cell wall analysist

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

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