Analysis of Carbohydrates

ANALYSIS OF

Ilyana A. Causing ENFT-3A

INTRODUCTION

• Carbohydrates in food Isolated Molecules Physically Associated or Chemically Bound

• Classification of Carbs depends on the number of monomers Glycoproteins – covalently bond to proteins Glycolipids – covalently bond to lipids

• Carbohydrates are either Digestible – provide energy Indigestible – do not provide energy

• Dietary Fiber and Lignin is indigestible But! Dietary Fiber is beneficial to human health

1. Helps reduce risk of certain types of cancer2. Coronary diseases3. Diabetes4. Constipation

INTRODUCTION

Why Determine the type and concentration of Carbohydrates?

Standards of IdentityNutritional Labelling

Detection of AdulterationFood Quality

EconomicFood Processing

Classification of Carbohydrates

1. Monosaccharide• Water-soluble crystalline compounds

2. Oligosaccharides• Low molecular weight polymers of monosaccharides

3. Polysaccharides• High molecular weight polymers of monosaccharides

Monosaccharide

• Aliphatic aldehydes or ketones• Most natural carbohydrates are

Hexoses – Glucose, Fructose, Galactose Pentoses – Arabinose, Xylose

• There reactive centers Carbonyl Group Hydroxyl Group

Oligosaccharides

• Covanlently bonded through glycosidic linkages• Common in food contains monomers of• Glucose• Fructose• Galactose

• Either be Disaccharides or Trisaccharides

Polysaccharides

• Majority of carbohydrates found in natureHomopolysaccharide• Same monosaccharides• Starch, Cellulose, Glycogen – GlucoseHeteropolysaccharide• Contain different type of monomers• Pectin, Hemicellulos, Gums

Method of Analysis

• Carbohydrate content can be measured after all other components are measured

• But! This can lead to erroneous results• Instead. Directly measure the carbohydrate content

Monosaccharide and Oligosaccharides

• Amount of preparation depends on the nature of the food• Aqueous solutions require little preparation• But! Physically Associated or Chemically Bound need to be isolated

Method of Isolation depends on the :1. Carbohydrate type2. Food Matrix Type3. Purpose of the Analysis

Prior to Analysis: Sample Preparation

Example: Sample Preparation

Dried (under vacuum)

Ground to a fine powder

Defatted(by solvent extraction)

To avoid thermal degradation

To enhance solvent extraction

Extracting Low Molecular Weight Carbohydrate

1. Boil a defatted sample with 80% alcohol solution• Mono & Oligo – soluble in alcoholic solutions• Poly & Dietary Fibers – insoluble

2. Separate the component by filtering the boiled solution• Filtrate and Retetante

3. The two fractions are dried and weighedNote!• Filtrate may contain small molecules (Amino acids, Organic Acids,

Pigments, Vitamins, Minerals) Have to be remove prior analysis by treatment of Clarifying Solutions or

Ion-Exchange Resins• Alcohol can be removed by evaporation under vacuum

Methods of Analysis

1. Chromatographic and Electrophoretic methods2. Chemical methods3. Enzymatic methods4. Physical methods5. Immunoassays

Chromatographic Methods

• Most powerful analytical techniques Analysis of Type and Concentration of Mono and Oligo

• Commonly used to separate and identify carb1. Thin Layer Chromatography, TLC 2. Gas Chromatography, GC3. High Performance Liquid Chromatography, HPLC

• Carbohydrates are separated base of their differential absorption characteristics

Electrophoretic Methods

• Carbohydrates are separated by electrophoresis after being derivitized – make them electrically charged

• Solution of derivitized carbs is applied to a gel and a voltage is applied across

• The carbohydrates are then separated base of their size– The smaller the size – faster it moves in an electrical field

Chemical Methods

• These methods are used to determine Mono & Oligo because most of them are reducing sugars

• Concentration of carbohydrates can be determined:1. Gravimetrically (Gravimetric Methods)2. Spectrophotometrically (Colorimetric Methods)3. Titration Methods

Note!• Non-reducing carb can be determined but they have to be

hydrolyzed

Enzymatic Methods

• They base on the ability of the enzymes to catalyze specific reactions• These methods are rapid, highly specific, sensitive to low concentrations• Little sample preparation is required

– Liquid Foods – can be tested directly– Solid Foods – dissolved in water

• Two commonly used methods1. Allow the reaction to complete and measure the concentration of the product

Concentration of the product – Concentration of the Initial Substrate2. Measure the initial rate of the enzymes catalyzed reaction

Rate - Substrate Concentration

Physical Methods

• These methods rely on being a change in physiochemical characteristics as its carbohydrate concentration varies

• Commonly Used1. Polarimetry2. Refractive Index3. Infrared4. Density

Immunoassays

• Specific for low molecular weight carbohydrates• Developed by

1. Attaching the carbohydrate to a protein2. Injecting it into an animal3. Animal develops antibodies specific for the carbohydrate molecule4. Antibodies are extracted and used for determining the concentration of the

carbohydrate• Immunoassays are extremely sensitive, specific, easy to use, and

rapid

Analysis of Polysaccharides and Fiber

Polysaccharides are classified according to their:1. Molecular Characteristics

– Type, Number, Bonding, Sequence of Monosaccharides2. Physiochemical Characteristics

– Water Solubility, Viscosity, Surface Activity3. Nutritional Function

– Digestible or Non-digestible

Analysis of Starch

• Starch is a mixture of Amylose and Amylopectin The two have different physiochemical properties

• Its important to determine 1. Concentration of EACH Component 2. Overall Starch Concentration

• Cannot be determined directly because is contained in a structurally and chemically complex food matrix

• Need to isolate from other components prior analysis

Starch

Prior to Analysis: Sample Preparation

In Natural foods• Starch granules is separated by drying, grinding, steeping in water,

filtration and centrifugationHow this worksStarch granules will move to the bottom because its water-insoluble and

have high densityIn Processed foods• The sample is dried, ground and dispersed in hot 80% Ethanol solution

then filtered or centrifuged How this worksMono&Oligo are soluble in ethanol solution and starch is insoluble

Important Note

• Starch is often present in a semi-crystalline form – It’s inaccessible to chemical reagents used in analysis

What to do?1. Disperse the sample in water and heat until the starch gelatinizes 2. Add Perchloric Acid or Calcium Chloride prior to heating to

facilitate solubilization

Starch

Analysis Methods: Starch

1. Addition of Enzymes– To breakdown the starch to glucose– Glucose concentration is analyzed and used to determine the starch

concentration2. Addition of Iodine

– Gravimetric method - formation of an insoluble starch-iodine complex (Collected, Dried and Weighed)

– Titrate method – determine by the amount of Iodine needed to precipitate the starch

3. Physical Methods– Density, Refractive Index or Polarimetry

Analysis of Fibers

• Plant Polysaccharide • Indigestible to humans• Resistant Starch – types of starch much like dietary fiber

Major Components1. Cellulose2. Hemicellulose3. Pectin4. Hydrocolloids5. Lignin

Major Components of Dietary Fiber

Cell Wall Polysaccharides– Cellulose is usually associated with Hemicellulose and Lignin– There associated determines the characteristic textural properties of edible plants

• Hemicellulose – soluble in dilute alkali solutions but insoluble in water• Pectins - soluble in hot water and capable of forming gelsNon Cell Wall Polysaccharides

– Include Hyrdocolloids (Guar Gum, Locust Bean Gum, Gum Arabic, Agar, Alginates, Caragenans)– Used as Gelling agents, Stabilizers and Thickeners

Lignin– Non-carbohydrate polymer– Usually associated with Cellulose and Hemicellulose

1. Lipid Removal2. Protein Removal3. Starch Removal4. Selective Precipitation of Fibers5. Fiber Analysis

Prior to Analysis: Sample Preparation

Dietary Fibers

Analysis Methods: Dietary Fiber

1. Gravimetric Methods– Crude Fiber Method– Total Insoluble and Soluble Fiber Method

2. Chemical Methods– Englyst-Cummings Procedure

Gravimetric Methods

Crude Fiber Method– Gives an estimate of indigestible fiber in foods– Through sequential extraction of defatted sample with 1.25% Sulphuric Acid and

1.25% NaOH– Insoluble residue is collected through filtration, dried, weighed, and ashed (correct

for mineral contamination)– Used to measure cellulose and lignin but not hemicellulose, pectin, and hydrocolloid

Gravimetric Methods

Total Insoluble and Soluble Fiber Method– Principle: Isolate the fraction of interest by selective precipitation then determine its

mass by weighingSteps1. A Gelatinized sample (dried and defatted) is digested with α-amylase,

amyloglucosidase and protease2. Addition of 95% Ethanol to precipitate all the fiber then is filtered, collected, dried,

and weighed3. Determine protein and ash content

Fiber = Residue Weight – Weight of (Protein + Ash)

Chemical Methods

Englyst-Cummings ProcedureSteps1. The defatted sample is heated in water to gelatinize the starch2. Addition of enzymes to digest the starch and proteins3. Addition of Pure Ethanol to precipitate the fiber4. Separation by centrifugation then washed and dried5. The fiber is hydrolyzed using Sulfuric Acid to breakdown into constituent

monosaccharides• The mass of fiber is assumed to be equal to the total mass of

monosaccharides