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7/29/2019 cell wall analysist
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Cell wall analysis
Dr Gbola Adesogan
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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)
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Van Soests 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)
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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(Fisher et al., 95)
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Plant anatomy vs. chemical fractions
(Minson, 1990)
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Van Soest (1991)
The NDF and ADF procedures described in the
original publications (including Agricultural
Handbook 379) are obsolete and of historical interest
only.
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What does NDF include
Desired contents
Structural cell wall componentscellulose, hemicellulose,
lignin,
Pectins (ignored)
Undesired contents
Silica, sand
Fiber-bound proteins
Starch
Lipids
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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
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NDF procedural differences
(Van Soest 91)
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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 cant use in sequential analysesleading to lignin determination / for in vitro digy.
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Pre / post treatments
Ash
Express results on OM (ash free) basis to
eliminate soil contamination
Decalin
Originally included to prevent foaming
Increases fiber yield
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Starch contamination
Can overestimate NDF
Starch removal simplifies filtration
Amylase treatment
Which amylase ? NotBacillus subtilis
May contain undesired enzymes - hemicellulase, 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
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Effect of amylase source on NDF
(Van Soest, 1991)
ND- S2 =Bacillus subtilis; ND-T3 heat stable bacteria
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Limitations of NDF method
Not nutrient-based
Doesnt give chemically/anatomically pure fractions
Fiber itself is not chemically, physically or nutritionally
uniform
Doesnt differentiate between polysaccharides sufficiently
Ignores pectin & glucans
Pectins uniquefiber which is
Not digested by mammalian enzymes Rapidly fermented by rumen microbes
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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
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Factors affecting ADF
Acid strength
Boiling time
Contaminants
(McLeod & Minson 72)
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ADF contaminantsSulphuric acid removes most of the digestible fiber
CTAB removes some protein
leaves fiber-bound protein
ADF residue contains pectin & hemicellulose except ifdetermined by sequence after NDF extraction
Express on OM basis to eliminate AIA
Determine ADIN to account for indigestible N
Dont use asbestos packed crucibles for filtering
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MADF
UK replacement for ADF
Longer boiling time,
Stronger acid
Gave a more accurate digy. prediction than ADFShouldnt be used to assay ADIN / heat damaged
protein since
MADF sample must be oven dried
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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 foroperation
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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 plantsstructural
In ruminantsdecreases energy density & digestibility
(McDonald et al., 95)
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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 withProtease pretreatment
ADF pretreatment
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Lignin methods
ADF lignin
ADF pretreatment followed by sulfuric acid orpermanganate solution
Dilute acid (1M H2SO4) at 100oC
Followed by Conc acid (12 M H2SO4 at 25o
C) 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
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Lignin methods
Gravimetric methods
Lignin is left as the residue after the digest
ADL underestimates lignin due to ligninsolubilization in the acid
Difference methods
Lignin is solubilized / oxidized and determined bydifference
Can use chlorite, permanganate etc.
Absorbance method
Lignin is solubilized (e.g. with acetyl bromide) and
then determined spectophotometrically
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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.
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Lignin methods
Pyrolysis mass spectroscopy
Pyrolysis thermal degradation of sample in an inertatmosphere or a vacuum.
mass spectrometer used to separate the components of
the pyrolysate on the basis of their mass-to-charge ratioCalorimetry
Based on compairing the actual gross energy of thesample to a calculated GE based on energy values ofthe 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)
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% 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)
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Lignin analysis methods
(Cherney, 2000)
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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 (Giger1985)
Better digestibility predictions from lignin expressed on afiber basis
Lignin/NDF
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References
Varel VH, Weimer PJ, et al. Accuracy of Klason lignin and acid detergent ligninmethods as assessed by bomb calorimetry J AGR FOOD CHEM 47 (5): 2005-2008MAY 1999
Abrams SM Sources of error in predicting digestible dry-matter from the acid-detergentfiber 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 Evaluationin 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 andklason lignin with digestibility of forage dry matter and neutral detergent fiber. J DairySci. 80: 1622-1628
http://www.cabi-publishing.org/Bookshop/Readingroom/browseA-Z.asp
http://tame.mimas.ac.uk/isicgi/CIW.cgi?PFq64oJYy4MAAFj1OXw_8D5E631F_PFq64oJYy4MAAFj1OXw-0&Func=Abstract&doc=6/1http://tame.mimas.ac.uk/isicgi/CIW.cgi?PFq64oJYy4MAAFj1OXw_8D5E631F_PFq64oJYy4MAAFj1OXw-0&Func=Abstract&doc=6/1http://tame.mimas.ac.uk/isicgi/CIW.cgi?PFq64oJYy4MAAFj1OXw_8D5E631F_PFq64oJYy4MAAFj1OXw-0&Func=Abstract&doc=8/1http://tame.mimas.ac.uk/isicgi/CIW.cgi?PFq64oJYy4MAAFj1OXw_8D5E631F_PFq64oJYy4MAAFj1OXw-0&Func=Abstract&doc=8/1http://www.cabi-publishing.org/Bookshop/Readingroom/0851993443/3443ch14.pdfhttp://www.cabi-publishing.org/Bookshop/Readingroom/0851993443/3443ch14.pdfhttp://www.cabi-publishing.org/Bookshop/Readingroom/0851993443/3443ch14.pdfhttp://www.cabi-publishing.org/Bookshop/Readingroom/0851993443/3443ch14.pdfhttp://www.cabi-publishing.org/Bookshop/Readingroom/0851993443/3443ch14.pdfhttp://tame.mimas.ac.uk/isicgi/CIW.cgi?PFq64oJYy4MAAFj1OXw_8D5E631F_PFq64oJYy4MAAFj1OXw-0&Func=Abstract&doc=8/1http://tame.mimas.ac.uk/isicgi/CIW.cgi?PFq64oJYy4MAAFj1OXw_8D5E631F_PFq64oJYy4MAAFj1OXw-0&Func=Abstract&doc=8/1http://tame.mimas.ac.uk/isicgi/CIW.cgi?PFq64oJYy4MAAFj1OXw_8D5E631F_PFq64oJYy4MAAFj1OXw-0&Func=Abstract&doc=8/1http://tame.mimas.ac.uk/isicgi/CIW.cgi?PFq64oJYy4MAAFj1OXw_8D5E631F_PFq64oJYy4MAAFj1OXw-0&Func=Abstract&doc=8/1http://tame.mimas.ac.uk/isicgi/CIW.cgi?PFq64oJYy4MAAFj1OXw_8D5E631F_PFq64oJYy4MAAFj1OXw-0&Func=Abstract&doc=8/1http://tame.mimas.ac.uk/isicgi/CIW.cgi?PFq64oJYy4MAAFj1OXw_8D5E631F_PFq64oJYy4MAAFj1OXw-0&Func=Abstract&doc=8/1http://tame.mimas.ac.uk/isicgi/CIW.cgi?PFq64oJYy4MAAFj1OXw_8D5E631F_PFq64oJYy4MAAFj1OXw-0&Func=Abstract&doc=6/1http://tame.mimas.ac.uk/isicgi/CIW.cgi?PFq64oJYy4MAAFj1OXw_8D5E631F_PFq64oJYy4MAAFj1OXw-0&Func=Abstract&doc=6/1