EXTRACTION AND CHARACTERISATION OF PECTIN FROM SWEET ...

Journal of Engineering Science and Technology Special Issue on SOMCHE 2014 & RSCE 2014 Conference, January (2015) 22 - 29 © School of Engineering, Taylor’s University

EXTRACTION AND CHARACTERISATION OF PECTIN FROM SWEET POTATO (IPOMOEA BATATAS) PULP DAYANG NORULFAIRUZ ABANG ZAIDEL1,*, NURUL NADIA ZAINUDIN1, YANTI MASLINA MOHD JUSOH1, IDA IDAYU MUHAMAD2 1

Faculty of Chemical Engineering, Universiti Teknologi Malaysia,81310 UTM Johor Bahru, Johor, Malaysia 2 IJN-UTM Cardiovascular Engineering Centre, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia *Corresponding Author: [email protected]

Abstract Sweet potato (Ipomoea batatas) pulp contains pectin, which acts as thickening and gelling agents in food application. This study investigates the yield and profile of the galacturonic acid (GalA) and rheological properties of the sweet potato pectin produced using acid extraction method. In this work, pectins were extracted using hydrochloric acid at different concentrations (0.05 M, 0.1 M, 0.15 M, 0.2 M, and 0.25 M) at pH 1.5 and temperature 90 °C for 1 hour. Hydrolysis of residual starch in the cell wall of sweet potato using heat stable αamylase and amyloglucosidase was employed prior to pectin extraction to eliminate starch contamination. Pectins were characterised for yield, GalA profile, degree of esterification (DE) and its rheological properties. Profile of GalA and DE were determined by using Fourier Transfer Infrared Spectroscopy (FTIR). Rheological properties of pectin were performed by addition of calcium ions (Ca2+) to investigate the viscosity changes due to gelation of pectin. The yield of pectin obtained was in the range of 5.0 – 6.0% (w/w) and the DE was affected by the acid concentrations used during extraction. For its rheological properties, viscosity of pectin increases as Ca2+ was added into the pectin solution indicating the gelation of pectin by the cross-linking formation between calcium ion and non-esterified GalA in pectic polysaccharides. Keywords: Degree of esterification, Pectin, Polysaccharides, Rheology, Sweet potato.

1. Introduction Sweet potatoes are mainly used to produce starch and starchy foods, which generates a considerable quantity of residues. The majorities of these residues are 22

Extraction and Characterisation of Pectin from Sweet Potato . . . . 23

directly thrown out and consequently pollute the environment. The peels are mostly waste materials resulting from the sweet potato processing industry and are normally discarded. These discarded peels may cause an environmental problem, particularly water pollution [1]. Thus, in addition to being fed to animals, the peels can be used in the production of pectin, which would then increase the potential return for the sweet potato processing industry. Sweet potato contains pectin, which has many important functions in plants. Commercially, pectin has wide applications in both the pharmaceutical and food industries, where it acts as thickening and gelling agents, regulates the thickness and mouth-feel of fruit drink powder when the powder is dissolved in cold water, and prevents the formation of cheesy milk layer in gelled milk dessert [1]. It is reported that sweet potato residues, a good resource of pectin production, contain about 15% pectin on dry matter basis [2]. Therefore, extractions of pectin from the residues not only lower the pollution but also reduce the waste of resources. In addition, pectin has proven to have beneficial effects on human health [3-5]. Extraction of pectins can lead to large variations in the chemical structure of the final material. The ease of pectin extraction ispartly affected by the degree of esterification [6]. Pectins are industrially extracted from citrus peels and apple pomace by hot acidified water. Extraction conditions of pH 1.5 to 3.0 and temperatures of 60 to 100 °C for 0.5 to 6 hours were varied to give a material that has the desired gelling capacity and degree of methylation [7]. This study was conducted to investigate the extraction parameters for sweet potato pectin such as concentration of solvent by using acid extraction method to yield highest production of pectin. The highest pectin yields were obtained by hot acid extraction, as reported previously [7, 8]. In this study, chemical properties of sweet potato pectin, such as galacturonic acid content, degree of esterification, molecular weight and rheological properties were investigated.

2. Materials and Methods 2.1. Materials Sweet potato (Red Sweet Potato) was obtained from local supermarket in Taman Universiti, Skudai. The cell wall materials had been prepared from sweet potato pulp as described previously [9]. Commercial citrus pectin was purchased from Sigma (USA) in order to compare its physicochemical properties with sweet potato. Heat-stable α-amylase (Termamyl 120 type LS) and amyloglucosidase (EC 3.2.1.3 from Aspergillusniger, 30-60 units per mg protein) for enzymatic hydrolysis and all reagents were obtained from Sigma (St. Louis, MO).

2.2. Sample preparation Sweet potatoes (5 kg) were washed with tap water and peeled. Pulp were then sliced into small pieces and macerated in a Waring blender (Scientific Industries, New York). The slurry was filtered through double layers of cheesecloth to separate the residue from the starch milk. The residue was rinsed thoroughly with running water until the water ran clear, and was then dried in a forced-air dryer (Whisper 500, Philip, Germany) at 45 °C for 18 hours. The resultant dried starch

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residue (500 g) was ground and passed through a 60-mesh (250 µm) sieve before used for enzymatic hydrolysis. Ground dried sweet potato starch residue (10 g) was suspended in distilled water (200 mL) and boiled for 5 minutes. The suspension was kept at 80 °C, and 0.5 mL of heat-stable α-amylase (Termamyl 120 type LS from Sigma Chemical Co, St. Louis, USA) was added, and then incubated for 30 minutes to hydrolyse the residual starch. The mixture was centrifuged at 3000 rpm for 10 minutes, supernatant was discarded and digestion of the residue was repeated with 0.5 mL amyloglucosidase (EC 3.2.1.3 from Aspergillusniger, 30-60 units per mg protein, Sigma Chemical Co, St. Louis, USA ), then incubated at 55 °C for 30 minutes at pH 4.5. The mixture was filtered using two layers of cheesecloth. The residue was washed with distilled water, methanol and acetone, successively, and air-dried for 48 hours.

2.3. Extraction of pectin Pectin was extracted according to previous method with slight modification [10]. Hydrochloric (HCl) was used as extracting solvents in this experiment. Samples (10 g) of dried ground cell wall materials were dispersed in 250 mL 0.05 M HCl. The dispersion was stirred and kept at 90 °C for 1 hour. After incubation, the suspensions were centrifuged at 10 °C for 15 minutes at 10000 rpm (Kubota Model, Germany). The liquid fraction containing extracted pectin materials was neutralised with 32% NaOH, then the same volume of 95% ethanol was added and the mixture was stirred for 5 minutes and then stored at 4 °C for 12 hours. The mixture was then centrifuged at 10000 rpm for 15 minutes and the pectin residue washed successively with 70, 80, 90% ethanol [11]. Finally the extracted pectin was dried in a freeze dryer (model HETO FD 4.0, United Kingdom) for 18 hours, ground and stored in a desiccator until further analysis. The extracted pectin was analysed for moisture content, pectin yield, detection of starch in pectin, galacturonic acid, degree of esterification and the molecular weight. The procedure as described above was repeated by adjusting the concentration of the acid to 0.1 M, 0.15 M, 0.2 M, and 0.25 M HCl. The experiment was duplicated for each concentration and 10 samples were extracted.

2.4. Analysis of pectin 2.4.1. Yield of pectin Pectin yield was calculated as the ratio of the weight of dried pectin obtained to dried cell wall materials [12].

2.4.2. Determination of the degree of esterification The degree of esterification (DE) of pectin was determined by Fourier transform infrared (FTIR) spectrometry (Perkin Elmer, USA) and enzymatically using pectate lyase [13]. For the enzymatic determination of DE, pectin samples were solubilised (2 mg/mL) in 50 mM Tris–HCl buffer, pH 8, and the pectin solution were mixed with 790 µL of 50 mM Tris–HCl buffer, 1 mM CaCl2, pH 8, and 10 Journal of Engineering Science and Technology



µL of enzyme (0.01 U in 50 mM Tris–HCl buffer, 1 mM CaCl2, pH 8). The reaction and blanks were conducted at 40 °C for 30 minutes and monitored at 235 nm. The amount of product was calculated using 235 = 4600 M/cm. The degree of esterification was calculated from the calibration curve of the pectin standards. All measurements were performed in triplicate.

2.4.3. Rheological properties of pectin Brookfield digital viscometer, model DV-II + Pro, with an attached UL adapter was used to describe the rheological characteristics of the blend solutions in this work. The viscosity was determined in 20 mL of the sample and the shearing time was 15 seconds. For the storage time measurements, solutions were kept at room temperature in glass bottles in a dark place until analysis. Each measurement was recorded as an average value of five readings when a constant shear rate was applied.

3. Results and Discussion 3.1. Yield of pectin The yield of pectin extracted from this study was shown in Table 1. The result shows that the yield of pectin decreased as the acid concentrations increased. This indicates that the conditions of extraction significantly affected pectin yields. It is expected that the highest acid concentration will provide the most efficient procedure. This indicates that pectins in sweet potato cell walls are mainly calciumbound low methoxyl pectin. From previous research, the yields of pectin extracted from cell wall materials of sweet potato using various conditions were between 7.2% and 29.3% of dry cell wall materials [14]. These pectins are not extractable with mild acid or alkaline [15]. A high yield of pectin obtained from extractions at pH 1 of acid extraction was reported previously [16]. It has been reported that, despite the presence of starch, lowering pH increased the level of galacturonic acid detected, with pH 1 resulting in the highest galacturonic acid content of pectin extracted [14]. This was attributed to the lower pH, leading to greater loosening of the cell wall matrix so that pectin could be extracted more easily. Table 1. Yield of pectin (%) as extracted by different acid concentrations. Acid Concentration 0.05 M HCl 0.10 M HCl 0.15M HCl 0.20 M HCl 0.25 M HCl

Pectin Yield (%) 74.43 60.73 46.12 20.38 18.05

3.2. Determination of the degree of esterification Although degree of esterification depends on the original sources and treatment, pectin extracted under acidic conditions usually contains around 60% methyl ester groups [16]. Lowering the pH to 1 also increased the degree of esterification of the pectin from no peak to 38.8% in the Beauregard variety sweet potato. This



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may be due to starch degradation at pH 1, leading to a proportional increase in galacturonic acid or pectin released from the residue [16]. Figure 1 shows the infrared spectra of the sweet potato pectin from extraction of various acid concentrations. Among the absorption bands common to all spectra at about 3410 cm-1 due to O-H stretching vibration, while absorption at 1640 cm-1 was due to C=O stretching vibration of salt-form carboxyl group. The intensity of the absorption at 1740 cm-1 declined with the decrease in the degree of esterification. The degree of esterification was calculated from absorbance intensities for 1630 and 1745 cm-1 band. The result of FTIR spectroscopy in this study proved that the sweet potato pectin has a very low degree of methyl-esterification. The carbohydrates show high absorbance between 1200 and 950 cm-1, which constitutes the ‘fingerprint’ region, specific for each polysaccharide. Structural features arising from particular conformations around the glycosidic bond of the pectin were observed in the 990-1100 cm-1 range.

Fig. 1. Infrared spectra of the sweet potato pectin from extraction at various acid concentrations.

3.3. Rheological properties of pectin The rheological characteristics of pectin solutions and Ca2+ pectin gels partially depend on the concentration regime of the pectin solutions [17]. In all cases, viscosity was almost independent of shear rate similar to the result reported for apple pectin [18]. Low methoxyl pectin needs Ca2+ to form gel meanwhile high methoxyl pectin can form gel itself by presence of sugar and water. The gelation properties of pectin have been explained by the formation of junction zones called “egg boxes” between two pectin chains in the presence of divalent ions such as calcium. Gelation of pectin confines sugar, water and others Journal of Engineering Science and Technology



in a 3-dimensional network formed through junction zones. The gel formation and gel properties of pectin are markedly affected by the DE, molecular weight of pectin, pH, temperature, and concentration of calcium. Therefore in this study, the effect of extrinsic parameter, such as concentration of calcium, on the breaking pressure of the pectin gel was evaluated. Since pectin extracted by using 0.05 M HCl gave the highest percentage yield, it has been used to check on pectin viscosity.

Breaking pressure of gel (N/m2)

Figure 2 shows the changes in breaking pressure with increasing calcium: pectin ratio. Calcium concentration is an important factor affecting the breaking pressure of the gel. As the calcium concentration increased from 10 to 25 mg/gpectin, the breaking pressure increased linearly. The maximum breaking pressure was obtained at 25 mg/g-pectin and the value was 6.45×104 N/m2. As the concentration exceeded 25 mg/g-pectin, the gels were short and brittle, and the breaking pressure value dropped sharply. In LM-pectin, junction zones of suitable length are believed to be required for producing stable gels. There is also a tendency to decrease the amount of liquid that can held in the gel network as the junction zones become longer in the presence of excess calcium. Therefore, calcium concentration is considered to be very important in gel formation. In this experiment, the breaking pressure of the gel dropped sharply as the calcium content exceeded 25 mg/g-pectin due to the reason described above. 7000 6000 5000 4000 3000 2000 1000 0 0

20 40 Concentration of Ca2+ (mg/mg-pectin)

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Fig. 2. Effect of Calcium (Ca2+) on breaking pressure of pectin.

4. Conclusions In this work, an investigation to find new parameter for pectin extraction from sweet potato pulp with highest percentage yield was carried out. Yield, degree of esterification and rheological properties such as their stiffness and stress relaxation was determined. Pectin could be prepared from sweet potato pulp by hydrochloric acid extraction method with yield of 74.43% from 0.05 M HCl. The maximum breaking pressure was obtained at 25 mg/g-pectin and the value was 6.45×104 N/m2.



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Acknowledgment The authors would like to acknowledge Universiti Teknologi Malaysia and Ministry of Higher Education Malaysia for the financial support under Fundamental Research Grant Scheme (R.J130000.7844.4F447) and Research University Grant (Q.J130000.2544.06H39).

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