The production of reducing sugars by acid of carbohydrate cereals powder, jams (total sugar content) and jams (reducing sugar content) were performed in order to study reducing sugar production. The study involved hydrolysis carbohydrate cereals powder and jams by immersing in boiling water at environmental temperature. Acid hydrolysis was carried out through reflux boiling for 20 minutes with 10ml 0f 1. 5M concentrations of diluted sulphuric acid. Reducing sugars concentrations were determined by spectrophotometry using the 3, 5 dinitrosalicylic acid (DNS) method. The first sample was operated as blank for zero.
The stock solution was prepared in order to find the unknown concentration of carbohydrate cereals powder, jam (total sugar content) and jams (reducing sugar content). The absorbance of stock solution were 0. 017,0. 030, 0. 038, and 0. 099 respectively and the absorbance of carbohydrate cereals powder, jams (total sugar content) and jams (reducing sugar content) were 0. 06, 0. 014 and 0. 138 respectively. After determining the specific absorbance of stock solution, the calibration curves was made to determine the unknown concentration of carbohydrate cereals powder, jams (total sugar content) and jams (reducing sugar content). . 00 INTRODUCTION Determination of total reducing is of great important in the food industries especially in industries where quality control is monitored. One method to determine the sugar concentration of reducing sugars is by heating with 3, 5 dinitrosalicylic acids (DNS) which produce a red-brown product (Miller1959) The reaction is direct, thus the method is preferred over the Benedict’s test method. The concentration of the coloured complex can be determined with the spectrophotometer at Absorbance 540 ( http://en. wikipedia. org/wiki/3,5-Dinitrosalicylic_acid) .
The sugar concentration of unknown sample can then be read off a calibration curve(standard curve) created using known sugar concentrations. The dilutions of a solution of known concentration are used to determine the concentration of unknown. Being familiar with the background information about reducing sugars and various methods used to identify them, Biotechnology students were provided with a Fructose sample solution and were required to find its concentration Maltose can be used as a standard for estimating reducing sugar in unknown samples.
Constructing a standard curve / graph for maltose helps us to estimate concentration of reducing sugars present in an unknown sample and for determining the activity of amylase enzyme in forthcoming experiments. The standard curve for maltose is usually constructed using 3, 5-Dinitro salicylic acid (DNS) as the reagent. Maltose reduces the pale yellow colored alkaline 3,5-Dinitro salicylic acid (DNS) to the orange- red coloured, 3 amino,5 nitro salicylic acid. The intensity of the colour is proportional to the concentration of maltose present in the solution as per Beer Lambert’s law; (http://amrita. lab. co. in/? sub=3&brch=63&sim=156&cnt=1). This intensity change in colour is measured using a colorimeter as the absorbance at 540nm wavelength. Wave length is set to 540 nm because it is the region where orange-red colour absorbs. A series of solutions containing varying concentrations of maltose are prepared in test tubes and a known quantity of DNS is added to each. These test tubes are then heated on a water bath for few minutes and their optical densities are measured using a colorimeter. A graph is then plotted with amount of maltose on X axis and the observed optical density at Y axis.
The plot thus obtained is called a standard maltose curve. This method tests for the presence of free carbonyl group (C=O), the so-called reducing sugars. This involves the oxidation of the aldehyde functional group present in, for example, glucose and the ketone functional group in fructose. Simultaneously, 3,5-dinitrosalicylic acid (DNS) is reduced to 3-amino,5-nitrosalicylic acid under alkaline conditions: Oxidation Aldehyde group ———-> carboxyl group reduction 3,5-dinitrosalicylic acid ———-> 3-amino,5-nitrosalicylic acid Because dissolved oxygen can interfere with glucose oxidation, ulfite, which itself is not necessary for the color reaction, is added in the reagent to absorb the dissolved oxygen. The above reaction scheme shows that one mole of sugar will react with one mole of 3,5-dinitrosalicylic acid. However, it is suspected that there are many side reactions, and the actual reaction stoichiometry is more complicated than that previously described. The type of side reaction depends on the exact nature of the reducing sugars. Different reducing sugars generally yield different color intensities; thus, it is necessary to calibrate for each sugar.
In addition to the oxidation of the carbonyl groups in the sugar, other side reactions such as the decomposition of sugar also compete for the availability of 3,5-dinitrosalicylic acid(http://www. ehow. com/about_5244996_general-use-dinitrosalicylic-acid. html). As a consequence, carboxymethyl cellulose can affect the calibration curve by enhancing the intensity of the developed color. Although this is a convenient and relatively inexpensive method, due to the relatively low specificity, one must run blanks diligently if the colorimetric results are to be interpreted correctly and accurately.
One can determine the background absorption on the original cellulose substrate solution by adding cellulose, immediately stopping the reaction, and measuring the absorbance, i. e. following exactly the same procedures for the actual samples. When the effects of extraneous compounds are not known, one can effectively include a so-called internal standard by first fully developing the color for the unknown sample; then, a known amount of sugar is added to this sample. The increase in the absorbance upon the second color development is equivalent to the incremental amount of sugar added.
The concentration of the colored complex can be determined with the spectrophotometer set at 540nm. The sugar concentration of unknown sample can then be read off a calibration curve (standard curve) created using known sugar concentrations. The dilutions of a solution of known concentration are used to determine the concentration of unknown. The figure illustrating how glucose react with DNS (Dinitrosalicylic acid) to give 3-amino-5-nitrosalicylic acid (red-brown) which is detected in spectrophotometer. The background information about the nature of sugar is very essential.
This method detects glucose, fructose and other monosaccharide only. Sucrose cannot be detected by this method directly. Therefore, the sample containing sucrose must be digested so that glucose and fructose products can be detected; (Lanthong et al. , 2006). Owing to the advantages of this method over other reducing sugar determination methods that are generally used nowadays in foods industry particularly Fehling’s method which poses many problems in automation due to analytical steps including precipitation; (Macias et al. 2001). In this experiment sugar concentration of various foods were determined from the calibration graph of standard food sample at various sugar concentrations. 3. 00 MATERIALS AND METHODOLOGY a)Materials Materials used were as mentioned in the practical hand out , practical number 5 B) Procedures Food preparation a)Solid food (cereals) preparation The food was ground into fine powder and 0. 2gm of this powder was weighed into a boiling tube. In the same test tube 10ml of 1. M sulfuric acid was added and heated in a boiling water bath for 20 minutes , stirring occasionally, to hydrolyses polysaccharides and other non reducing sugars. The solution was left to cool and carefully 12Ml of 10% NaOH was added the content was mixed well then filtered into a 100ml volumetric flask finally distilled water was added to make a volume of 100ml. From 100ml, 1ml was measured and transferred into a test tube, into the same test tube 2ml of water and 1ml of DNS reagent was added.
The mixture contents were then heated into water bath for 5 minutes this was done to allow reaction between glucose and dinitrosalicylic acid. b) Jam -total sugar content 1. 0gm of Jam was weighed accurately into a boiling tube; in the same boiling tube 10ml of 1. 5M sulfuric acid was added this mixture was heated in a boiling water bath for 20 minutes, stirred occasionally, to hydrolyze non reducing sugars. The mixture was left to cool and carefully 12ml of 10% NaOH was added then mixed well, the mixture was filtered into 100ml volumetric lask distilled water was added to it to make a volume of 100ml mixed well by repeated inversion. From this solution 10mls were taken into 250ml conical flask then diluted with distilled water to make a volume of 250ml and again the content was mixed well by repeated inversion From 250ml, 1ml was measured and transferred into a test tube, into the same test tube 2ml of water and 1ml of DNS reagent was added. The mixture contents were then heated into water bath for 5 minutes this was done to allow reaction between glucose and dinitrosalicylic acid. c) Jams-reducing sugar content gm of jam was accurately weighed into o conical flask, 50ml of distilled water was added then the mixture was warmed and stirred for 10 minutes, the content was filtered into 100ml volumetric flask and the residues were washed using small amount of distilled water then the mixture was diluted using distilled water to make a volume of 100ml,from this solution 10 ml were taken into another conical flask and diluted to make a volume of 250ml and also mixed well by repeated inversion. From 250ml, 1ml was measured and transferred into a test tube, into the same test tube 2ml of water and 1ml of DNS reagent was added.
The mixture contents were then heated into water bath for 5 minutes this was done to allow reaction between glucose and dinitrosalicylic acid. d) Preparation of standard glucose solution 2. 5 mL, 5 mL, 7. 5 mL and 10 mL of standard glucose solution (1 mg/ mL) was measured into the flat bottom flask followed by addition of distilled water to make a volume of 10 mL. This concentration was obtained from given concentrations 0. 25, 0. 5, 0. 75 and 1. 0 respectively of glucose per mL by using C1V1=C1V2 formula.
Then, 10 mL of distilled water was measured into another flat bottom flask and used as the blank. Four clean and dried test tubes were labeled according to the concentration above and one test tube for the blank solution and make total of five test tubes. Then 1 mL of solution from each flat bottom flask was measured into five labeled the test tubes. By using micropipette, 1 mL of of DNS solution was measured into each five test tubes. All test tubes were heated in boiling water bath for 5 minutes to allow reaction between glucose and DNS to occur. The mixture was allowed to cool.
By using pipette, 16 mL of distilled water was added into the test tubes accurately to make a volume of 20 mL. Then, the absorbance of the samples was detected using spectrophotometer machine. Absorbance measurements Standard glucose solution 5 dry and clean test tubes were prepared (labeled blank, 0. 25, 0. 5, 0. 75 and 1. 0) each test tube was added with 1ml distilled water, the test tubes were then added with 1ml of DNS and 2. 0ml of distilled water the mixture was then heated in a water bath for 5 minutes, left to cool finally the absorbance was determined and recorded . 00 EXPERIMENTAL RESULTS Table 1 for standard glucose solution (concentration and their respective absorbance) Concentration Absorbance (A) 0. 00 0. 250. 017 0. 50. 03 0. 750. 038 1. 00. 099 A graph of Absorbance (A) against concentration (g/ml) of standard glucose solution Finding concentration of unknown using the equation from the above graph (y=0. 0876x-0. 007) From the equation Y= absorbance X=unknown concentrate Therefore, To find the concentration of cereal with absorbance 0. 06 A Y= 0. 0876x-0. 007 X=y+0. 007/0. 0876 =0. 06+0. 007/0. 0876 = 0. 648(mg/ml) To find the concentration of Jam B with absorbance of 0. 014 A Y= 0. 0876x-0. 007 = 0. 014+0. 007/0,0876 =0. 2397(mg/ml) To find the concentration of Jam C with absorbance of 0. 138A = 0. 138+0. 007/0. 0876 =1. 655(mg/ml) Calculations of percentage of cereal and Jams a) % available carbohydrate in cereals is given by the formula below Glucose = C*10/W Where C=Concentration in mg of glucose per 20ml W=weight of cereal used in (g) In this case C= 0. 7648(mg/ml) W=0. 2g Then, % carbohydrate in cereal =0. 7648*10/0. =38. 24% b) % sugar content of Jam B = C*250/W But, C=0. 2397(mg/ml) W=1g Then, %=0. 2397*250/1 =59. 93% c) % sugar content of Jam C C=1. 655(mg/ml) W=3g = 1. 655*250/3 =137. 92% Table 2 showing the concentration obtained from the graph of the standard solution and absorbance of foods (cereal, Jam B and Jam C) Food sampleAbsorbance( A)Concentration (mg/ml)Percentage (%) Blank 0. 00. 000 Cereal 0. 060. 76538. 24 Jam B0. 0140. 239759. 93 Jam C0. 1381. 6555137. 92 5. 00 DISCUSSION The direct relationship between absorbance and concentration is that the ncrease in absorbance is direct proportional to the increase in concentration. The relationship is clearly seen in the graph above as the absorbance increases the reducing sugar concentration also increase. From the results above among cereal, jam total sugar content and jam reducing sugar content, the Jam reducing sugar content was observed to have high content of reducing sugar this may be probably due to the fact that it has huge amount of reducing sugar compared to cereal which might have other contents in it like wise to Jam total sugar content.
The hydrolysis of cellulose to glucose only occurs at economically viable yields when a catalyst is used. The three main catalyst classifications are enzymatic, concentrated acid and dilute acid catalysts. In this experiment the diluted acid was used (H2SO4) during preparation of food for measuring amount of reducing sugar, this is because acid acts as a catalyst for sugar decomposition. During acid hydrolysis, an increase of time reaction improves reducing sugars production .
In analyzing the relationship between sugar degradation and acid concentration, suggests that treatment are prone to degradation with increased acid concentration. Although enzymatic catalysts have advantages than acid. They are the high specific (example no by–products), enzymes operate under mild conditions, are environmentally friendly and a small amount of enzyme results in high yields. In using enzymatic hydrolysis however, pre–treatment is necessary to open up the structure and to provide access for the enzyme to the active sites.
Pre–treatment is usually preformed by energy intensive physical methods, high temperature and pressure or the uses of a chemical solvent. The Nelson-Somogyi and 3,5-dinitrosalicylic acid assays for reducing sugars are widely used in measurements of carbohydrase activities, but among the simplest one is 3,5-dinitrosalicylic acid assays. This is because other methods requiring high volumes of samples, demanding in chemicals, involve several steps, labor-intensive, time-consuming and thus much manual work. The type of side reaction depends on the exact nature of the reducing sugars.
Different reducing sugars generally yield different color intensities; thus, it is necessary to calibrate for each reducing sugar. Although this is a convenient and relatively inexpensive method, due to the relatively low specificity, one must run blanks diligently if the colorimetric results are to be interpreted correctly and accurately. When the effects of extraneous compounds are not known, one can effectively include a so-called internal standard by first fully developing the color for the unknown sample; then, a known amount of sugar is added to this sample.
The increase in the absorbance upon the second color development is equivalent to the incremental amount of sugar added. The concentration of the colored complex was determined with the spectrophotometer at absorbance 540nm. The sugar concentration of unknown sample was then be read off, a calibration curve(standard curve) was created using known sugar concentrations. The dilutions of a solution of known concentration were used to determine the concentration of unknown. . 00 CONCLUSION A sound, simple and quick analytical DNS method has been developed and applied in routine determination of reducing sugars using microtiter plates. The investigated parameters (linearity, limit of detection, limit of quantification, precision and accuracy) confirm that this method is adequate, reliable and suitable for the study of Reducing Sugar in food, beverage and drug samples. 7. 00 REFFERENCES Miller, Gail Lorenz (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar”. Anal. Chem. 31 (3): 426–428. doi:10. 1021/ac60147a030 http://en. wikipedia. org/wiki/3,5-Dinitrosalicylic_acid on 5th May 2013 http://amrita. vlab. co. in/? sub=3&brch=63&sim=156&cnt=1 on 5th May 2013 http://www. ehow. com/about_5244996_general-use-dinitrosalicylic-acid. html on 6th May 2013 UNIVERSITY OF DAR ES SALAAM COLEGE OF NATURAL AND APPLIED SCIENCES (CoNAS) DEPARTMENT OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY