Isolation and Characterization of Galactomannan from Sugar Palm Nut (Arenga pinnata) Endosperm

Rafael B. Navarro University of the Philippines Los Baos Los Baos, Laguna, Philippines 4024 rafael.berroya.navarro@gmail.com
ABSTRACT Galactomannan gum was extracted from sugar palm nut (Arenga pinnata) endosperm by homogenization and precipitation with ethanol. The gum extract was further purified by precipitation with Fehlings solution and the gum content of the fruit had an average of 4.7 0.2 % by mass and the gum appeared as hard translucent white solid. The isolated gum was characterized physically and chemically. It was shown that the gum is insoluble in organic solvents such as acetone, ethanol, isopropanol, and butanol but soluble in different aqueous solutions each having 5% hydrochloric acid, 5% sulfuric acid, 5% sodium hydroxide, 5% potassium hydroxide, 1 M and 0.1 M sodium chloride respectively. Benedicts test revealed the presence of reducing sugars in the gum isolates but the bluish green precipitate roughly indicated levels of reducing sugars to be below 1% by mass. The total sugar content determined using phenol-sulfuric acid assay was revealed to be 90.4 % by mass while the total reducing sugar content determined using 3,5-dinitrosalicylic acid assay was revealed to be about 11.8 %. The water-holding capacity of the gum was not successfully performed because of the problem of gelatinization of the polysaccharide with water. In summary, the purification of galactomannan from the fruit was a success in terms of high yield; however, there were contaminants of reducing sugars in the sample as detected in the spectrophotometric assays. Microbial contamination, extremely low pH, and exposure to extreme heat must be avoided to ensure the integrity of the gum sample to prevent large discrepancies in the chemical composition of the gum. Keywords: Galactomannan, Sugar Palm, Fehlings reagent, Benedicts test, Precipitation, Spectrophotometry 1. INTRODUCTION Sugar palm or kaong (Arenga pinnata) in Tagalog is a feather palm native to Southeast Asia and India. It thrives along stream banks at low to middle altitudes and it is a source of sap that contains high amounts of sugars which can be fermented to produce wine and vinegar (Helen et al 2003; Lustca and Lerio 1997; Torio et al 2006). The endosperm of the sugar palm nut is commonly made into sweets in the Philippines and Indonesia; additionally, the endosperms were found to contain galactomannans which have potential use in the food and pharmaceutical industries (Chudzikowski 1971). Galactomannans are polysaccharides which are primary components of commercially available natural gums such as guar gum, fenugreek gum, and locust bean gum. These polysaccharides consist of (14)-linked -Dmannopynarose which serve as the backbone with branchpoints at 6-positions linked to -D-galactopyranose. Galactomannans are synthesized in seeds by the enzymes, GDP-D-mannose mannosyltransferase and UDP-D-galactose galactosyltransferase working together (Grant Reid et al 1987). In addition, the ratio of the number of mannose units with respect to the number of galactose varies according to the source of the natural gum (Torio et al 2006). These polysaccharides have a wide range of applications in food and pharmaceutical industry particularly as gelling agent, thickening agent, emulsifying agent, and binding agent (Roberts 2011). Important parameters needed to consider the gum for industrial use can be determined through physical characteristics such as viscosity and water-holding capacity and chemical properties such as purity, total sugar content, average molecular weight, and mannose to galactose ratio. This study aimed to isolate galactomannans from sugar palm nut endosperm and to chemically and physically characterize the purified polysaccharide in order to survey its potential applications as an additive to neutraceuticals and pharmaceuticals. In order to isolate the polysaccharide, precipitation with ethanol and with Fehlings reagent was employed. The solubility in various solvents was tested and the water-holding capacity of the gum was determined. The total sugar content and the total reducing sugar content of the gum were also determined spectrophotometrically. 2. MATERIALS AND METHODS 2.1. Plant Material The sugar palm nut endosperms were acquired from bottled sweetened kaong available in the supermarket. The

endosperms were washed with tap water repeatedly to remove the sucrose-rich syrup. 2.2. Isolation and Purification of Galactomannans Thirty grams of sugar palm endosperms with 100 mL of distilled water was homogenized using an osterizer. The resulting suspension was centrifuged at 4C at 6000 rpm for ten minutes. The supernatant was mixed with an equal amount of 95% ethanol and the solution was continuously stirred for thirty minutes at room temperature. The white precipitates were isolated through centrifugation at 4C at 6000 rpm for fifteen minutes and then washed with 95% ethanol. The precipitate was air dried under the hood overnight and prepared for further purification procedure (Kooiman 1971). The dried gum was reconstituted with distilled water and Fehlings solution was added to the suspension. The resulting precipitate was filtered and mixed with distilled water. One milliliter of 2 M hydrochloric acid was added to the suspension and stirred to dissolve all precipitates. An equal amount of 95% ethanol was added afterwards to reprecipitate the gum. The precipitates were isolated through centrifugation at 4C for fifteen minutes at 6000 rpm and then washed again with 95% ethanol. The precipitate was again air dried under the hood overnight and weighed afterwards. 2.3. Solubility Test of the Gum Extract Approximately 0.5 grams of the purified gum extract was added to 50 mL of each organic solvent namely acetone, ethanol, isopropanol, butanol and of each aqueous solutions including 5% hydrochloric acid, 5% sulfuric acid, 5% sodium hydroxide, 5% potassium hydroxide, 1 M and 0.1 M sodium chloride. 2.4. Water-holding Capacity of the Gum Extract Precisely 0.5 g of the purified gum extract was suspended in 15 mL distilled water and stored overnight at approximately 2C. The suspension was centrifuged for 30 minutes at 4C at 10,000 rpm. To determine percent water retention, the amount of water retained in the precipitate was compared to the total amount of water in the solution which is 15 mL. 2.5. Sugar Content of the Gum Extract Benedicts test was employed to qualitatively detect the presence of reducing sugars in the gum extract solution. The total reducing sugar was quantitatively determined using DNS method. One gram of the gum extract was suspended in 100 mL distilled water. Three milliters of dinitrosalicylic acid solution was added to 1 mL o the gum solution and then heated in boiling water bath for 15 minutes. After cooling, the total reducing sugar was determined spectrophotometrically at 490 nm wavelength using galactose solution to calibrate standard curve. To determine the total sugar content of the gum extract, one gram of dried sample was reconstituted with water. An aliquot was treated with 5% phenol solution and concentrated sulfuric acid. After ten minutes of occasional shaking, the solution was cooled and the total sugar content

of the sample was determined spectrophotometrically at 550 nm wavelength using galactose solution to calibrate standard curve. 3. RESULTS AND DISCUSSION 3.1. Gum Content of Sugar Palm Nut Endosperm The purified gum was characterized as opaque white fibers as it precipitated out of the solution and when air dried, it became a hard translucent white solid substance. The mass of the crude gum isolated from the nut had an average of 2.37 0.06 grams (4.95 0.05 %) while the mass of the purified gum had an average of 2.24 0.05 grams. The gum content of the nut had an average of 4.7 0.2 %.

Figure 1. Purified gum isolates. Trials 1 (left) and 2 (right). The galactomannans were easily precipitated out of the solution with the help of ethanol because the polysaccharide is insoluble in the organic solvent. The ethanol which is strongly miscible with water, when added to the aqueous solution, prevents the interaction of galactomannans with water thus precipitating them out. Fehlings solution is widely used to purify galactomannans (Cunha et al 2009; Cerqueira et al 2008). In theory, the axial and equatorial hydroxyl groups in the C2 and C3 position of each mannopyranose units contribute to the complex formation with cupric ions (protected by tartrate ions to prevent formation of cupric hydroxide precipitation) in Fehlings reagent (Chawla 2003). The complexation reaction thus precipitates out the galactomannans and it was reported that this procedure has relatively high yield compared to other purification methods for galactomannans. However, this method also provides copper contamination which is toxic for consumption (Cunha et al 2007). 3.2. Solubility of the Gum in Solvents and Solutions The gum was found to be insoluble in organic solvents which include acetone, ethanol, isopropanol, butanol whereas the gum was soluble in aqueous solutions which include 5% hydrochloric acid, 5% sulfuric acid, 5% sodium hydroxide, 5% potassium hydroxide, 1 M and 0.1 M sodium chloride. The insolubility of the gum in organic solvent provided advantage, especially in the purification procedure. Unlike water, organic solvents like ethanol have very weak polarity that it cannot penetrate through the intramolecular hydrogen bonding in the hydroxyl group-rich polysaccharide,

galactomannan; thus, galactomannans become insoluble in ns the said solvent. On the other hand, ionic solutions including solutions of extreme pH and high ionic strength have available ions that help in the solubilization of galactomannans. 3.3. Water-holding Capacity of the Gum Isolate holding The experiment for determining the water e water-holding capacity of the gum was unfortunately a failure due to the gelatinization of gum. The gelatinous suspension can no longer be separated from the water component therefore it must be avoided when doing the proce procedure. 3.4. Carbohydrate Chemistry of the Gum Isolate 3.4.1. Benedicts test. Benedicts test roughly indicated presence of reducing sugars in the gum suspension through the change in color of the solution from clear blue to cyan plus formation of bluish green precipitate This means that precipitate. the solution contained about 1% o below in terms of amount of reducing sugars (Chawla 2003). The test involves the reduction of cupric ions into tion cuprous ions which develops into cuprous oxide which is a brick red-colored precipitate. The small amount of reducing colored sugars detected can be attributed to free monosaccharides in the solution. Although galactomannans are composed o of mannose and galactose, the units are bonded that their ring form cannot break open to expose their aldehyde functional groups. The purpose of citric acid is to prevent the formation of cupric hydroxide precipitates in the reagent while the sodium carbonate makes the solution moderately alkaline te (Benedict 1908Brummer and Cui 2005) edict 2005). 3.4.2. Phenol-Sulfuric Acid Method The phenol-sulfuric Method. acid method indicated that the total sugar cont content in the sample is about 90.4 %. Figure 2 shows the standard curve used in the phenol-sulfuric acid assay to determine the total sulfuric sugar content of the gum isolate. Torio et al (2006) reported that the total sugar content of mature seed endosperms had ~70%.

furan derivatives will then undergo a condensation reaction co with phenol to produce dark colored complexes. In this case, galactomannans which are the primary components of the gum firstly hydrolyze to form monosaccharides. The monosa monosaccharides particularly hexoses like mannose and galactose became dehydrated by the concentrated acid to form hydroxymethyl furaldehyde which then formed a fo complex with phenol that absorbs UV light at a maximum wavelength of 490 nm (Dubois et al 1956; Rao and ois Pattabiraman 1990). The large discrepancy can be attributed to contamination of sucrose since the sample obtained was immersed in sucrose-rich syrup and the washing step to rich eliminate the syrup might probably be inefficient. Dinitrosalicylic Method 3.4.3. 3,5-Dinitrosalicylic Acid Method. The amount of total reducing sugar determined by DNS method was 11.8 %. Figure 3 shows the standard calibration curve used in the . experiment. Torio et al (2006) also reported that the total reducing sugar of mature endosperms of sugar palm nuts erms was ~4%.

Figure 2. Standard calibration curve for DNS method. The DNS method tests for the presence of free carbonyl group which is common in reducing sugars like aldoses and ketoses that tautomerize to form aldoses. The reaction involves the oxidation of the aldehyde group in mannose, galactose and glucose, and of the ketone group in the case of fructose. Under alkaline conditions, the oxidation of der reducing sugars result into the reduction of 3,53,5 dinitrosalicylic acid to 3-amino,5-nitrosalicylic acid. Phenol nitrosalicylic was added to enhance the color intensity of the reaction (Miller 1959). In this experiment, the large discrepancy of t the total reducing sugar from the reported value by Torio et al. can be attributed to the contamination of acid and contaminati microorganisms that hydrolyze the polysaccharides to form monosaccharides which significantly contribute to the reduction of DNS. 4. CONCLUSION AND RECOMMENDATIONS MENDATIONS The purification of galactomannan from the sugar palm nut endosperm was successful because of the high yield although there seem to be contaminants of reducing sugars in the sample detected spectrophotometrically. To prevent contamination, it is recommended that degradation of recommende galactomannans should be avoided. Samples must be free from concentrated acids, microorganisms, and exposure to extreme heat to ensure integrity of the sample. For a

Figure 2. Standard calibration curve for phenol phenol-sulfuric acid assay. In theory, carbohydrates undergo chemical reactions that lead to the formation of furan derivatives. Initially, the concentrated acid leads to the dehydration of carbohydrates which then leads to form furan derivatives. The resulting ds

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