CARBOHYDRATE ANALYSIS

1. CARBOHYDRATE ANALYSIS SFA3023 FOOD ANALYSIS

2. INTRODUCTION • Classification of carbohydrates (CHO) • Monosaccarides • Disaccharides • Oligosaccharides • Polysaccharides • Digestible • Non-digestible

3. Sample Preparation for mono-, di- and oligosaccharides • Need to isolate the CHO first before the measurement is made. • Preliminary method commonly used to many isolation techniques • Food are dried under vacuum to prevent thermal degradation. • Ground to a fine powder to enhance solvent extraction. • Defatted by solvent extraction.

4. Most commonly used method for isolation • Boil a defatted sample with an 80% alcohol solution to extract low molecular weight CHO from foods. • Mono-, di- and oligosaccharides are soluble in alcoholic solution while the other components not. • The soluble part are separated from the insoluble part by filtration method.

5. Treating the filtrate with clarifying agents or by passing it through one or more ion-exchange resins – to remove minor components such as amino acids; • Clarifying agents: • Example: heavy metal salts • Function: to form insoluble complexes with interfering substances that can be removed by filtration or centrifugation.

6. 2. Ion-exchange: • Mono-, Di- and Oligosaccharides are polar. • Therefore possible to separate by using the combination of a positively and negatively charged columns.

7. METHODS OF ANALYSIS • CHO content can be determined by calculating the % remaining after all the other components have been measured: % CHO= 100 - % moisture - % protein - % of lipid - % mineral • Can lead to erroneous results due to experimental errors in any of the other methods.

8. MONOSACCHARIDES AND OLIGOSACCHARIDES Chromatographic Methods • HPLC • Advantages: rapid, can tolerate a wide range of sample conc., precise and accurate and requires no prior derivatization • Disadvantages: require micron-filter filtration prior to injection • Stationary phase used: Ion exhange chromatography, reverse phase chromatography and normal phase chromatography

9. 2. GC • Most be converted to volatile derivatives • Involve two preparation steps • Reduction of aldehyde groups to primary alcohol groups • Conversion of the reduced sugar into a volatile peracetate ester or pertrimethylsilyl ether derivative • These steps must be complete 100% to ensure the measurement is accurate and precise.

10. Chemical methods • Based on the fact that many of mono-, di-, and oligosaccharides are reducing agents that can react with other components to yield precipitates or colored complexes. • Non-reducing CHO can be determined after hydrolysis • 3 categories of chemical method: titration, gravimetric and colorimetric.

11. Titration Methods • Example: Lane-Eynon method • Use to determine the conc. of reducing sugar in a sample • Disadvantages: • The results depend on the precise reaction times, temp and reagent conc. → must be carefully controlled • Cannot distinguishing bet different types of reducing sugar • Cannot directly determine of non-reducing sugars • Susceptible to interference from other types of molecules that act as reducing agents

12. Procedure A known amount of boiling copper sulfate solution and a methylene blue indicator CHO solution Color changes: blue to white

13. B. Gravimetric Methods • CHO is heated in the presence of an excess of copper oxide and alkaline tartrate (to keep Cu 2+ ion in solution) under controlled conditions → leads to the formation of a copper oxide precipitate: Reducing sugar + Cu 2+ + base → oxidized sugar + CuO2 (precipitate) • This method has similar disadvantages as Lane-Eynon method • However, it is more reproducible and accurate.

14. The concentration of precipitate present can be determined • Gravimetrically – filtration, drying and weighing • Titrimetrically – redissolving the precipitate and titrating with a suitable indicator

15. C. Colorimetric Methods • Can be used to determine total sugar (reducing and non-reducing sugars) due to the presence of oxidizing agent (sulfuric acid) • Example of methods: Anthrone method and Phenol – Sulfuric Acid method • The concentration of CHO is measured at specific absorbance using spectrophotometer.

16. Anthrone Method Procedure Sample + sulfuric acid + anthrone reagent Boiled until a blue-green color is yielded Measured the solution absorbance at 620 nm

17. Phenol-Sulfuric Acid Method Procedure CHO solution is added into a test tube Phenol + sulfuric acid are added into the CHO solution containing test tube Yellow – orange color is formed Measured at 420 nm

18. Somogyi – Nelson Method • Determine total reducing sugar • Is based on the reduction of Cu2+ to Cu+ ions by reducing sugars. • Cu+ then reduced an arsenomolybdate complex which produce blue color that is measured spectrophotometrically.

19. D. Enzymatic Methods • Relies on enzyme ability to catalyze specific reactions • Rapid, highly specific and sensitive to low concentrations • Little sample preparation needed • Liquid foods – directly tested • Solid foods – need to be dissolved in water first • Two most common methods • Allowing complete reaction and measure the product conc. • Measuring the initial rate of enzyme catalyzed reaction

20. i. D-glucose/D-Fructose • Glucose is converted to glucose-6-phosphate (G6P) by enzyme hexakinase and ATP • G6P is oxidized by NADP+ in the presence of G6P-dehydrogenase (G6P-DH) G6P + NADP+ → gluconate-6-phosphate + NADPH + H+ • The amount of NADPH formed is proportional to the G6P conc. and the absorbance can be measured at 340 nm • Fructose needs to be converted to glucose first before the analysis.

21. ii. Maltose/Sucrose • Maltose and sucrose are broken down into their constituent monosaccharides by α-glucosidase enzyme • Conc. of glucose and fructose are determined using the previous methods • Problem: oligosaccharides are also converted to monosaccharides by α-glucosidase enzyme

22. E. Physical Methods i. Polarimetry • A device that measures the angle that plane polarized light is rotated on passing through a solution • The conc. of CHO in an unknown sample is determined by measuring its angle of rotation and comparing it with the calibration curve.

23. ii. Refractive Index (RI) • Is velocity of light in a vacuum divided by the velocity of light in the material • RI of CHO solution increases with increasing conc. • Temp (20°C) and w/length (589.3 nm) dependent • Used routinely in industry to determine sugar conc. of syrups, honey, molasses, tomato products and jams

24. iii. Density • Density of an aqueous solutions increases as CHO conc. increases • Routinely used in industry for determination of CHO conc. of juices and beverages.

25. iv. Infra Red • A material absorbs infrared due to vibration or rotation of molecular groups. • Measurements are normally carried out by measuring intensity of an infra red wave reflected from the surface of a sample. • Advantages: non-destructive and rapid.

26. F. Immunoassays • Low molecular weight CHO are developed by attaching the CHO of interest to a protein and then injecting it into an animal • Antibodies specific to CHO molecule is developed then and can be extracted for determining the specific CHO concentration. • Advantages: extremely sensitive, specific, easy to use and rapid

27. POLYSACCHARIDES • Digestible • Important source of energy. E.g. starch • Non-digestible • Cellulose, hemicellulose and pectins

28. ANALYSIS OF STARCH • Starch properties • Insoluble in water • High density • It is therefore possible to separate from other soluble and less dense materials. • Methods of starch separation for processed foods; • Is similar to isolation of mono- and oligosaccharides using 80% hot ethanol solution • Take the sediment as starch components due to insolubility of starch in ethanol

29. For semi-crystalline starch, the sample can be dispersed in water and heated to a temp where the starch gelatinizes. • Addition of perchloric acid or calcium chloride to the water prior to heating facilitates to solubilization of starch.

30. Methods of starch determination • Specific enzyme is added to the starch solution to breakdown the starch to glucose. The glucose concentration is then analyzed using the methods described previously. • Iodine can be added to the starch to form an insoluble starch-iodine complex that can be determined gravimetrically by collecting, drying and weighing the precipitate formed or titrimetrically by determining the amount of iodine required to precipitate the starch

31. Analysis of Fibers • Fiber is also known as resistant starch • The basis of many fiber analysis techniques is therefore to develop a procedure that mimics the processes that occur in the human digestive system. • Major components of dietary fiber • Cell wall polysaccharides • Non cell wall polysaccharides

32. Lipid removal Fiber analysis Protein removal Selective precipitation of fibers Starch removal Sample Preparation and Analysis

33. Gravimetric Methods • Crude Fiber Method • Gives an estimate of indigestible fiber in foods • Determine by sequential extraction of a defatted sample with 1.25% H2SO4 and 1.25% NaOH • The insoluble residue is collected by filtration, dried, weighed and ashed to correct the mineral contamination of the fiber residue • Crude fiber measures celllulose and lignin in the sample but does not determine hemicelluose, pectins and hydrocolloidsbecause they are digested by the alkali and acid

34. Total, insoluble and soluble fiber method • The basic principle: to isolate the fraction of interest by selective precipitation and then to determine its mass by weighing • A gelatinized sample of dry, defatted food is enzymatically digested with α-amylase, amyloglucosidase and protease to break down the starch and protein components. Footnote: During gelatinization, starch granules are swell, loss their crystallinity and birefringence and become much more susceptible to enzyme-catalyzed hydrolysis.

35. Total fiber content of the sample is determined by adding 95% ethanol to the solution to precipitate all the fiber. • The solution is then filtered and the fiber is collected, dried and weighed. • Water - soluble and water - insoluble fibers can be determined by filtering the enzymatically digested sample.

36. Soluble fiber in the filtrate solution and the insoluble fiber trapped in the filter • The soluble component is precipitated from solution by adding 95% alcohol to the filtrate and is then collected by filtration, dried and weighed.

37. Official method for determine fiber content and is widely used in food industry • Disadvantages: tends to overestimate the fiber content of foods containing high concentrations of simple sugars such as dried fruit, possibly because they get trapped in the precipitates formed when the ethanol is added.

38. Chemical Methods • Englyst-Cummings Procedure • A defatted food sample is heated in water to gelatinize the starch • Enzymes are then added to digest the starch and proteins • Pure ethanol is then added to the solution to precipitate the fiber which is separated from the digest by centrifugation and is then washed and dried

39. The fiber is then hydrolyzed using a concentrated sulfuric acid solution to break it down into its constituent monosaccharides • The concentration is then determined using the previous methods mentioned. • Total mass of fiber in the original sample is assumed to be equal to the total mass of monosaccharides present.

40. The concentration of insoluble and soluble dietary fiber can also be determined by this method using similar separation steps as the total, insoluble and soluble gravimetric method mentioned above. • However, it does not provide information about the lignin content.