5. cell wall analysis
TRANSCRIPT
Cell wall analysis
Dr Gbola Adesogan
Van Soests’ Scheme
Based on nutritional availability of forage components
– I.e. ‘nutritive entities’ that had the same true digy
in all feeds & forages.
Devd for forages, reputed to be variable & difficult to
use with other feeds (Mertens, 2001)
Van Soest’s scheme
Lignin
CTAB + H2SO4
H2SO4/ KMnO4
NFC
Sample
Starches
Pectin
WSCs
B-glucans
NDF
ADF
ADIN
EDTA & Na lauryl sulphate
Kjeldahl
Hemicellulose
AIAAshing
Sodium lauryl + ethylenediaminetetraacetic acid (EDTA)
Cetyltrimethylammonium (CTAB)
Prox. analysis Chemical component Van Soest Analysis
CP Protein
NPN
EE Lipids
Pigments NDS
Sugars
Organic acids
NFE Pectin
Hemicellulose
Alkali soluble ligninLignin
Alkali insoluble lignin NDF
CF Fiber bound N ADF
Cellulose
Detergent insoluble minerals
Ash Detergent soluble minerals (Fisher et al., ’95)
Plant anatomy vs. chemical fractions
(Minson, 1990)
Van Soest (1991)
The NDF and ADF procedures described in the
original publications (including Agricultural
Handbook 379) are obsolete and of historical interest
only.
What does NDF include
Desired contents
– Structural cell wall components – cellulose, hemicellulose,
lignin,
– Pectins (ignored)
Undesired contents
– Silica, sand
– Fiber-bound proteins
– Starch
– Lipids
Factors affecting NDF values
Processing
– Subsampling
– Sample quantity
– Grinding
– Drying
Method
–Reagent quantity
–Boiling duration
–Reflux time & temperature
–Filtration method and vessel
–Soaking
–Weighing method
NDF procedural differences
(Van Soest ’91)
Pre or post treatments
Lipid removal
– >10% of lipid can be problematic
– Acetone dissolves fats
– Ethanol also used to dissolve lipids
Protein removal
– For high protein (>30%) feeds
– Proteases
– Sulfite
• Originally included to NDF bound nitrogen e.g. keratin
• Still useful for high protein forages
• Solubilizes lignin – can’t use in sequential analyses leading to lignin determination / for in vitro digy.
Pre / post treatments
Ash
– Express results on OM (‘ash free’) basis to
eliminate soil contamination
Decalin
– Originally included to prevent foaming
– Increases fiber yield
Starch contamination
Can overestimate NDF
Starch removal simplifies filtration
Amylase treatment
• Which amylase ? Not Bacillus subtilis
• May contain undesired enzymes - hemicellulase, ß glucanase & protease activity
• High temperature treatments reduce undesired ‘activities’
• Enzyme activity variations
For concs, combine amylase pretreatment with
– 2 Ethoxyethanol effective; but is a health risk,
• Replace with triethylene glycol
– Urea treatment
Effect of amylase source on NDF
(Van Soest, 1991)
ND- S2 =Bacillus subtilis; ND-T3 heat stable bacteria
Limitations of NDF method
Not nutrient-based
– Doesn’t give chemically/anatomically pure fractions
• Fiber itself is not chemically, physically or nutritionally
uniform
– Doesn’t differentiate between polysaccharides sufficiently
Ignores pectin & ß glucans
– Pectins unique – fiber which is
• Not digested by mammalian enzymes
• Rapidly fermented by rumen microbes
ADF
Though often used to predict digestibilty
Van Soest claims
– No valid theoretical basis to link ADF to digy.
– ADF is a preparative residue for isolating:
• Cellulose
• Lignin
• Maillard products
• Silica
• AIA
• ADIN
Desirables
Undesirables
Factors affecting ADF
Acid strength
Boiling time
Contaminants
(McLeod & Minson ’72)
ADF contaminantsSulphuric acid removes most of the digestible fiber
CTAB removes some protein
– leaves fiber-bound protein
ADF residue contains pectin & hemicellulose except if determined by sequence after NDF extraction
Express on OM basis to eliminate AIA
Determine ADIN to account for indigestible N
Don’t use asbestos –packed crucibles for filtering
MADF
UK replacement for ADF
– Longer boiling time,
– Stronger acid
Gave a more accurate digy. prediction than ADF
Shouldn’t be used to assay ADIN / heat damaged
protein since
– MADF sample must be oven dried
ANKOM NDF
Ideal for ‘difficult to filter’ samples
– E.g. Silage or soil contaminated samples
Precise
– Eliminates most elements of Technician variability.
– Consistent with conventional and alternative method results.
Efficient
– Reduces labor
– Processes up to 24 samples at a time.
Safety
– Eliminates handling of Hot chemicals.
Space Saver
– Instrument requires little space for operation
Lignin
What is lignin
– Non- CHO substance that resists digestion
– not a well defined, individual compound
– Complex, cross-linked polymer containing phenylpranoid units derived from
• Coumaryl alcohol
• Coniferyl alcohol
• Sinapyl alcolhol
Functions
– In plants –structural
– In ruminants – decreases energy density & digestibility
(McDonald et al., ’95)
Methods of lignin analysis
Most based on lignin insolubility in 72% H2SO4
– (I.e Klason lignin)
Overestimates lignin due to co-precipitation of
– Protein
– Malliard products
– Cutin
– Tannins
Protein contamination can be reduced with
– Protease pretreatment
– ADF pretreatment
Lignin methods
ADF lignin
– ADF pretreatment followed by sulfuric acid or permanganate solution
– Dilute acid (1M H2SO4) at 100oC
– Followed by Conc acid (12 M H2SO4 at 25oC)
– Residue is lignin
Klason lignin
– Pretreatment with ethanol, amylase & amyloglucosidase
– Acid hydrolysis conc (12M H2SO4) at 39oC followed by
– (0.4M H2SO4)
– Residue is lignin
Lignin methods
Gravimetric methods
– Lignin is left as the residue after the digest
– ADL underestimates lignin due to lignin solubilization in the acid
Difference methods
– Lignin is solubilized / oxidized and determined by difference
– Can use chlorite, permanganate etc.
Absorbance method
– Lignin is solubilized (e.g. with acetyl bromide) and then determined spectophotometrically
Lignin methods
Saponification method
– Lignin is determined by cleavage of the
ester linkages in lignin
Ball milling
– Pulverized sample amongst ball bearings for long
periods of time. A portion of the lignin can then be
extracted with certain solvents.
Lignin methods
Pyrolysis mass spectroscopy
– Pyrolysis thermal degradation of sample in an inert atmosphere or a vacuum.
– mass spectrometer used to separate the components of the pyrolysate on the basis of their mass-to-charge ratio
Calorimetry
– Based on compairing the actual gross energy of the sample to a calculated GE based on energy values of the chemical components in the samples
– Calculated GE = (Protein x 5700kcal/kg) + (Carbohydrate x 4000 kcal/kg) + (lipid x 9500 kcal/kg) + (lignin x 8000 kcal/kg)
% GE recovered
Sample Actual GE
Kcal/kg
Calculated
GE from ADL
Calculated
GE from
Klason lignin
Alfalfa 4493 80.8 97.1
Clover 4463 76.2 87.6
Corn silage 4497 78.4 93.7
Oat straw 4182 73.8 96.7
(Jung & Vagel, 1996)
Lignin analysis methods
(Cherney, 2000)
Problems with lignin assays
No standardized method
Reasons
– Complex unknown structure
– No standardized method
– Variation in lignin content with forages
• Leading to poor predictions of digy.
No current method accurately measures pure lignin (Giger 1985)
Better digestibility predictions from lignin expressed on a fiber basis
– Lignin/NDF
References
Varel VH, Weimer PJ, et al. Accuracy of Klason lignin and acid detergent lignin methods as assessed by bomb calorimetry J AGR FOOD CHEM 47 (5): 2005-2008 MAY 1999
Abrams SM Sources of error in predicting digestible dry-matter from the acid-detergent fiber content of forages ANIM FEED SCI TECH 21 (2-4): 205-208 OCT 1988
D.J.R. Cherney Characterization of Forages by Chemical Analysis. Forage Evaluation in ruminant Nutrition. Eds Givens, Owens & Ohmed. CABI
Van Soest, P.J., Robertson, J.D. and Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber and non-starch polysaccharide in relation to animal nutrition. J. Dairy Sci. 74, 3583-3597.
Jung HJG, Mertens, D R and Payne, A J. 1997. Correlation of acid detergent lignin and klason lignin with digestibility of forage dry matter and neutral detergent fiber. J Dairy Sci. 80: 1622-1628
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