CARBOHYDRATE ANALYSIS

SFA3023 FOOD ANALYSIS

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

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 1. Food are dried under vacuum to prevent thermal degradation. 2. Ground to a fine powder to enhance solvent extraction. 3. Defatted by solvent extraction.

 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.

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; 1. Clarifying agents: Example: heavy metal salts Function: to form insoluble complexes with interfering substances that can be removed by filtration or centrifugation.

2. Ion-exchange:

Mono-, Di- and Oligosaccharides are polar. Therefore possible to separate by using the combination of a positively and negatively charged columns.

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.

MONOSACCHARIDES AND OLIGOSACCHARIDES

Chromatographic Methods 1. 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

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.

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.

A. Titration Methods Example: Lane-Eynon method Use to determine the conc. of reducing sugar in a sample Disadvantages:
1. The results depend on the precise reaction times, temp and reagent conc. must be carefully controlled 2. Cannot distinguishing bet different types of reducing sugar 3. Cannot directly determine of non-reducing sugars 4. Susceptible to interference from other types of molecules that act as reducing agents

Procedure
A known amount of boiling copper sulfate solution and a methylene blue indicator

CHO solution

Color changes: blue to white

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.

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

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.

Anthrone Method Procedure

Sample + sulfuric acid + anthrone reagent
Boiled until a blue-green color is yielded

Measured the solution absorbance at 620 nm

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

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.

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 Allowing complete reaction and measure the product conc. Measuring the initial rate of enzyme catalyzed reaction

Two most common methods

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.

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

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.

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 (20C) and w/length (589.3 nm) dependent Used routinely in industry to determine sugar conc. of syrups, honey, molasses, tomato products and jams

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.

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.

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

POLYSACCHARIDES
Digestible
Important source of energy. E.g. starch

Non-digestible
Cellulose, hemicellulose and pectins

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

 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.

Methods of starch determination

1. 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. 2. 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

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

Sample Preparation and Analysis

Lipid removal

Fiber analysis Protein removal

Starch removal

Selective precipitation of fibers

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

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 -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 enzymecatalyzed hydrolysis.

 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.

 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.

 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.

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

 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.

 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.