Carbohydrates - Food Analysis, PDF

 Carbohydrates  Carbohydrates are one of the most important components in many foods. Often write "CHO" At least 90% of the carbohydrate in nature is in the form of polysaccharides Loading… Carbohydrates may be present as isolated molecules or...

 Carbohydrates  Carbohydrates are one of the most important components in many foods. Often write "CHO" At least 90% of the carbohydrate in nature is in the form of polysaccharides Loading… Carbohydrates may be present as isolated molecules or they may be physically associated or chemically bound to other molecules.. This carbohydrates have a wide range of solubilities Some carbohydrates are digestible by humans and therefore provide an important source of energy,. supplying 4 Cal/g  carbohydrate (Fibre, Starch ,Sugars) glycogen , dextrin , cellulose Starch are the only polysaccharides that humans can digest and used as a source of calories whereas others are indigestible polysaccharides and therefore do not provide energy,. Non digestible polysaccharides can be divided into soluble and insoluble in water classes Indigestible carbohydrates form part of a group of substances known as dietary fiber  Carbohydrate Starch and Cellulose Starch contains -D-glucose linkage Cellulose has a -D-glucose linkage Loading… we store starch in muscle tissues as glycogen, peeled off‫ تتحلل‬by enzymes when needed cellulose is primary component of plant tissue, largely indigestible,must have enzyme, “cellulase to cellulose analysis  Sugars means all mono-saccharides and di-saccharides (Lactose, maltose, sucrose)present in food There are two types of sugars. 1) Reducing Sugars Reducing sugars are sugars which have free aldehyde or ketone group. These sugars can react with other compounds and provide electrons to them. i.e glucose, maltose, lactose, galactose, and many others 2) Non-Reducing Sugars. Non-reducing sugars are sugars which don’t have free aldehyde or ketone group. i.e sucrose and  Monosaccharides can also be classified as ketoses: contains a carbonyl group, in addition to having one or more hydroxyl groups. aldose : contains an aldehyde group in addition to the hydroxyl groups  Carbohydrate Carbohydrate Classification Usually Carbohydrate Classification by the number of sugar units in the molecule: monosaccharide (glucose, fructose, galactose) - Oligosaccharides) disaccharides (2 units ) such as maltose (2 glucose units) sucrose (glucose + fructose) Trisaccharides(3 units of sugar) such as raffinose polysaccharides (long chain polymers of monosaccharide most important polysaccharides are glycogen , dextrin , starch and cellulose  Sample preparation Factors affecting on sample preparation: Specific raw material Ingredient Food product being analyzed Specific carbohydrate being determined,. However, some generalities can be presented, see the following 0 Sample preparation: general steps Drying Grinding Fat extraction Using 95:5 v/v chloroform - methanol in a Soxhlet extractor , Goldfish, Mojonnier) (we can use other solvent methods). Prior extraction of lipids makes extraction of carbohydrates easier and more complete.  methods of analysis carbohydrates A large number of analytical techniques have been to measure the total concentration of carbohydrates and type of carbohydrates present in foods. indirectly measure :The carbohydrate content of a food can be determined by calculating the percent remaining after all the other components have been measured: Loading… %carbohydrates = 100 –( %moisture + %protein + %lipid + Ash % % saccharides determination =100 –( %moisture + %protein + %lipid + Ash % + Fiber % this method can lead to erroneous results due to experimental errors in any of the other methods, directly measure usually better to directly measure the carbohydrate content for accurate measurements.  Chemical methods A number of chemical methods used to determine monosaccharides and oligosaccharides are based on the fact that many of these substances are reducing agents that can react with other components (such ascopper sulfate (II) )to form precipitates or colored complexes which can be quantified  Fehling method The Fehling method is an example The test is commonly used for reducing sugars ‫السكريات ا&ختزلة‬ required solutions are : Fehling solution, a standard solution of Reducing sugar , 3- titration by standard solution 1- Fehling solution is a chemical reagent used to differentiate between aldehyde and Ketone functional groups , and this is as a test for reducing sugars and non reducing sugars Two solutions are required Fehling's "A" uses 7 g CuSO4.5H2O (blue copper(II) sulfate dissolved in 100ml distilled water containing 2 drops of dilute sulfuric acid Fehling's "B" uses 35g of potassium tartrate (also known as Rochelle salt) and 12g of NaOH dissolved in 100 ml of distilled water (is a clear and colorless solution  Equal volumes of the two mixtures are mixed to get the final Fehling's solution, which is a deep blue colour. In this final mixture  2- preparation a standard solution of Reducing sugar Concentration is a general measurement unit stating the amount of solute present in a known amount of solution Express the wt.(g) concentration in g / L Conc.= (1) VL Express the concentration in mg / ml wt.(mg) Conc.= (2) VmL  2- preparation a standard solution. How many grams of of Reducing sugar as glucose or dextrose would be dissolved to make up to 250ml in a standard flask = concentration = mg Reducing sugar. volume (per ml) mg ascorbic acid = v x c = 250 *0.005 = or. Weigh out accurately about 5g of pure anhydrous glucose, in water and make up to in a standard flask = 1L X 5= 5g dissolve at 1000 ml  How many grams of glucose would be dissolved to make 1 liter of a 0.5M glucose solution? moles solute (NUMBER of gram MOLES of solute )= Morality X volume solution (liters)= = 0.5M x 1 L= 0.5mole/liter material MASSES = MOLAR mass x number of moles -= 180 x0.5=90g Or material masses= molar mass x number of moles X volume solution (liters 0.5 mole/liter x 180 grams/mole x 1 liter = 90 g Or 1 moles of glucose has---- 180g 0.5MOL =90G  3- titration by standard solution of reducing sugars with Fehling's solution occur reduction of the deep blue solution of copper sulfate (II) (‫ أيونات النحاس الثنائي ) النحاسيك‬TO copper sulfate (I) ‫ أيون نحاس أحادي غير الذائب ذو اللون البني )النحاسوز‬to gives a red precipitate of insoluble copper oxide a because reducing sugars (such as Glucose – Fructose –galactose Lactose) containing Aldehyde or ketone functional group Aldehydes are oxidized, giving a positive result and conversion copper sulfate when heated to gives cuprous. , ‫لدهيد‬%‫عند تفاعله مع ا‬ ‫يتحول إلى أيون نحاس أحادي ذو اللون البني )النحاسوز‬but ketones do not react. 4-titration by standard solution of reducing sugars with Fehling's solution+ sample  “Add 10 ml of Fehling's Proceduresolution to an empty flask and a methylene blue as indicator then. Dilute with 10ml of distilled water Place the flask on hote plate at 60° C.for 1-2 min(Boil very gently Slowly titration with a standard solution of Reducing sugar (standard dextrose) \ The end titration test is indicated by a red precipitate of insoluble copper oxide. Record volume of a standard solution consumed in the titration = 9 ml  Repeat the previous steps with 1ml of the unknown sample juice and record results as follows (Add 10 ml of Fehling's solution to an empty flask,and add 1ml of the unknown sample add a methylene blue as indicator then Place the flask on hote plate then titration with a standard solution and record results ) = 7.5 ml The end titration test is indicated by a red precipitate of insoluble copper oxide Fehling's test.mp4  The test is sensitive enough that even 1 mg of glucose will produce the characteristic red colour of the compound From the results of your titration you should be able to calculate an equivalent amount of glucose that reacts with Fehling’s solution  Calculation The original sample weight 5 gram= prepared solution50ml If 10ml of of Fehling's solution consumed 9 ml of standard solution of Reducing sugar - weight of sugar in 1 ml = conc standard solution x volume standard solution = 5mg\ml x9ml= 45 mg 10ml of of Fehling's solution with 1ml sample consumed 7.5 ml - weight= conc x volume= 5mg\ml x7.5 ml = 37.5mg 45mg - 37.5mg = 7.5mg weight of sugar in 1 ml 1ml ----7.5 50ml-----s = 50x7.5\1ml =375 mg / 1000 =. 0375 g = weight \ solu weight x 100=.0375\ 5g x100 =7.5%  The Lane- Eynon method is an example of a titration method of determining the concentration of reducing sugars in a sample. Lane and Eynon method is based on the principle of reduction of Fehling’s solution by reducing sugars. add the carbohydrate solution being analyzed (sample) to a flask containing a known amount of boiling copper sulfate solution and a methylene blue as indicator. + and titration a standard solution of Reducing sugar) the flask.change from blue to white.  The reducing sugars in the carbohydrate solution react with the copper sulfate present in SAMPLE The volume of sugar solution required to reach the end point is recorded.  estimation of reducing sugars by Lane and Eynon method is Chemicals and Reagents Fehling’s solution A: Dissolve 69.28 g copper sulphate (CuSO4.5H2O) in distilled water and dilute to 1000 ml. Filter and store in amber colour bottle. Fehling’s solution B: Dissolve 346 g Rochelle salt (Potassium sodium tartrate: KNa C4H4O6. 4H2 O) and 100 g NaOH in distilled water. Dilute to 1000 ml. Filter and store in amber colour bottle. Neutral lead acetate solution: Prepare 20% neutral lead acetate solution.:  Potassium oxalate solution: Prepare 10% potassium oxalate (K2C2O4. H2O) solution. Methylene blue indicator: Prepare 1% methylene blue solution in distilled water Standard invert sugar “ Weigh 10g of sucrose into 1 litre flask, add 100ml water and 5ml concentrated HCl for hydrolysis. Allow to stand for 3 days at 20-250C or 7 days at 15oC inversion to take place and then make up to volume.  The first step in the estimation of reducing sugars by Lane and Eynon method is the determination of Factor for Fehling’s solution. Fehling factor is the quantity of invert sugar in grams required to completely reduce the Fehling’s solution (usually 5 ml each of Fehling’s A and B solutions THE SECOND STEP :estimation of reducing sugars Procedure Standard invert sugar solution Standardization of standard invert sugar: ). Accurately weigh 4.75g of AR grade sucrose. Transfer to 500 ml volumetric flask with 50 ml distilled water. and few drops of Phenolphthalein indicator. Neutralize with Loading… 20% NaOH until solution turns pink. Acidify with 1N HCl adding it drop wise until pink color disappears. and allow to stand for 24 hr.. Mix well and transfer 25 ml to a 100 ml volumetric flask and make up to volume (1 ml = 2.5 mg of invert sugar).\  Standardization of the Fehling’s Solution for Invert Sugar () Preliminary titration: ) Mix 5ml Fehling A + 5ml Fehling B solution in 250ml conical flask. Add 25-50ml water and heat the flask Methylene blue indicator. Add standard invert sugar solution from the burette drop AND Continue the addition of the sugar solution drop until the blue colour disappears to a brick-red end point. note the titre value of invert solution, repeat the titration and calculate factor for Fehling’s solution as under:-. Titre = V1 = ------ ml Factor for Fehling solution((g of invert sugar) ) = TITRE X2.5 /1000 = 0,0025 X V x 20 =0.05  Preparation of sample 1- Weigh accurately 10 g sample and transfer to 250 ml volumetric flask. Add about 100 ml water and neutralize with 1 N NaOH solution, add phenolphthalein. Add 10 ml neutral lead acetate solution, shake and let stand for 10 min. Add potassium oxalate solution in small amounts(few drops ) until there is no further precipitation. to remove excess of lead acetate. Make volume to 250ml with water and filter.  OR Take 10- g of sample (fresh fruit or fruit product) and grind in a pestle and mortar, blend in blender, add 100 ml water. Neutralize solution with 1 N NaOH. Boil gently for 1 hour with stirring. Replace water lost during evaporation, cool and transfer to 500 ml volumetric flask. Make volume 500 ml and filter through whatman filter paper.  ii) Procedure for estimation of reducing sugars Final titration:. Pipette 10ml of mixed Fehling’s solution (5 ml each of Fehling A and B) and few ml of water into 250ml conical flask. Heat the flask containing mixed Fehling’s solution and add the sample solution( clarified sample) (about 0.05 to 1.0 ml.) on hot plate Complete the titration by adding 2 to 3 drops of sugar solution , until the indicator is decolourized. At the end point, the boiling liquid assumes the brick red colour At the end point, note the readings and calculate the reducing sugars.  Weight of the sample = W = ------- g Dilution volume for the sample = V2 = ------ ml Volume of clarified sample solution required for Fehling’s reaction (titre) = V3 = ------- ml  Problem: 10 g of fruit jam was made into 500 ml after neutralization with 1 N NaOH and filtered. From the filtrate 100ml of aliquot was clarified using lead acetate and potassium oxalate and volume made to 250 ml and filtered. 10 ml Fehling’s solution (A+B) was titrate against clarified sample. The titre value was 16 ml. Calculate the reducing sugar when factor for Fehling’s solution is 0.05. Solution: Given 10g made to 500ml 100 ml aliquot made to 250 ml Titre 16ml.....Factor for Fehling’s solution 0.05  Calculations: Reducing sugar (%) mg of invert sugar × dilution × 100 = --------- --------------------------------------- titre × weight of the sample × 100 Or % Reducing Sugar = Factor for Fehling solution × Volume made × 100 = -------------------------------------------------------- Titre × wt of sample × ml of aliquot  % Reducing Sugar = Factor× vol. made for 10g to 500ml × vol made 100ml to 250 ml × 100 -------------------------------------------------------- Titre × wt of sample (10) × ml of aliquot 100 0.05 × 500 × 250 × 100 = ------------------------- = 39.06% 16 × 10 × 100  The disadvantages of this method are (i) the results depend on the reaction times, temperatures and reagent concentrations used and so these parameters must be carefully controlled; (ii) it cannot distinguish between different types of reducing sugar, (iii) it cannot directly determine the concentration of non- reducing sugars, (iv) it is sucseptible to interference from other types of molecules that act as reducing agents. determination of mono- and oligosaccharides by use 80% alcohol solution Sample preparation: general steps 0  Determination of mono- and oligosaccharides by use 80% alcohol solution One of the most commonly used methods of extracting low molecular weight carbohydrates from foods is to boil a defatted, dried sample ‫غلي‬ ‫ عينةمنزوع الدهن مجفف‬with an 80% alcohol solution. Monosaccharides and oligosaccharides are soluble in alcoholic solutions, whereas proteins, polysaccharides and dietary fiber are insoluble. in hot 80% ethanol..  The soluble components can be separated from the insoluble components by filtering the boiled solution and collecting the filtrate (the part which passes through the filter) and (the part retained by the filter).  The aqueous alcohol‫ائي‬$‫ الكحول ا‬of the ethanol extract is removed under reduced pressure‫تحت ضغط منخفض‬ using a rotary evaporator. The residue is dissolved in a known, measured amount of water. These two fractions can then be dried and weighed to determine their concentrations  The disadvantages of this method are In addition, to monosaccharides and oligosaccharides various other small molecules may also be present in the alcoholic extract that could interfere with the subsequent analysis e.g., ‫مع‬ ‫تحليل &حقة‬ amino acids, organic acids, pigments, vitamins, minerals etc. It is usually necessary to remove these components prior to carrying out a carbohydrate analysis. This is commonly achieved by treating the solution with clarifying agents( are used to remove suspended solids from liquids) or by passing it through one or more ion-exchange.  clarifying agents inducing such as aluminium chlorohydrate aluminium sulphate calcium oxide calcium hydroxide iron(II) sulphate (ferrous sulphate) iron(III) chloride (ferric chloride)  Munson and Walker method is an example of a gravimetric method of determining the concentration of reducing sugars in a sample. Carbohydrates(reducing sugar ) are oxidized in the presence of heat and an excess of copper sulfate and alkaline tartrate under carefully controlled conditions which leads to the formation of a copper oxide precipitate: reducing sugar + Cu2+ + base oxidized sugar + CuO2 (precipitate) The concentration of precipitate present can be determined gravimetrically (by filtration, drying and weighing), The amount of precipitate formed is directly related to the concentration of reducing sugars in the initial sample or(by redissolving the precipitate and titrating with a suitable indicator).  The Phenol - Sulfuric Acid method is an example of a colorimetric method that is widely used to determine the total concentration of sugar present in foods.. In this method, the concentrated sulfuric acid and heat breaks down any polysaccharides, oligosaccharides, and disaccharides to monosaccharides These compounds then react with phenol (Phenol, 80%, 1 ml) to produce a yellow-gold color. (as a result of the interaction between the carbohydrates and the phenol ) that can be measured spectrophotometrically  After that the absorbance is measured at 490 -420 nm.. This method it is necessary to prepare a calibration curve using a series of standards of known carbohydrate concentration The sulfuric acid causes all non-reducing sugars to be converted to reducing sugars, so that this method determines the total sugars present  Reagents Glucose std solution, 100 mg/L. Phenol, 80%, wt/wt in H2O, 1 ml** - Prepare by adding 20 ml deionized distilled (dd) water to 80 g of redistilled reagent grade phenol (crystals) Sulfuric acid concentrated