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16 Permanganate Lignin, Cellulose and Silica (Van Soest and Wine 1968)

16 Permanganate Lignin, Cellulose and Silica (Van Soest and Wine 1968)

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Nutritional Evaluation of Forages

Dissolve KMno4 and Ag2SO4 in distilled water. Keep out of direct sunlight. Add

silver sulphate to dehalogenate the reagent.

1. Lignin buffer solution: 1 L

Ferric nitrate, monohydrate: 6.0 g

Silver nitrate: 0.15 g

Acetic acid, glacial: 500 mL

Potassium acetate: 5 g

Tertiary butyl alcohol: 400 mL

Distilled water: 100 mL

Dissolve ferric nitrate nonhydrate (Fe(NO3)3.9H2O and silver nitrate in distilled

water. Combine with acetic acid and potassium acetate. Add tertiary butyl

alcohol and mix.

2. Combined permanganate solution:

Combine and mix saturated potassium permanganate and lignin buffer solution

in the ratio of 2:1 by volume, before use. Unused mixed solution that may be

kept about a week in a refrigerator in the absence of light solution is usable if

purple and containing no precipitate. Old solutions that assume a reddish colour

should be discarded.

3. Demineralizing solution: 1 L

Oxalic acid dehydrate: 50 g

95% Ethanol: 700 mL

Concentrate HCl (12 N): 50 mL

Distilled water: 250 mL

Dissolve oxalic acid dehydrate in 95% ethanol. Add concentrate HCl and

distilled water and mix.

4. Ethanol 80%: 1 L

95% Ethanol: 845 mL

Distilled water: 155 mL

5. Hydrobromic acid, reagent grade


1. Dry sample at less than 65 C and grind through 20–30 mesh (1 mm) serene.

Prepare ADF according to standard procedure.

2. Use a 1.0 g sample; except on samples containing a high amount of lignin (15%

or more) use 0.5 g sample.

3. Place previously weighed crucibles in a shallow enamel pan containing cold

water to a depth of about 1 cm. Fibre in crucible should not be wet.

4. Add about 25 mL of combined saturated potassium permanganate and lignin

buffer solution (2:1 by volume) to the crucible in the enamel pan containing cold

water. Adjust level (2–3 cm) of water in pan to reduce flow of solution out of

crucible. Place a short glass rod in each crucible to stir contents, to break lumps

and to draw permanganate solution up on sides of crucible to wet all particles.


Permanganate Lignin, Cellulose and Silica


5. Allow crucible to stand at 20–25 C for 90 Ỉ 10 min; add more mixed permanganate solution if necessary. Purple colour must be present at all times.

6. Remove crucible to filtering apparatus. Suck dry. Do not wash. Place in a clean

enamel pan and fill crucible no more than half full with demineralizing

solution. Demineralizing solution may be added directly to crucible in case

filtering is difficult. Care must be taken to avoid spillage by foaming. After

5 min, suck dry on filter and refill half full with demineralizing solution. Repeat

after second interval if solution is very brown. Rinse sides of crucible with

solution from a wash bottle with a fine stream. Treat until fibre is white. Total

time required 20–30 min.

7. Fill and thoroughly wash crucible and contents with 80% ethanol. Suck dry and

repeat two times. Wash twice in similar manner with acetone. Suck dry.

8. Dry at 100 C overnight and weigh. Calculate lignin content as loss in weight

from acid-detergent fibre.

9. Ash at 500 C for 3 h, coal and weigh calculate residual ash as the difference

between this weight and original tare of crucible. Calculate cellulose by weight

loss upon ashing.

10. A presumptive analysis for silica may be obtained by hydrobromic acid treatment of the ashed permanganate lignin of ADF residue. This determination has

its greatest value when the residual ash is greater than 2%. Ash and weigh and

then add enough drops of 48% HBr (not more than 4 mL) to moisten all

particles. Allow to stand 1–2 h. Add more drops of HBr if much red colour

forms. Suck off excess acid on vacuum and wash once with acetone. Use no

water. Dry and ash briefly at 500 C, cool and weigh. Report silica as the

difference between this weight and the original tare.


Empty wt. of crucible ¼ ——— g

Wt. of dried sample ¼ ——— g

Wt. of acid-detergent fibre ¼ ——— g

Wt. of permanganate fibre residue ¼ ——— g

Wt. of crucible and ash ¼ ——— g

Wt. of crucible + ash, after hydrogen bromide washing ¼ ——— g


1. Lignin percent on DM basis

wt. of acid detergent fibre À wt. of permanganate fibre residue



wt. of oven dried sample taken for ADF analysis

2. Cellulose percent on DM basis

wt. of crucible and permanganate fibre À wt. of crucible and ash



wt. of oven dried sample taken for ADF analysis

3. Silica percent on DM basis

wt. of crucible ỵ ash after HBr washingị Empty wt. of crucible


wt. of oven dried sample taken for ADF analysis




Nutritional Evaluation of Forages

Estimation of Non-Starch Polysaccharides

Non-structural carbohydrate (NSC) minus starch and sugars equals non-starch

polysaccharide (NSP). Starch and sugars can be measured directly. The net fraction

can reasonably be calculated by difference using one of two formulae:

1. NSC ẳ 100 NDF ỵ protein ỵ fat ỵ ashị

2. NSC ẳ 100 NDF NDF proteinị ỵ protein ỵ fat ỵ ash

The second equation deletes neutral-detergent insoluble protein, which is the

slowest to be degraded and should therefore be excluded. The NSPs do not generally

include native hemicelluloses and celluloses that are ordinarily a part of the lignified

cell wall matrix, which recovers hemicellulose and cellulose. So the determination

of organic matter, starch, protein, fat, NDF and lignin will help to estimate NSP

indirectly or by directly estimating different components of NSP in the animal

feedstuffs. It includes cellulose, hemicellulose, b-glucans, pentosans, pectins,

galactomannans and L-galactosides.


Determination of Starch


Powdered feed sample is treated with alcohol to solubilize free sugars, lipids, most

pigments and cuticular waxes. The residue rich in starch is solubilized with perchloric

acid and the extract is treated with anthrone–sulphuric acid to determine glucose.

Reagents and Chemicals

1. Anthrone–sulphuric acid: Add 0.2 g anthrone in 100 mL cold 95% H2SO4 stored

at 4 C (prepare fresh)

2. 80% ethyl alcohol–water : 95% ethyl alcohol ¼ 1.68 L (make final volume 2 L

with distilled water)

3. 52% perchloric acid: 270 mL 72% perchloric acid + 10 mL distilled water.

4. Standard glucose: Stock solution (1 mg/mL): 0.1 g glucose in 100 mL distilled

water, 0.1 g benzoic acid as preservative and working solution (10–100 mg);

dilute 2 mL stock to 100 mL with distilled water.



1. To 0.2 g sample add a few drops of 80% ethyl alcohol and 5 mL of distilled water.

2. Stir thoroughly and add 25 mL hot 80% ethyl alcohol.

3. Mix well and centrifuge, discard the supernatant and add 30 mL hot 80% ethyl


4. Mix well and then centrifuge and discard the supernatant.


Determination of Digestibility by In Vitro and In Sacco Techniques


Table 14.2 Protocol for starch estimation

Test tube no.


1 (blank)






Distilled water

Std. glucose

Conc. of glucose (mg)

Reagent 1

























5. Repeat washing twice more or until a test with anthrone is negative.

6. Collect all the washings in a volumetric flask for the determination of free sugars.

7. To the residue add 5 mL of distilled water; cool in ice water bath and then add

6.5 mL diluted perchloric acid reagent while stirring. Stir and keep for 20 min

with occasional stirring and then again add 20 mL of water.

8. Centrifuge and transfer aqueous solution to a 100 mL volumetric flask.

9. Repeat solubilization with perchloric acid reagent for 30 min at 0 C and

transfer the content to the volumetric flask and make final volume with water.

10. Filter the content and store the filtrate at 0 C.


1. Dilute 5–10 mL filtered starch solution to 250 mL with water (10–40 mg starch,

mL extract)

2. Take 2.5 mL diluted extract in a test tube.

3. Cool in water bath and add 5 mL anthrone reagent.

4. Mix thoroughly and heat in boiling water bath for 7.5 min.

5. Cool and read OD at 630 nm.

6. Arrange tubes in triplicate for standard as given below and draw the standard

curve (Table 14.2).


Starchmg%ị ẳ

Concentration of glucose mg=mLị dilution factor  100  0:9

g sample taken ðDM basisÞ Â 1; 000

where 0.9 ¼ conversion factor of glucose to starch.


Determination of Digestibility by In Vitro

and In Sacco Techniques

The in vitro digestibility of feeds for ruminants can be measured by fermenting them

with rumen liquor and then treating with pepsin. This is also known as two-stage

in vitro method. During the first stage, finely ground sample of feed is incubated for

48 h with buffered rumen liquor in a tube under anaerobic conditions. In the second



Nutritional Evaluation of Forages

stage, the microbial activities are stopped by acidifying with hydrochloric acid to pH

2.0 and then digested by incubating with pepsin for another 24 h. The insoluble

residue is filtered off, dried and ignited, and its organic matter is subtracted from the

feed to obtain digestible organic matter.


In Vitro Dry Matter Digestibility (Tilley and Terry 1963)







Conical flask/tube – 100 mL

Water bath

Cork with Bunsen valve


Carbon dioxide gas cylinder


1. Phosphate–carbonate buffer

Na2HCO3: 9.80 g

Na2HPO4.2H2O: 7.00 g

KCl: 0.57 g

NaCl: 0.47 g

MgSO4.2H2O: 0.12 g

CaCl2: 0.04 g

Mix the above chemical except CaCl2 in 800 mL distilled water in 1 L volumetric

flask, stir to dissolve and make the volume to 1 L. Just before use, add CaCl2, keep

at 39 C and pass CO2 through the solution.

2. 6 N HCl: Add 530.3 mL conc. HCl in 400 mL distilled water and make the

volume to 1 L after cooling the solution.

3. Pepsin powder (1:3,000).


1. Take 0.50 g finely ground (particle size < 1 mm) sample in 100-mL conical


2. Add 40 mL CO2 saturated phosphate–carbonate buffer and 10 mL strained

rumen liquor.

3. Pass CO2 through the contents for 10 s and put a stopper (cork fitted with Bunsen

valve) on the flask/tube immediately.

4. Incubate the flask/tube at 39 C with periodic shaking.

5. After 48 h of incubation, add 2 mL 6 N HCl and 0.1 g pepsin powder.

6. Incubate the tubes for another 24 h.

7. Filter the contents through filter paper (no. 54) or sintered crucible (G1).


Determination of Digestibility by In Vitro and In Sacco Techniques


8. Dry the residue at 100 C overnight and weigh.

9. Run parallel blank with phosphate–carbonate buffer and rumen liquor without

feed sample.


DM disappearance ¼ Wt. of sample À ðwt. of residue of test

À wt. of residue of blankị

DM digestibility %ị ẳ

DM disappearance


Wt. of sample (DM basis)


1. Collection of rumen liquor

(a) Feed normal diet twice daily for about 7 days to the donor animal.

(b) Draw homogenous rumen liquor samples from different parts of rumen by

suction using plastic tube about 30 mm in diameter with several 9 mm holes

in the lower 2 in. portion.

(c) Filter rumen liquor through four layers of cheese cloth.

(d) Transport rumen liquor to the laboratory in an insulated jug with temperature

maintained at 39 C.

2. Rumen liquor should be collected 3 h of post-feeding.

3. The animal should not be given water between 1 and 3 h post-feeding.


Modified Method of In Vitro Dry Matter Digestibility

This is the modification of the two-stage in vitro method developed by Tilley and

Terry (1963) and is completed in two stages

(a) Fermentation with rumen liquor and

(b) Extraction with neutral-detergent solution











Concial flask/tube – 100 mL

Water bath

Cork with Bunsen valve

Filter paper/crucible


Carbon dioxide gas

Spoutless beaker

Hot plate




Nutritional Evaluation of Forages






Mc Dougall’s buffer

Sodium hydroxide (1 N)

Casein hydrolysate

Reducing agent: 625 mg of cystine–hydrochloride dissolved in 95 mL distilled

water and 4 mL 1 N NaOH. Add 625 mg anhydrous sodium sulphite.

5. Resazurin solution (0.1%, w/v)

6. Neutral-detergent solution

Sodium lauryl sulphate: 30.00 g

Disodium ethylene diamine tetra acetate (EDTA) dihyrate: 18.61 g

Sodium borate decahydrate: 6.81 g

Disodium hydrogen phosphate: 4.56 g

2-Ethoxy-ethanol (Ethylene glycol): 10.00 mL

Make up volume 1 L with distilled water

Put EDTA and sodium borate decahydrate (Na2BO7.10H2O) in a beaker of 2 L

capacity. Add 400 mL distilled water, shake and heat until dissolved. Add sodium

lauryl sulphate and 2-ethyoxy-ethanol. Take disodium hydrogen phosphate

(Na2HPO4) in a separate beaker and add 400 mL distilled water and heat until

dissolved. Mix both the reagents and make the volume to 1 L. The pH of the solution

should be in between 6.9 and 7.1.


1. Take 0.5 g finely ground sample in 100-mL Erlenmeyer flask.

2. Prepare fermentation medium by adding in the order: 2.5 g casein hydrolysate

and 1.5 mL of resazurin solution in 1,000 mL of McDougall’s buffer. Keep the

medium on magnetic stirrer and bubble CO2 for 30–40 min. Add reducing agent

(50 mL) and pass CO2 gas till it becomes colourless.

3. Transfer 40 mL medium in each flask and add 10 mL rumen liquor.

4. Bubble CO2 for 5 min.

5. Seal the flasks with Bunsen valve and incubate at 39 C for 48 h with shaking.

6. Wash the flask with 100 mL NDS in 500 mL spoutless beaker to make total volume

to 150 mL. Reflux the sample for 1 h at 100 C and then filter on previously weighed

sintered glass crucible. Wash the sample with hot water to remove the detergent


7. Dry the crucible at 100 C for 24 h and record weight.

8. If samples are not to be processed immediately for NDS extraction, preserve

the contents of the flask by adding 1 mL toluene and store in refrigerator till



Wt. of crucible ¼ W

Wt. of sample on DM basis ¼ W1


Quantification of Tanins in Foliage


Wt. of residue ặ crucible ẳ W2

Wt. of residue left ¼ W2 À W ¼ W3

Net digestible dry matter ¼ W1 À W3 ¼ W4






Quantification of Tanins in Foliage

Tannins are generally defined as naturally occurring polyphenolic compounds of

high enough molecular weight to form complexes with proteins. These are classified into two groups based on their structural types: (a) hydrolysable tannins and

(b) condensed tannins. Methods for quantification of tannins may be based on the

chemical properties of tannins or their capability to bind substrates, particularly

proteins. The methods for the quantification of tannins could be described in three


1. Chemical methods

(a) Determination of total phenolics: The method is based on the fact that

phenolics are reducing agents. It may be noted that all tannins are phenolics,

but not all phenolics are tannins.

(b) Determination of total tannins: It is partly chemical, based on reducing

property of tannins and partly physical because tannins are measured as

the reduction in phenolics that occur when a binding agent (polyvinyl

polypyrrolidone, PVPP) is added to the extract.

(c) Determination of condensed tannins (proanthocyanidins): It is based on

oxidative depolymerization of condensed tannins in butanol–HCl reagent.

The presence of iron is considered to increase the reproducibility and

sensitivity of the assay.

(d) Determination of gallotannins: It is based on hydrolysis of gallotannins to

gallic acid under acidic conditions and measurement of the released gallic

acid by reaction with rhodanine or by HPLC.

2. Protein precipitation/binding methods

(a) Determination of protein-precipitable phenolics: It is based on the formation of tannin–protein complexes (tannins in the plant extract and the

protein, bovine serum albumin [BSA]). Tannins present in the complex are

determined using ferric chloride assay for total phenolics. Iron forms a

complex with phenols to give a pink chromatophore which is measured


(b) Filter paper–protein Ponceau S dye assay: The protein–tannin complexes

are formed on a sheet of filter paper, and the protein bound to the complex

is measured by dyeing it with Ponceau S dye. This dye is specific for proteins



Nutritional Evaluation of Forages

and does not bind tannins. The colour of the dye bound to proteins is eluted

and measured spectrophotometrically.

(c) Radial diffusion assay: In this method, tannin molecules migrate through

agarose gel which is impregnated with the protein, BSA. The tannin–protein

complex is formed in the gel which appears as a ring. The diameter of the

ring is a measure of protein precipitation/binding capacity of tannins.

3. Tannin bioassay

The samples under investigation (or tannins) are incubated in the absence and

presence of a tannin-complexing agent, polyethylene glycol (PEG), in the

in vitro rumen fermentation system containing rumen microbes. The affinity of

PEG for binding to tannins is very high; it even breaks the already formed

tannin–protein complexes and releases proteins from the complex. In the in vitro

rumen fermentation system, release of gas and production of microbial mass are

measured. The difference between these parameters observed in the absence and

presence of PEG is a measure of tannin activity in relation to their effects on

rumen fermentation.


Method for Extraction of Tannins

The aim is to quantitatively diffuse phenolics present in the plant material to liquid

phase. For the extraction process, a suitable solvent is required. Generally, aqueous

methanol (50%) and aqueous acetone (70%) are common choices. The latter

has been reported by various workers to be better in extracting phenolics from

tree leaves. One can try both these solvents for extraction, and then based on the

efficiency of extraction of phenolics (using Folin–Ciocalteu method) and/or condensed tannins (using butanol-HCl method), one can decide the solvent to be used

for a particular plant material.

Dried (finely ground) plant material (200 mg) is taken in a glass beaker of

approximately 25 mL capacity. Ten millilitres of aqueous acetone (70%) is added

and the beaker is suspended in an ultrasonic water bath and subjected to ultrasonic

treatment for 20 min at room temperature. The contents of the beaker are then

transferred to centrifuge tubes and subjected to centrifugation for 10 min at

approximately 3,000 Â g at 4 C (if refrigerated centrifuge is not available, cool

the contents by keeping the centrifuge tube on ice and then centrifuge at 3,000 Â g

using an ordinary centrifuge). Collect the supernatant and keep it on ice. Transfer

the pellet left in the centrifuge tube to the beaker using two portions of 5 mL each of

70% aqueous acetone and again subject the contents to ultrasonic treatment for

20 min centrifuge and collect supernatant as described above. Folin–Ciocalteu

method is used for determination of total phenols in the two supernatants.

• Very long extraction at too high a temperature may lead to degradation and

loss of phenolics.


Measurement of Total Phenolics and Tannins Using Folin–Ciocalteu Method


• Pigments and fat can be removed from the dried leaf sample by extracting with

diethyl ether containing 1% acetic acid before extracting tannins. Freshly

prepared extract should be used for tannin analysis.

• Tubes/container containing the extract should be kept on ice till the analysis

is complete.


Measurement of Total Phenolics and Tannins

Using Folin–Ciocalteu Method

The method for total phenol is useful in order to know the efficiency of extraction

of phenolics in solvents. This method can be coupled with the use of insoluble

matrix, PVPP (binds tannin-phenolics), for measurement of tannins. The results can

be expressed as tannic acid equivalent. The nature of tannic acid varies from one

commercial source to the other.


1. Folin–Ciocalteu reagent (1 N): Dilute commercially available Folin–Ciocalteu

reagent (2 N) with an equal volume of distilled water. Transfer it in a brown

bottle and store in a refrigerator (4 C). It should be golden colour. Do not use it

if it turns olive green.

2. Sodium carbonate (20%): Weigh 40 g sodium carbonate (Â10 H2O), dissolve

it in about 150 mL distilled water and make up to 200 mL with distilled water.

3. Insoluble polyvinyl pyrrolidone (PVPP)

4. Standard tannic acid solution (0.1 mg/mL): Dissolve 25 mg tannic acid (TA)

obtained from 25 mL distilled water and then dilute 1:10 in distilled water

(always use a freshly prepared solution).

Analysis of Total Phenols

Take suitable aliquots of the tannin-containing extract (initially try 0.02, 0.05 and

0.1 mL) in test tubes, make up the volume to 0.5 mL with distilled water and add

0.25 mL of the Folin–Ciocalteu reagent and then 1.25 mL of the sodium carbonate

solution. Vortex the tubes and record absorbance at 725 nm after 40 min. Calculate

the amount of total phenols as tannic acid equivalent from the above calibration

curve. Express total phenolic content on a dry matter basis (x%).

Estimation of Tanins

Removal of tannin from the tannin-containing extract: PVPP binds tannins. Weigh

100 mg PVPP in a 100 Â 12-mm test tube. Add to it 1.0 mL distilled water and then

1.0 mL of the tannin-containing extract (100 mg PVPP is sufficient to bind 2 mg of

total phenols; if total phenolic content of feed is more than 10% on a dry matter

basis, dilute the extract appropriately). Vortex and keep the tube at 4 C for 15 min,



Table 14.3 Calibration curve for estimation of tanins

Tannic acid




Tube (0.1 mg/mL), mL water (mL) reagent (mL)

Blank 0.00























Nutritional Evaluation of Forages



solution (mL)







Absorbance Tannic

at 725 nm

acid (mg)













vortex it again, then centrifuge (3,000 Â g for 10 min) and collect the supernatant.

This supernatant has only simple phenolics other than tannins (the tannins would

have been precipitated along with the PVPP); the procedure for binding of tannins

to PVPP is presently being modified, and the modification is to bind tannins to

PVPP at pH 3 since PVPP binds maximally to tannins at this pH. Measure the

phenolic content of the supernatant as mentioned above (take at least double the

volume (preferably three times) used for total phenol estimation, because extract

has already been diluted twofold and expect to lose tannin-phenols though binding

with PVPP). Express the content of non-tannin phenols on a dry matter basis (y%).

Preparation of calibration curve

See Table 14.3.


Determination of Condensed Tannins


• Butanol–HCl reagent (butanol–HCl 95:5; v/v): Mix 950 mL n-butanol with

50 mL conc. HCl (37%).

• Ferric reagent (2% ferric ammonium sulphate in 2 N HCl): Make 16.6 mL of

conc. HCl up to 100 mL with distilled water to make 2 N HCl. Dissolve 2.0 g

of ferric ammonium sulphate in this volume of 2 N HCl. This reagent should be

stored in a dark bottle.


In a 100 nm  12 mm glass test tube, pipette 0.50 mL of the tannin extract diluted

with 70% acetone. The quantity of acetone should be large enough to prevent the

absorbance (550 nm) in the assay from exceeding 0.6. It will depend on the quantity

of condensed tannin expected in the sample, and occasionally will need to be

determined by trial and error. To the tubes add 3.0 mL of the butanol–HCl reagent

and 0.1 mL of the ferric reagent. Vortex the tubes. Cover the mouth of each tube with

a glass marble and put the tubes in a heating block adjusted at 97–100 C (or in a

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