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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