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Application of sorghum starch in fructose syrup production

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https://www.eduzhai.net/ International Journal of Food Science and Nutrition Engineering 2017, 7(4): 70-74 DOI: 10.5923/j.food.20170704.02 Utilization of Sorghum (Feterita) Starch in Production of Fructose Syrup Elamin A. Elkhalifa*, Nadia K. A. Abdalla, Sarah, A. M. Abdelkareem Department of Food Engineering and Technology, Faculty of Engineering and Technology, University of Gezira, Wad Medani, Sudan Abstract The experiments of this research were conducted for the isolation of starch from sorghum grains (Feterita) by wet milling process and subsequent use for production of fructose syrup by enzyme hydrolysis. Chemical composition and percentage of starch, amylose, water soluble amylose, total soluble sugars and reducing sugars were determined. Isolated starch was cooked by heating and liquefied by α–amylase and saccharified by amyloglucosidase. The glucose syrup produced was treated with isomerase to produce fructose syrup. The percentages of moisture, protein, fat, ash, crude fiber and total carbohydrate of Feterita were 4.9, 12.8, 2.5, 1.7, 1.8 and 76.3, respectively. Feterita grains contained 49% starch. Amylose and water soluble amylose were 32.2% and 5.6, respectively. Total soluble sugars were 2.9%. In hydrolysate reducing sugars, total soluble solids (TSS) and glucose contents increased successively during hydrolysis of starch. The glucose content was increased from 38.14 mmole/L to 360.35 mmole/L whereas glucose conversion to fructose reached 50%. Keywords Sorghum, Feterita, Starch, Hydrolysate, α-amylase, Amyloglucosidase, Isomerase 1. Introduction In the Sudan, starch is available from varieties of cheap sources (sorghum, maize, millet, cassava, sweet potato and potato) and the cheapest source is Feterita. The enzyme technology of glucoamylase is applied to the starch conversion process, giving added economic benefits to produce dextrose and dextrose syrups. In this process high starch concentrations can be used which requires less steam energy in subsequent evaporation steps. The conversion of glucose to fructose via-the action of enzyme glucose isomerase remains one of the most important transformations used in industry [1]. The chemical isomerization of glucose to fructose is possible especially under condition of high temperature and alkaline pH [2-4]. However, chemical isomerization is not employed commercially because of the usual production of non-metabolizable materials such as psicose, formate and colored materials that are costly to remove [5]. The technique for the production of high fructose syrup (HFS) was first developed in Japan and later improved in United States [6]. In the U.S. corn starch is treated with the enzymes α–amylase and amyloglucosidease to produce glucose. The latter is then treated further with glucose isomerase to produce significant fructose content and hence greater sweetening capacity [5]. In the United States ten million tons * Corresponding author: benkhalifa_99@yahoo.com (Elamin A. Elkhalifa) Published online at https://www.eduzhai.net Copyright © 2017 Scientific & Academic Publishing. All Rights Reserved of HFS are produced annually and used to replace sucrose in the majority of its uses [1]. Since 2000, production of high fructose corn syrup (HFCS) has declined by about 10 percent, with 2015 production totaling 8.5 million tons [7]. Fructose play an important role in the diet of the diabetics as it is only slowly absorbed by the stomach and intestinal tract and hence dose not influence the blood glucose level [8]. In modern developments of starch industry, the starch can be converted easily to glucose and then further to HFS using immobilized enzymes process that can be recovered for reuse. Considering this, successful HFS industry can be developed in the Sudan adding economic value to the high production of sorghum crop in the country. The objectives of this study to utilize enzymes of α–amylase, amyloglucosidase and glucose isomerase to convert the Feterita starch to fructose syrup. 2. Materials and Methods 2.1. Materials Sorghum grains (Feterita) were purchased from Wad Medani local market. α–amylase, amyloglucosidase and isomerase enzymes were obtained from NOVO Nordisk A/S Denmark. The standard glucose kits were obtained from MDSS GmbH, Hannover, Germany. 2.2. Proximate Analyses The percentages of moisture, ash and fiber were determined using A.O.A.C. methods [9], while the protein content was determined by A.A.C.C. methods [10] and the International Journal of Food Science and Nutrition Engineering 2017, 7(4): 70-74 71 fat content was determined by A.O.C.S. methods [11]. Total carbohydrates were obtained by difference between the sum of the other major compositions, namely moisture, protein, fat, ash and fiber from 100 percent. 2.3. Starch Isolation The starch isolated from Feterita grains by steeping first in an anti-bacterial mercuric chloride solution overnight and then by wet grinding in sodium chloride solution according to the procedure of Badenhuizen [12]. The isolated starch percentage was calculated using the following formula: Yield of starch fraction (g) X100 Weight of grains sample (g) 2.3.1. Amylose Amylose content in Feterita starch was released by treatment with diluted alkali according to the procedure of Williams, et al. [13]. The extracted amylose content was determined against amylose standard with iodine reagent at wavelength 600 nm. 2.3.2. Water-Soluble Amylose Water-soluble amylose content in Feterita starch using hot water was determined against amylose standard with iodine reagent at wavelength 600 nm according to the procedure of Juliano et al. [14]. 2.4. Total Soluble Sugars Total soluble sugars content in defatted Feterita flour was extracted with aqueous ethyl alcohol, followed by treatment with phenol- sulphuric acid to produce golden yellow color. The absorbance was measured at 490 nm against glucose standard with different concentrations [15]. The percentage of total soluble sugars was calculated using the following formula: Cstd X Aextract X 1gm X DF X 100 Astd 1.000.000 0.1gm sample Where: Cstd = Conc. of standard (µg) Astd = Absorbance of standard Aextract = Absorbance of 1ml sample extract DF = Dilution factor (100 ml) 2.5. Conversion of Sorghum Starch to Fructose Glucose was produced by enzymatic hydrolysis and then converted to fructose syrup, under optimum temperature, pH and incubation period required for the activity of the three enzymes as described by the method of Cheetham [16]. Starch α-amylase amyloglucosidase Dextrin + Oligosaccharides glucose units isomerase Fructose + glucose 2.5.1. Determination of pH Electrometric method employing pH-meter with glass electrode (assembly) was used for pH measurements. The pH-meter was adjusted with standard buffer solutions. The pH of the hydrolysate after each enzyme treatment was recorded. 2.5.2. Determination of total Soluble Solids (TSS) The Abbe refractometer was adjusted at 20°C to give zero reading using distilled water. 2-3 drops of starch slurry was transferred by a glass rod to the instrument. The reading was recorded in Brix to represent TSS [17]. The procedure was also used for determining TSS in hydrolysate sample. 2.5.3. Determination of Reducing Sugars Determination of reducing sugar using Lane and Eynon method. This method was used for determination of reducing sugars and other substances [18]. The percentage of reducing sugars was calculated using the following equation: Reducing sugars % = 100 Ff VC Where: Ff = the correction factor; V = the volume (ml) of the test solution used in the titration; C = the concentration (g/100 ml) of the sample in the test solution. 2.5.4. Determination of Glucose Content The glucose content in starch mixture and hydrolsate sample was determined according to Tinder [19]. GOD-PAP enzymatic colorimetric method sold as a Kit was adopted for determination of glucose (MDSS GmbH, Hannover, Germany). The red Quinoneimine formed is proportional to the amount of glucose present in the sample as presented in the following reaction: Glucose + 2 H2O + O2 GOD Gluconic acid + H2O2 2 H2O2 +Phenol PAP + 4-amino-antipyrine 4H2O + Quinoneimine The amount of glucose was calculated as follows: Glucose concentration (mg/dl) = Absorbance of sample X conc. standard Absorbance of standard 72 Elamin A. Elkhalifa et al.: Utilization of Sorghum (Feterita) Starch in Production of Fructose Syrup 2.5.5. Determination of Fructose Content Fructose content was determined by amount of glucose which was converted to fructose by isomerase. Fructose content was calculated by subtracting the unconverted glucose in the enzymic reaction from the total glucose in the sample. 3. Results and Discussion 3.1. Chemical Composition of Sorghum (Feterita) The results obtained for the study of sorghum analysis are presented in Table 1. Table (1). Proximate analysis of sorghum grains (Feterita) amylose content were found to be 32.2% and 5.6%, respectively. Buddair [24] reported that Feterita starch had the highest amylose content than other sorghum varieties (Dabar and Tetron). Rooney and Saldivar [25] reported that the amylose content in starch was 30%. This variation in result may be due to the genetic makeup of the sorghum varieties. The percentage of total soluble sugars was found 2.9% as reported in Table 2. 3.3. Starch Hydrolysate All polysaccharides can be hydrolyzed with acids or enzymes to yield monosaccharides. The results of pH, total soluble solids (TSS) and reducing sugars in starch slurry and hydrolysate samples are presented in Table 3. Moisture 4.9 Protein 12.8 Fat Ash 2.5 1.7 Crude fiber % 1.8 Carbohydrate 76.3 The moisture content reported in this investigation was found to be 4.9%. The estimated value of protein content was 12.8% which was lower than the value of 13.4 reported by Eggum et al. [20]. This variation may be related to differences in genetically and environmental conditions, sorghum has low fat content, the fat content of studied Feterita was found 2.5%, which was less than Eggum et al. [20] who reported that fat content of Feterita was 4.1%. These differences may be related to genetic and climate variation. The ash content of food stuffs represent the inorganic residue remaining after the organic matter has been burned. The ash content of Feterita was found 1.7%, which was less than Eggum et al. [20] who reported that ash content of Feterita 2.07%. The fiber content of studied Feterita was found to be 1.8% which was less than Eggum et al. [20] who found that fiber content of Feterita was 2.1%. This difference may be also to environmental and genetic variations. The carbohydrate of Feterita was 76.3% which was within the range reported by other workers [20-22]. Table (2). The percentages of starch amylose, water soluble amylose and total soluble sugars in sorghum (Feterita) Sample Feterita grains Starch Components % Amylose Water soluble amylose 49 32.2 5.6 Total soluble sugar 2.9 3.2. The Isolated Starch The percentage of starch was found 49%. This result was higher than the result of Abd Elnour [23] who reported that the percentage of starch in Ferterita was found to be 44.2% in grains without decortication then increased to 57.05% in decorticated seeds once, and increased to 61.8% when Feterita was decorticated twice. Amylose and water soluble Table (3). pH, total soluble solids and reducing sugars in Feterita starch hydrolysate Treatment Starch slurry 30% solids Starch slurry (α–amylase) Starch hydrolysate (amyloglucosidase) Starch hydrolysate ( isomerase) Steps of hydrolysis - pH TSS Reducing Bx sugars % 6.80 0.0 0.0 Liquefaction 5.60 25.5 10.2 Saccharification 4.41 28.8 22.2 Isomerization 7.50 36.0 42.2 The result showed that in starch slurry the TSS and reducing sugars were not reported. The pH of starch slurry was found 6.8. In liquefaction step the pH was adjusted at pH 5.6. After hydrolysis of starch slurry with α–amylase for 2 hours, the TSS and reducing sugars were found 25.5°Bx and 10.2%, respectively. In Saccharification step using amyloglucosidase hydrolysis for 48 hours, the pH was adjusted at 4.41 and temperature set at 60°C. The TSS was increased to 28.8°Bx and a double increase in reducing sugars to 22.2% compared to liquefaction step was obtained. Hence, reducing sugars and total soluble solids increased due to hydrolysis of starch by amyloglucosidase enzyme. In the isomerization step the incubation conditions for isomerase were set at pH 7.5 and temperature at 60°C for 2 hours. The total soluble solids was increased to 36.0°Bx and reducing sugars content in the hydrolysate was substantially increased to 42.2% compared to the previous Saccharification step. In this stage of hydrolysis part of glucose is converted to fructose which has greater reducing power toward Fehling’s reagent Shallenberger and Birch [26]. Glucose content in starch slurry was found 0.0938 mmole/l (Table 4). When α–amylase was added to the starch slurry, starch was broken down to soluble dextrin and oligosaccharide and glucose content increased to 38.14 mmole/l. While the hydrolysis continued by the addition of International Journal of Food Science and Nutrition Engineering 2017, 7(4): 70-74 73 amyloglucosidase enzyme, the glucose concentration in the hydrolysate was substantially increased to 360.35 mmole/l due to hydrolysis of dextrin to glucose by amyloglucosidase [3] Scallet, B.L; Shieh, K; Ethernthal, I. and Slapshak, L. (1974). Studies in the isomerization of D-glucose. Die Starke, (26): 405-408. enzyme. In the isomerization step the incubation conditions [4] Barker, S.A; Somers, P.J. and Hatt, B.W. (1975). Methods in for isomerase enzyme was set at pH 7.5 and 60°C for 2 hours, more than half of glucose content was converted into high fructose syrup manufacture (Novo Industry A/S Denmark). United States Patent 3, 875, 140. fructose. Accordingly glucose content was reduced to 172.75 mmole/l (Table 4). The isomerization of glucose gives fructose, the most sweet natural sugar. Fructose syrup [5] Blanchard, P.H. and Geiger, E.O. (1984). Production of high fructose corn syrup in the USA. Sugar Technology Reviews, Elsevier Science Publishers, Amsterdam, 1-94. competes with sucrose of cane sugar in many food [6] White, P.J. and Pollak, L.M. (1995). Corn as a food source in applications. the United States: Part II. Processes, products, composition and nutritive value. Cereal Foods World 40 (10): 756-762. Table (4). Glucose and fructose contents of sorghum (Feterita) starch hydrolysate [7] United States Department of Agriculture, USDA. (2017). U.S. Treatment Steps of hydrolysis Glucose content mmole/l Fructose content mmole/l Sugar Production. Economic Research Service. [8] Davis, E.A. (1995). Functionality of sugars: Physiological interactions in foods. American Journal of Clinical Nutrition Starch slurry 30% solids - 0.0938 - 62 (1): 170-177. Starch slurry (α–amylase) Liquefaction 38.14 - Starch hydrolysate (amyloglucosidase) Saccharification 360.35 - [9] A.O.A.C. (1984). Official Methods of Analysis 14th edition. Association of Official Analytical Chemists, Washington, D.C., USA. Starch hydrolysate (isomerase) Isomerization 172.75 187.6 [10] A.A.C.C. (1983). Approved methods of analysis. American Association of Cereal Chemists, St. Paul, USA. 4. Conclusions [11] A.O.C.S. (1981). Official Tentative Methods of Analysis 3rd edition, Association Oil Chemists, Society, Champaign, IL 61920, USA. Feterita grain is relatively rich in protein and carbohydrates compared to other sorghum varieties. The starch isolated from Feterita grains had high amylose and water soluble amylose contents. Addition of isomerase enzyme converted the starch hydrolysate to a mixture of glucose and fructose whereas glucose conversion to fructose reached 50%. Feterita starch can be processed through enzymatic conversion to produce fructose syrup. Further research is needed to isolate the starch through dry milling process. Also further investigation is needed to study the physical and chemical properties of fructose syrup produced from Feterita starch. [12] Badenhuizen, N.P. (1964). General Method for Starch Isolation pp. 14-15. In: Methods in Carbohydrate Chemistry, vol. V (R.L. Whistler, R.J. Smith, J.N. Bemiller, and M.L. Wokform (eds.). Academic Press, New York, USA. [13] Williams, V.R; WU, W.T; Tsai, H.Y. and Bates, H.G. (1958). Varietal differences in amylose content of rice starch. J. Agric. Food Chem. 6: 47-48. [14] Juliano, B.O; Contano, A.V. and Vidal, A.J. (1968). Note on a limitation of the starch-iodine blue test for milled rice amylose. Cereal Chem. (45) : 63-65. [15] Dubois, M; Gilles, K.A; Hamilton, J.K; Rebers, P.A. and Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: 350-356. ACKNOWLEDGEMENTS The authors express their sincere gratitude to Dr. Nagla Gasmelseed, in Nuclear Medicine Institute for her assistant and Mr. Hassan Ansari the head technicians in Food Analysis Laboratory. [16] Cheetham, P.S.J. (1987). The Application of Enzymes in Industry, pp 274-379. In: A. Wiseman (ed.). Handbook of Enzyme Biotechnology. Eilis Horwood Limited, Chichester, England. [17] A.O.A.C. (2000). Official Methods of Analysis 17th edition. Total soluble solids and titratable acidity of fruits and fruits products. Association of Official Analytical Chemists, Washington, D.C., USA. REFERENCES [1] Housler, H. and Stutz, A.E. (2001). D-xylose (D-glucose isomerase and related enzymes in carbohydrate synthesis). Topics current chemistry, (215 L): 77-114. [2] Poulsen, P; Borge, R. and Zittan, I.E. (1977). Process of isomerazing glucose. (Novo Industry A/S Denmark). United States Patents 4, 025, 289. [18] International Commission Uniform Methods of Sugar Analysis (ICUMSA). (1998). Determination of reducing sugars by Lane Eynon constant volume procedure Official Method Book with first supplement, ICUMSA Publications Department C/O British Sugar Technical Center. Norwich Research Park, Coney, Norwich NR4, 7UB, England. [19] Tinder, P. (1969). Glucose-GOD-PAP-enzymatic colorimetric method. Ann. Clin Biochem. 6: 24. [20] Eggum, B.D; Monawar, L; Bach Knudesn, K.E; Munk, L. and 74 Elamin A. Elkhalifa et al.: Utilization of Sorghum (Feterita) Starch in Production of Fructose Syrup Axleel, J. (1983). Nutritional quality of sorghum and sorghum foods, from Sudan. Journal of Cereal Science (10): 127-137. [21] Reichert, R.D. (1982). Sorghum dry milling. In: Sorghum in the eighties. Proceeding of the International Symposium on Sorghum. ICRISAT, Patancheru, A.P. India. [22] Elkhalifa, E.A. (2000). Sorghum-based Traditional Foods of Sudan. Preparation and Quality Aspects. Proceeding of the 1997 International Conference on Traditional Foods. p p 117- 127, CFTRI, Mysore. India. [23] Abd Elnour, M.K. (2001). The Effect of Decortications on Wet-milling and Starch Quality of Sorghum and Millet Grains. M.Sc. Thesis, University of Khartoum, Khartoum, Sudan. [24] Buddair, A.A. (1977). Chemical Studies on Sorghum Grains and their Products. M.Sc. Thesis, University of Khartoum, Sudan. [25] Rooney, L.W. and S.O. Saldivar. (1991). Sorghum Hand Book of Cereal and Technology. (K.J. Iorenz and K. Kalp (eds.)), Marcel Dekkar, New York, USA. [26] Shallenberger, R.S. and Brich, G.G. (1975). Sugar Chemistry. The AVI Publishing Company, Inc. West Port, Connecticut, U.S.A.

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