.50* 0.94
0.170 0.450 0.450 0.450
9.0 3.8 1.5 4.5
E-6 E-2 E-2 E-2
Coeff
Std. Err.
p
17.78* 9.15* 5.85* 8.45* 0.96
0.535 1.414 1.414 1.414
6.0 7.5 2.6 9.4
E-5 E-3 E-2 E-3
*Significative at significance level of 0.05.
(see Table 2). Beyond what we can assume that normality, independence and randomness of the residuals were satisfied. From these results it is possible to observe that for all acids analyzed the highest yields were obtained when the highest field strength (628 W) and exposure time (9 min) were used (Figs. 2e4). Tartaric acid was the best extracting agent (Table 1), with yields between 15.32 and 30.29%. For acetic acid, yields remained between 9.43 and 12.91% and for nitric acid between 9.5 and 13.1%. The results showed that not only concentration, but also acid type influenced the extracted pectin yields (Yapo, 2009a, 2009b) and that the use of tartaric acid in place of strong mineral acids is an efficient alternative as the former performed better in terms of extraction yield. These results are in accordance to study conducted by Yapo (2009a, 2009b), wherein, 3e14% yields were obtained for extractions carried out by using citric, nitric and sulfuric acids at different concentrations (10 mM and 30 mM) and pH (1.8 and 2.5). Similar pectin yields (8e13.1%) were obtained by Fishman, Chau, Hoagland, and Hotchkiss (2006), while studying the effect of microwaveassisted extraction under pressure on lime flavedo, albedo and pulp. The extractions were carried out at 630 W of power in a solution of HCl (pH 2.0) with heating times ranged from 1 to 10 min. However, although the tartaric acid has afforded the highest yields, pectin extracted with this agent had some undesirable qualities, such as: low molar mass and lower uronic acid content. On the other hand, pectin extracted with acetic acid showed a high molar mass and high uronic acid content. Therefore, as they are
both mild acids further studies are needed to explain what are the mechanisms of action, during the extraction process, of such acids and thus to understand the differences in the properties of pectin obtained. Preliminary extraction studies performed on a condensing thermostatic bath (conventional process), conducted at pH 2.0, 90 C and 40 min, resulted in 19.40%, 10.20% and 12.94% yields for extractions carried out with tartaric acid, acetic acid and nitric acid respectively (results not shown). Comparing these results with those from Table 1, an increased yield of pectin was observed for extractions conducted under microwave heating, especially while employing organic acid as an extraction medium. This effect may be due to the changes occurring in plant tissues when subjected to microwaves. The electromagnetic field energy is converted to heat, especially in polar substances, causing formation of intense vapors inside the capillary porous structure of the plant material, thereby leading to modification in their physical properties (Kratchanova, Pavlova, & Panchev, 2004). In the present study, an increase in temperature from microwaves may have caused rupture of the parenchymal cells of the plant material. The damage to the plant tissue increased with increasing intensity of the microwave field, which is explained by an increase in intracellular spaces. The images of scanning electron microscopy for the passion fruit peel flour, before (a) and after (b) undergoing the extraction process can be seen in Fig. 5. It can be clearly seen that extraction under microwave heating promotes an increase in capillary porosity of the passion fruit peel, so the cell will split and the pectin and other inclusion can release itself.
Fig. 2. Response surface showing the effect of extraction time (coded values) and power (coded values) on yield of pectin for extractions carried out with acetic acid.
Fig. 3. Response surface showing the effect of extraction time (coded values) and power (coded values) on yield of pectin for extractions carried out with nitric acid.
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F.L. Seixas et al. / Food Hydrocolloids 38 (2014) 186e192 Table 3 Monosaccharide composition and DE of pectins extracted from passion fruit peels using acetic acid, tartaric acid and nitric acid compared with the commercial citrus pectin. b
Monosaccharide (%)
Acetic acid
Tartaric acid
Nitric acid
Commercial
Rhamnose Fucose Arabinose Xylose Mannose Galactose Glucose Uronic acid
4.0 0.5 9.5 2.5 1.8 6.8 12.4 62.5
5.9 0.6 8.1 2.5 1.7 9.2 13.5 58.5
2.6 0.3 4.1 1.6 0.7 3.6 4.8 82.3
3.1 0.1 6.8 0.5 0.9 12.7 0.0 75.8
DEa
64.56 (1.90)
50.00 (1.48)
64.15 (1.65)
70.0 (0.55)
a
Mean (standard deviation) (3 repetitions). Neutral monosaccharides were determined by GLC and uronic acid content was obtained by colorimetric method. b
Fig. 4. Response surface showing the effect of extraction time (coded values) and power (coded values) on yield of pectin for extractions carried out with tartaric acid.
Zhongdong, Guohua, Yunchang, and Kennedy (2006) extracted pectin from orange skin and also observed that microwave radiation has the stronger destructive effect on the organization structure of orange skin, and the swelling effect of the microwave event forces of the cells to split. Thus, if one compares the extraction process under microwave heating with the conventional extraction process it is observed that in addition higher yields it is possible to get significant energy savings, since the conventional process lasts for about an hour, while microwave process has duration of only a few minutes. 3.2. Characterization of pectin Pectin extracted under the conditions that generated the highest yield (9 min and 628 W), for the three types of acid, were characterized and compared with commercially available citrus pectin (Vetec) for its degree of esterification, chemical composition and molar mass distribution. 3.2.1. Degree of esterification Table 3 enlists the values of the degree of esterification (DE) of pectins isolated from passion fruit peels which were in the range of 50.00e64.56%. The DE values were influenced by the kind of acid used in the extraction. The highest DE were observed for pectins
obtained with acetic acid (64.56%) and nitric acid (64.15%) while the extraction with tartaric acid afforded pectins with the lowest DE (50.00). The DE values for pectins extracted with acetic acid and nitric acid were close to that found for commercially available citrus pectin (70%). The pectins obtained in the present work had a medium to high degree of esterification (above 50%) and hence can be considered to be of great economic interest, as per the conventional classifications (Thakur et al., 1997). It is of interest to note that unlike conventional methods of extraction, microwave extraction, produces high DE pectin (Fishman et al., 2006). Pinheiro et al. (2008) extracted pectins from passion fruit peel using citric acid at different concentrations and extraction times. They obtained pectins with DE ranging from 27.52% to 78.59%. Using a surface response methodology these authors found the satisfactory condition for extraction of high DE pectin with citric acid (0.086% w/v citric acid for 60 min). The extraction with acetic acid for 9 min under microwave heating resulted in a pectic fraction with DE 64.56. Yapo (2009b) used pure lemon juice and citric acid to extract pectins from passion fruit peel at 80 C for 90 min and obtained polysaccharides with DE 52e73%. In the present work, high DE pectins with DE w65% were isolated from passion fruit peel using lower times of extraction (9 min), showing the advantage of this method of extraction when compared with the traditional ones. 3.2.2. Monosaccharide composition The monosaccharide composition of pectins extracted from passion fruit peels with different acids, compared with the commercial citrus pectin is shown in Table 3. Since the commercial
Fig. 5. Scanning electron microscopy for the passion fruit peel flour before (a) and after (b) extraction under microwave heating (magnification 200).
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pectin is standardized using glucose, the glucose content of the sample (w26%) was not considered in the monomeric composition of the polymer. The pectins contain uronic acid as the main component (58.5%e82.3%). Among the neutral monosaccharides, glucose, galactose, arabinose and rhamnose were found in higher amounts. Galactose, arabinose and rhamnose arise from rhamnogalacturonan region. The commercial sample showed a greater level of galactose (12.7%) compared to samples obtained experimentally (3.6%e9.2%). Pectins extracted with nitric acid and acetic acid which had the highest DE also displayed the highest uronic acid contents, 82.3% and 62.5%, respectively. These levels of uronic acid are higher than those found by Yapo (2009b) for pectins extracted from passion fruit peel using pure lemon juice and citric acid which ranged from 63.8% to 78.3%. However, lower uronic acid content (58.5%) was observed for pectins extracted with tartaric acid. As established by FAO and EU, a minimum of 65% of galacturonic acid is required to be considered as pectin (Willats et al., 2006). Thus, the polysaccharides extracted with nitric and acetic acid from passion fruit peel and commercial citrus pectin (Vetec) can be classified as pectin. Value below the required standard was found for pectin extracted from passion fruit peel with tartaric acid. The ratio of uronic acid to rhamnose, an indicator of the proportion of homogalacturonan and rhamnogalacturonan I in the polymer, remained between 20 and 32 during extractions with acetic and nitric acid, respectively. The results suggest that these pectin samples consist mainly of linear regions of galacturonic acid. These results are in accordance to the data recently published for pectins extracted from yellow passion fruit peel where galacturonic acid to rhamnose ratios ranged from 14 to 35 (Yapo, 2009a). However, for pectin extracted with tartaric acid, the uronic acid to rhamnose ratio was 10. 3.2.3. Molar mass The pectins were analyzed by high-performance size-exclusion chromatography (HPSEC) equipped with multi-angle laser light scattering (MALLS) and refractive index (RI) detectors. The results of dn/dc, average molar mass (Mw) and polydispersity index (Mw/ MN) calculated from HPSEC-MALLS are given in Table 4. The highest Mw values were found for pectins extracted with nitric acid and acetic acid, 4.966 105 g/mol and 4.625 105, respectively. The pectin extracted with tartaric acid had the lowest Mw, 2.298 105 g/mol, which was lower than that calculated for the commercial sample (3.073 105 g/mol). These results are similar to those found by Fishman et al. (2006) for pectin extracted from lime under 3 min of microwave heating (3.1e5.15 105 g/mol). The polydispersity index (Mw/MN) of the polymers ranged from 2.492 for pectin extracted with acetic acid to 1.250 for pectin extracted with nitric acid. The highest Mw/MN for pectin extracted with acetic acid indicating a broader range of polymer size distribution in this sample. The differential molar mass distribution for pectins obtained with different acids under microwave heating is shown in Fig. 6. It is possible to observe that the samples shown a uniform distribution of sizes. The curves show how much material (differential Table 4 Results from HPSEC-MALLS for pectins extracted from passion fruit peels under microwave heating using nitric acid, acetic acid and tartaric acid, compared with the commercial citrus pectin. Pectin sample
dn/dc
Mw (g/mol)
Acetic acid Nitric acid Tartaric acid Commercial
0.127 0.124 0.128 0.142
4.625 4.966 2.298 3.073
105 105 105 105
191
Fig. 6. Curves of differential weight fraction as a function of molecular weight of pectins extracted from passion fruit peels under microwave heating using nitric acid, acetic acid and tartaric acid, compared with the commercial citrus pectin.
weight fraction) is contained in any molar mass interval. It is possible to observe that the pectins obtained by extracting with nitric acid present a sharp peak, with the narrowest distribution, in accordance with its lower Mw/MN. By the other side, pectin extracted with acetic acid showed the wider distribution of molecules, between 2 104 g/mol and 2 106 g/mol. Comparing the organic acids used in this work, it is noteworthy that the curve for the pectin extracted with tartaric acid was shifted to lower molar mass values. This is due to the fact that tartaric acid has two terminal carboxyl groups, thus, its higher hydrolytical power was greater than that of acetic acid which has only one terminal carboxylic. Therefore, despite the use of tartaric acid provide higher yields, your higher hydrolytical power degrades the pectin extracted. 4. Conclusions The greatest extraction yields in pectin were obtained when highest field strength and exposure time was employed. The type of acid used also affected the yield of extraction and the composition and molar mass of pectin obtained. The hydrolytic power of tartaric acid makes it more active for extraction and also more active for degrading pectin than the other acids investigated. By using the response surface analysis, we can conclude that extraction time significantly affects the yield of pectin obtained using all acids. The microwave power significantly affects the yield only in the case of extractions carried out with nitric and tartaric acids. The pectin extracted from the peels of passion fruit exhibited medium to high degree of esterification (50.00%e64.56%). Pectins obtained by the use of acetic acid and nitric had a yield of approximately 13.0% and showed the highest DE, molar mass and uronic acid contents. Highest yields were obtained the use of tartaric acid (30%), however, the extracted pectin had some undesirable qualities, such as: low molar mass and lower uronic acid content (58.5%) and DE (50%). Acknowledgment The authors gratefully acknowledge the Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico (CNPq) of Brazil for the financial support.
Mw/MN 2.492 1.250 1.638 1.414
0.026 0.010 0.021 0.016
References Biermann, C. J. (1989). Hydrolysis and the other cleavage of glycosidic linkages. In C. J. Biermann, & G. D. McGinnis (Eds.), Analysis of carbohydrates by GLC and MS (pp. 27e41). Florida: CRC Press.
192
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Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Analytical Biochemistry, 54, 484e489. Bochek, A. M., Zabivalova, N. M., & Petropavlovskii, G. A. (2001). Determination of the esterification degree of polygalacturonic acid. Russian Journal of Applied Chemistry, 75, 796e799. Canteri-Schemin, M. H., Fertonani, H. C. R., Waszczynskyj, N., & Wosiacki, G. (2005). Extraction of pectin from Apple pomace. Brazilian Archives of Biology and Technology, 48, 259e266. Carpita, N., & McCann, M. C. (2000). The cell wall. In B. Buchanan (Ed.), Biochemistry and the molecular biology of plants (pp. 52e108). Fishman, M. L., Chau, H. K., Hoagland, P. D., & Hotchkiss, A. T. (2006). Microwaveassisted extraction of lime pectin. Food Hydrocolloids, 20, 1170e1177. IBGE, Instituto Brasileiro de Geografia e estatística. Available in
May, C. D. (1990). Industrial pectins: sources, production and applications. Carbohydrate Polymers, 12, 79e99. Pagán, J., Ibarz, A., Llorca, M., Pagán, A., & Barbosa-Cánovas, G. V. (2001). Extraction and characterization of pectin from stored peach pomace. Food Research International, 34, 605e612. Pinheiro, E. R., Silva, I. M. D. A., Gonzaga, L. V., Amante, E. R., Teófilo, 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, 5561e5566. Rolin, C., & Pectin. (1993). In R. L. Whistler, & J. N. BeMiller (Eds.), Industrial gums (3rd ed.) (pp. 145e180). Academic Press: San Diego. Thakur, B. R., Singh, R. K., & Handa, A. K. (1997). Chemistry and uses of pectin e a reviews. Critical Reviews in Food Science, 37, 47e73. Vriesmann, L. C., Teófilo, R. F., & Petkowicz, C. L. D. O. (2011). Optimization of nitric acid-mediated extraction of pectin from cacao pod husks (Theobroma cacao L.) using response surface methodology. Carbohydrate Polymers, 84, 1230e1236. Willats, W. G. T., Knox, J. P., & Mikkelsen, J. D. (2006). Pectin: new insights into an old polymer are starting to gel. Food Science Technology, 17, 97e104. Wolfrom, M. L., & Thompson, A. (1963a). Acetylation. Methods in Carbohydrate Chemistry, 2, 211e215. Wolfrom, M. L., & Thompson, A. (1963b). Reduction with sodium borohydride. Methods in Carbohydrate Chemistry, 2, 65e68. Yapo, B. M. (2009a). Biochemical characteristics and gelling capacity of pectin from yellow passion fruit rind as affected by acid extractant nature. Journal of Agricultural and Food Chemistry, 57, 1572e1578. Yapo, B. M. (2009b). Lemon juice improves the extractability and quality characteristics of pectin from yellow passion fruit by-product as compared with commercial citric acid extractant. Bioresource Technology, 100, 3147e3151. Yapo, B. M., & Koffi, K. L. (2006). Yellow passion fruit rind e a potential source of low-methoxyl pectin. Journal of Agricultural and Food Chemistry, 54, 2738e2744. Zhongdong, L., Guohua, W., Yunchang, G., & Kennedy, J. F. (2006). Image study of pectin extraction from orange skin assisted by microwave. Carbohydrate Polymers, 64, 548e552.