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Ilyana A. Causing ENFT-3A


• 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 cancer
2. Coronary diseases
3. Diabetes
4. Constipation
Why Determine the type and concentration of Carbohydrates?

Standards of Identity
Nutritional Labelling
Detection of Adulteration
Food Quality
Food 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

• Aliphatic aldehydes or ketones

• Most natural carbohydrates are
 Hexoses – Glucose, Fructose, Galactose
 Pentoses – Arabinose, Xylose
• There reactive centers
 Carbonyl Group
 Hydroxyl Group

• Covanlently bonded through glycosidic linkages

• Common in food contains monomers of
• Glucose
• Fructose
• Galactose
• Either be Disaccharides or Trisaccharides

• Majority of carbohydrates found in nature

• Same monosaccharides
• Starch, Cellulose, Glycogen – Glucose
• Contain different type of monomers
• Pectin, Hemicellulos, Gums
Method of Analysis

• Carbohydrate content can be measured after all other components are

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

Prior to Analysis: Sample Preparation

• 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 type
2. Food Matrix Type
3. Purpose of the Analysis
Example: Sample Preparation

To avoid thermal degradation
(under vacuum)

Ground to a fine powder To enhance solvent extraction

(by 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 weighed
• 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
• Alcohol can be removed by evaporation under vacuum
Methods of Analysis

1. Chromatographic and Electrophoretic methods

2. Chemical methods
3. Enzymatic methods
4. Physical methods
5. Immunoassays
Chromatographic Methods

• Most powerful analytical techniques

 Analysis of Type and Concentration of Mono and Oligo
• Commonly used to separate and identify carb
1. Thin Layer Chromatography, TLC
2. Gas Chromatography, GC
3. High Performance Liquid Chromatography, HPLC
• Carbohydrates are separated base of their differential absorption
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
• 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 methods
1. Allow the reaction to complete and measure the concentration of the product
Concentration of the product – Concentration of the Initial Substrate
1. 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 Used
1. Polarimetry
2. Refractive Index
3. Infrared
4. Density

• Specific for low molecular weight carbohydrates

• Developed by
1. Attaching the carbohydrate to a protein
2. Injecting it into an animal
3. Animal develops antibodies specific for the carbohydrate molecule
4. Antibodies are extracted and used for determining the concentration of the
• 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 Monosaccharides
2. Physiochemical Characteristics
– Water Solubility, Viscosity, Surface Activity
3. 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

Prior to Analysis: Sample Preparation

In Natural foods
• Starch granules is separated by drying, grinding, steeping in water, filtration and
How this works
Starch granules will move to the bottom because its water-insoluble and have high
In Processed foods
• The sample is dried, ground and dispersed in hot 80% Ethanol solution then filtered
or centrifuged
How this works
Mono&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
Analysis Methods: Starch

1. Addition of Enzymes
– To breakdown the starch to glucose
– Glucose concentration is analyzed and used to determine the starch concentration
2. 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 Components
1. Cellulose
2. Hemicellulose
3. Pectin
4. Hydrocolloids
5. 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 gels
Non Cell Wall Polysaccharides
– Include Hyrdocolloids (Guar Gum, Locust Bean Gum, Gum Arabic, Agar, Alginates, Caragenans)
– Used as Gelling agents, Stabilizers and Thickeners
– Non-carbohydrate polymer
– Usually associated with Cellulose and Hemicellulose
Dietary Fibers

Prior to Analysis: Sample Preparation

1. Lipid Removal
2. Protein Removal
3. Starch Removal
4. Selective Precipitation of Fibers
5. Fiber Analysis
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
– 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
1. A Gelatinized sample (dried and defatted) is digested with α-amylase, amyloglucosidase and
2. Addition of 95% Ethanol to precipitate all the fiber then is filtered, collected, dried, and weighed
3. Determine protein and ash content

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

Chemical Methods

Englyst-Cummings Procedure
1. The defatted sample is heated in water to gelatinize the starch
2. Addition of enzymes to digest the starch and proteins
3. Addition of Pure Ethanol to precipitate the fiber
4. Separation by centrifugation then washed and dried
5. 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