Non-structural carbohydrate

(NSC) analysis

Dr Gbola Adesogan

Department of Animal Sciences

University of Florida

Nutritional CHO utilization concepts

Plant Cell StructureCell contents

Organic acids

Mono- &

Oligosaccharides

Starch

Fructans

Middle lamella

Pectic substances

-glucans

Cell wall

Hemicellulose

Cellulose

Lignin

(Hall, 2001)

CHO analysis

According to Prox. Analysis

Total CHO = CF + NFE

• Where NFE = (100-(CP+CF+Fat+Ash+Moisture))

• Problematic & subject to ‘estimated by difference’

errors

According to the Dairy NRC (2001)

Total CHO = NDF + Non-fiber CHO (NFC)

• Where NFC = (100 – (NDF+CP+Fat+Ash))

• Lumps together contrasting CHO

• Subject to ‘estimation by difference’ errors

Would all these carbs digest at the same

rate and have the same effect ???

Fermentation rates of NFC fractions

Sugars (80-350%/h)

organic acids

(0-5%/h)

Starch (3-30%/h)

Time

Pectin (20-40%/h, except soyhulls @ 4%/h)

(Hall, 2001)

NSC components are broken down

in different ways

(Hall, 2001)

Fermentation profiles of feeds

0

1

2

3

4

5

6

7

8

1 13 25 37 49 61 73 85 97

Time, h

Psi

Corn

Citrus pulp

hay

Krueger & Adesogan, 2004

Gas p

roduction,

psi

NSC concentrations of some feeds(Hall, 2001)

Challenges to the Dairy NRC (2001)

Different CHO should not be lumped together

as ‘NFC’ Since they ferment at different rates

and have different fates and effects on the

animal

Deduction

There is a need for better definition of dietary non-fiber carbohydrates than NFC

We need to be able to fractionate NFC into its components and measure each of them to improve animal nutrition

CHO classification & fractionation

CHO Classification scheme

Non fiber CHO (NFC)

– Sugars

– Starch

– Glycogen

– Pectins (soluble fiber)

– Fructans

Structural CHO (fiber)

– Cellulose

– Hemicellulose

– Lignin (Not really CHO)

Rather than just considering NDF and NFC as the CHO

fractions (NRC, 2001), they should be broken down as

follows:

Note: The term NFC is a misnomer for a fraction that

contains pectin. Hence, some prefer to use the term

non-structural CHO (NSC) instead of NFC.

Using ethanol to fractionate CHO

*Hall, 2002

80% ethanolLow Molecular Weight CHO

(monosacc & disacc)

Polysaccharides

(High Molecular weight CHO

e.g. pectin, fructan, starch, cellulose)

University of Florida

Suggested NSC Fractionation

SchemeSample

NDF

ND reagent

NFC (Starch, monosacc, oligosacc, pectin, fructans)

Starch

HS amylase

Monosacc, oligosacc,

pectin, fructans

Monosacc, oligosacc,organic acids

80% ethanol

pectin, fructans

High mol. wt

NSC

Low mol.

NSC wt

Fractionating CHO with ethanol

(Hall, 2001)

Pectin

analysis

Alternatives to ethanol for partitioning

NSC

Water – Extracts monosacc, oligo sacc, organic acids and

these are collectively called sugars

– However

• Hot water solubilizes starch & some dextrins

Acetate/phosphate buffer– Also extracts sugars

– However

• Extracts some polysaccharides

• Their respective pH modifies what they extract e.g. low pH depolymerizes pectin

Plant Carbohydrates

Cell

Contents

Cell

Wall

HemicellulosesPectic

Substances

-glucans

FructansStarchesMono+Oligo-

saccharides

Organic

Acids

Cellulose

ADF

NDSF

NDSC

Galactans

Non-Starch Polysaccharides

NDF

NFC

Dextrins

(Hall, 2001)

Methods for analyzing CHO

Low Molecular Wt CHO analysis

Includes reducing sugars i.e. mono & oligosacc that are extracted with 80% ethanol or water

– (organic acids not really CHO)

Methods include

– Reducing sugar analysis

– Condensation reactions

– Enzymatic reactions

– Chromatographic techniques

Simpler, faster

•Costlier,

•handles more samples

•No stds for oligosacc.

Reducing sugar assays

Principle– Carbonyl group reduces alkaline solutions of metallic

salts

Issues– Must hydrolyze all CHO into monosacc completely

e.g. unhydrolyzed sucrose undetected.

Interference– Protein

– Natural reducing agents e.g. in molasses

– Alcohol

Enzymatic assays

Principle

– Sample glucose is detected with a specific enzyme e.g.

glucose oxidase - peroxidase

1. Glucose oxidase allows glucose oxidation producing H2O2

• β,D glucose + O2 →GO → D-gluconic acid + H2O2

2. Peroxidase allows H2O2 to react with and change a

chromogen (e.g. 4-aminoantipyrine (4AA)) from a reduced,

colorless form to a colored, oxidized form.

• H2O2 + 4AAH2 → P → 2 H2O + 4AA

3. The color depth is proportional to the sample glucose conc.

Issues

– Only detects glucose; can’t be used for other monosacc.

Condensation assays

Principle– Phenolic acid + CHO + strong acid = chromogen

– Chromogen quantified

Issues– Sample hydrolysis is not necessary

– CHO quantified depends on the partitioning solvent, phenolic acid, solution temp & acid strength

Interference– Cellulolisic lint

Examples– Phenol sulfuric assay

– Anthrone assay

High Molecular Wt NSC analysis

Includes

1. Starch

• Methods include

– Polarimetry (not used for forages)

(http://www.rudolphresearch.com/polarimetry.htm)

– Enzymatic assays

2. Soluble fiber (pectin or pectic substances)

Polarimetric starch analysis

Sensitive, nondestructive technique.

Principle

– Based on measuring the optical activity exhibited

by starch in the compound

Starch is an optically active substance with its

own specific rotation

Optically active compounds rotate linearly

polarized light as it passes through them.

Widely used by the pharmaceutical industry.

Enzymatic starch analysis

Principle

– Specific enzymes hydrolyze starch to glucose

– Glucose determined colorimetrically

Steps

1. Gelatinization

2. Enzymatic hydrolysis

3. Quantification of end products

Crystalline

Non-

crystalline

Gelatinization

Starch analysis contd.

1. Gelatinization

– Achieved by boiling (@ 90-100oC) in H2O

– Destroys crystalline structure by dissolving H bonds,

facilitating subsequent degradation

2. Enzymatic hydrolysis

Must use enzymes that target only (1 4) & (1 6)

E.g. heat stable amylase and amyloglucosidase for

respective linkages

Optimal pH for amylase = 7; for amyloglucosidase =

4.5-5; buffers are used to achieve these values

3. End product quantification

End product = glucose (or other reducing sugars if

other enzymes are used)

Soluble fiber

Includes, non starch, non-NDF

polysaccharides e.g. pectin, fructans & gums

Soluble in what?????????????????

– NDF (NRC 2001)

– Phosphate buffer (AOAC)

Phosphate soluble fiber (AOAC

method)

Principle/ Steps– Solubilize CHO in phosphate,

– hydrolyze with amyloglucosidase

– Filter off insoluble residue

– Precipitate soluble fiber with 78% ethanol

Issues– Ethanol may incompletely precipitate soluble fiber

and also precipitate other compounds

– Phosphate depolymerizes some pectin

– Does not analyze individual NDSF like pectins or gums

Neutral detergent soluble fiber (NRC, 2001)

Principle /steps

– Extract sample with 80% ethanol, filter & weigh

the residue (contains starch, soluble fiber and

NDF)

– Determine NDF and starch separately and

substract these from the weight of the residue

Issues

– Subject to ‘estimated by difference’ errors

– Does not analyze individual NDSF like pectins or

gums

References

Hall, M. B. 2003, Challenges with nonfiber

carbohydrate methods. J. Anim. Sci. Vol. 81.

2003.