Non-structural carbohydrate
(NSC) analysis
Dr Gbola Adesogan
Department of Animal Sciences
University of Florida
Nutritional CHO utilization concepts
Plant Cell Structure
Cell 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)
Pectin (20-40%/h, except soyhulls @ 4%/h)
Starch (3-30%/h)
organic acids
(0-5%/h)
Time
(Hall, 2001)
NSC components are broken down
in different ways
(Hall, 2001)
Fermentation profiles of feeds
8
Corn
6
Citrus pulp
5
Psi
Gas production, psi
7
4
3
hay
2
1
0
1
13
25
37
49
61
73
85
97
Time, h
Krueger & Adesogan, 2004
(Hall, 2001)
NSC concentrations of some feeds
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
Rather than just considering NDF and NFC as the CHO
fractions (NRC, 2001), they should be broken down as
follows:
Non fiber CHO (NFC)
– Sugars
– Starch
– Glycogen
– Pectins (soluble fiber)
– Fructans
Structural CHO (fiber)
– Cellulose
– Hemicellulose
– Lignin (Not really CHO)
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
80% ethanol
Low Molecular Weight CHO
(monosacc & disacc)
Polysaccharides
(High Molecular weight CHO
e.g. pectin, fructan, starch, cellulose)
University of Florida
*Hall, 2002
Suggested NSC Fractionation
Scheme
Sample
ND reagent
NDF
NFC (Starch, monosacc, oligosacc, pectin, fructans)
HS amylase
Monosacc, oligosacc,
pectin, fructans
Starch
80% ethanol
pectin, fructans
High mol. wt
NSC
Monosacc, oligosacc,
organic acids
Low mol.
NSC wt
Fractionating CHO with ethanol
Pectin
analysis
(Hall, 2001)
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
Organic Mono+Oligo- Starches Fructans Pectic
Hemicelluloses Cellulose
Acids saccharides Dextrins
Substances
Galactans
ADF
-glucans
NDSF
NDF
Non-Starch Polysaccharides
NDSC
NFC
(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
Simpler, faster
•Costlier,
– Chromatographic techniques •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
Gelatinization
Noncrystalline
Crystalline
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.