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40 Estimation of Indoleactic Acid (Knegt and Bruinsma 1973)

40 Estimation of Indoleactic Acid (Knegt and Bruinsma 1973)

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Estimation of Ethylene


diethyl ether each time the lipid fraction is discarded. Adjust the aqueous layer to

pH 3 by adding about 3 ml of 2.8 M phosphoric acid. Extract IAA with 10 ml

diethyl ether.

The 10 ml diethyl ether is then extracted with 10 ml cold 50 mM, K2HPO4

solution. The pH of the solution is adjusted to 3 with phosphoric acid (0.28 M) and

the IAA is passed into a final 10 ml diethyl ether. The ether is then evaporated in a

few minutes under reduced pressure. Dissolve the residue in a known volume (5 ml)

of cold redistilled methanol.


1. Pipette out 1 ml of the above methanolic extract each in four different test tubes.

2. To each tube add 1 ml of methanol containing 0, 10, 20 or30 ng of IAA,


3. Dry the contents in each tube completely under reduced pressure and cool

to 0 C.

4. To each flask add 0.2 ml ice-cold trifluoroacetic acid-acetic anhydride reagent

and mix.

5. Place the tubes on ice for exactly 15 min to ensure the complete conversion of

IAA into indole-a-pyrone. Stop the reaction adding 3 ml water.

6. A blank may be prepared occasionally by adding first 3 ml water to one of four

aliquots and 0.2 ml reagent after 15 min.

7. Take the readings in a spectrophotofluorimeter with excitation at 440 nm and

emission at 490 nm for low concentration samples.

8. Calculate the amount in unknown sample.


Estimation of Ethylene

Ethylene, the ripening hormone is usually estimated for different physiological



The ethylene evolved is measured in a gas chromatography based on the adsorption

principle on activated silica gel or poropak.






Conical Flasks with facility to seal with rubber gaskets

Air-tight syringes

Ethylene gas




Methods for Nutritional Quality Evaluation of Food Materials







Place the fruits in conical flasks.

Seal the mouth with rubber septum or gasket.

Incubate for 1 h at 20 C.

Withdraw gas samples with hypodermic syringes and inject into GLC.

For standard, inject pure ethylene into empty conical flasks or cylinder of same

volume and satisfy identical assay conditions. Remove the same volume of

internal atmosphere as that of sample from the flask, inject into GLC and

measure ethylene peak height.


The quantity of ethylene produced is expressed as ml ethylene per hour per kg


Chapter 14

Nutritional Evaluation of Forages


Preparation of Plant Extract for Analysis

The general problem in the study of natural plant products is that their nature and

amount are dependent on various factors, which must be controlled as far as

possible. Some of these factors are (i) stress; the metabolic state of the plant may

change when it is stressed in any manner. This can be a problem before as well as

after harvesting a plant part for analysis. As cells die (the senescent process), the

cellular integrity is lost and as a result the enzymes come in contact with substrates

to which they are not normally exposed in living cells. In addition, it also increases

the oxidation process, which is a problem with phenolics since these are prone to

oxidation. On oxidation, phenolics oxidize to quinones and then polymerization

reaction could follow. If a plant is cut and dried under “near ambient” conditions,

which generally requires a large time to dry, the nature and content of phenolic

compounds can change. In order to avoid these changes, the metabolic activities of

the cells need to be curbed immediately. The next important step is to bring the

chemical constituent into solutions for their measurement.

1. Collection, drying and storage of plant material

Leaf age and stage of development affect levels and nature of phenolics.

Therefore, it is important to define the stage of maturity of plant and leaf as

close as possible before collecting leaf material for analysis. When the collection

site is close to the laboratory, the material can be transported to the laboratory in

fresh state. The fresh material should be kept on ice and transported under dark

conditions. Transportation of large amount of leaves in plastic bags should be

avoided, since temperature in the bag could rise leading to sweating and wilting,

which can change the nature and level of phenolics. If liquid nitrogen is

available, the better option is to freeze the sample and then freeze-dry the

material without thawing it. Thawing can rupture cell membranes leading to

changes in phenolics. If the material is frozen using a freezer, make sure that the

material is not thawed during transport. Solid carbon dioxide should be used to

transport such material. Once the material is dried, it should be kept in a dry

R. Katoch, Analytical Techniques in Biochemistry and Molecular Biology,

DOI 10.1007/978-1-4419-9785-2_14, # Springer Science+Business Media, LLC 2011




Nutritional Evaluation of Forages

place (preferably in a desiccator) in the dark. The freeze-dried material generally

is hygroscopic. Light is also known to change the nature of phenolics. After

freeze-drying, the cell structure is broken and the enzymes are in the native state.

With the absorption of water, enzymes and phenolics can react, which can

produce drastic changes in phenolics. The freeze-drying, though considered to

be one of the safest method for preservation of phenolics, can lead to drastic

changes if the storage conditions are not appropriate.

If a lyophilizer is not available, the plant material has to be dried under far

from ideal conditions. The sample can be dried at about 50–52 C using a forced

air oven. This will hasten the process of drying, and the enzymes present in

the plant sample will not have much time to react with phenolics. Drying at

temperatures higher than 55 C should be avoided, since it can lead to inactivation of phenolics or could decrease their extractability in solvent and effect the


2. Grinding of sample

Fresh material, when frozen using liquid nitrogen, can be ground using “Polytron”

homogenizers. Phenolics are generally extracted in aqueous organic solvents.

The moisture present in the fresh material needs to be taken into account while

preparing organic solvents for extraction.

It is suggested to grind the sample after drying the sample. About 500 g of the

plant material should be ground first to pass a 2-mm screen. All the ground

material including those parts remaining inside the mill should be taken, mixed

well and approximately 100 g of this sample is again ground to pass through a

0.5-mm screen. For fibre analysis, and in situ nylon bag and in vitro gas

techniques, the sample passed through 2-mm screen should be taken. At any

stage of the grinding, the sample temperature does not rise above 40 C.


Proximate Analysis of Forages (Van Soest 1963)

Proximate analysis was given by Henneberg and Stohmann in 1865 who were then

working at Weende experiment station in Germany. This scheme of analysis is

commonly referred to as the “Weende analysis” and forms the basis for description

of quality composition of feed and forages. This scheme partitions biological

materials into fractions which are referred for the nourishment of animal body.

The Weende proximate analysis as a chemical procedure is shown in the following

Fig. 14.1.

This scheme groups together a variety of substances having common chemical

characteristics and hence not an analysis of a lone nutrient of the feed. Except for

water, each component represents a combination of substances, some of which are

nutrition or a combination of nutrients, while others may not be of any nutrition

value to the animal. Most prominent points of significance and drawbacks of the

scheme are enumerated below.


Proximate Analysis of Forages



DRY AT 100 ± 2˚c



IGNITE AT 550 - 600˚C























Fig. 14.1 Weende proximate analysis

1. Moisture

Feeds containing more than 14% moisture cannot be stored in bulk for the

danger of mould formation as well as spontaneous combustion. Calculation of

relative cost of nutritional value involves consideration of moisture content.

While ensiling, the moisture of forages has to be brought nearer to 70%. Feeds

with high moisture may restrict the intake of other nutrients. Feeding standards

do not include water requirements and therefore need correction for this omission.

Moisture is usually determined as loss in weight by oven drying to a constant

weight just above the boiling point of water at atmospheric pressure. Such drying

sometimes results in a loss of heat labile substances such as volatile fatty acids.

In addition, some sugars decompose at above 70 C. The loss of these during

oven drying is also taken as moisture and inflates its value.

2. Crude protein

Knowing the protein (CP) content of a feed gives an idea about the class of feed

to which it belongs. Crude protein determination involves multiplication of

estimated nitrogen value usually by a factor 6.25 basing on the assumption

that most of the feeds contain 16% nitrogen. It is an estimate of total protein

without any consideration of its quality. It does not distinguish the nitrogen

contribution from true protein and non-protein nitrogenous substances such as

urea, uric acid and ammonium salts. While analysing faecal nitrogen, conversion

factor for feed protein may not apply for portion of nitrogen of metabolic origin.



Nutritional Evaluation of Forages

3. Ether extract

In addition to as a non-specific source of energy, ether extract (EE) provides

essential fatty acids. Knowing the fat content in milk is of commercial significance and milk without this component would not be useful as the early diet of

mammals. Rancid feeds are objectionable and fat portion of feeds is most

unstable. Storing high fat, especially with greater unsaturation, is a problem

and chemical changes during storage may result in undesirable substances such

as amines.

Ether extract is estimated by extraction with fat-soluble solvents and consists

of glycerides of fatty acids, free fatty acids, cholesterol, lecithins, chlorophyll,

alkali substances, volatile oils, resins, carotenoids, fat-soluble vitamins, etc.

Some of which like chlorophyll, alkalies, volatile oils and resins are not classed

as nutrients. While analysing faeces, the soaps formed in the intestinal tract from

free fatty acids and calcium are not completely removed when extracted with

ether. It gives erroneously high values for the digestibility of ration fat.

4. Crude fibre

Crude fibre represents the insoluble residue of a feed left after successive

boiling with dilute acid and alkali, the nutritional significance of which is

not clear and precise. As per original supposition of Weende analysis, crude

fibre is the indigestible portion of total carbohydrate of the feed. Sometimes

it is misleading index of overall digestibility of a feed as in a number of cases the

crude fibre is digested as high as soluble carbohydrates, normally referred

to as nitrogen-free extract, especially in case of ruminants. Moreover, crude

fibre estimation simulates monogastric digestion than that of the ruminants.

5. Ash

The inorganic residue left after ignition at 550–600 C represents total ash.

It does not discriminate the proportion of mineral matter and sand or silica due

to either contamination or adulteration. Some volatile mineral elements, such as

iodine and selenium, are lost on ashing.

Despite various limitations, Weende analysis thus forms a basis for chemical

description of feeds, body tissues and biological excreta to find digestibility and

utilization and in the feeding standards of different categories of animal species.

6. Nitrogen-free extract

Nitrogen-free extract (NFE) is a non-cellulose portion of feed carbohydrates and

is a non-specific source of energy to the animal. This fraction makes up about

40% of dry weight of forage feeds and 70% of basal feeds. The proportion of

NFE will be inversely related to protein content in concentration feeds.

NFE is the difference between actual sample weight and sum of weights of

water, ether extract, crude protein, crude fibre and ash. Hence, its numerical

value is affected by analytical errors of these five as well as by the lack of

precission of crude fibre determination in separating functional categories of

carbohydrates as it is a mixture of all the starches and sugars plus some

hemicellulose and much of the lignin.


Estimation of Dry Matter (DM)



Estimation of Dry Matter (DM)


A known quantity of sample is dried in hot air oven for 8–12 h at 100 Æ 2 C to a

constant weight and the loss of weight is expressed as moisture percentage from

which dry matter percent is calculated.


DM in Bulk Samples

The representative samples collected as per described standard procedure are

brought to the laboratory in closed containers such as polyethylene bags and are

immediately subjected to moisture estimation.


1. Physical balance

2. Hot air oven

3. Aluminium tray or Glass Petri dish


1. Weigh representative sample of approximately required quantity in a clean dry

preweighed aluminium tray or glass Petri dish.

2. Dry the weighed sample in hot air oven at 100 Ỉ 2 C for 8–12 h (overnight) to a

constant weight.

3. Weigh quickly after cooling at the room temperature without undue exposure to

atmosphere for prolonged period to avoid absorption of moisture.

4. The difference in weight is expressed as moisture percent from which DM

percent can be calculated.

5. Store the oven-dried sample, after grinding in laboratory grinder (1-mm size), in

air tight containers for further estimation of various proximate principles.


Moisture %ị ẳ

b aị c aị


b aị

Dry matter %ị ẳ 100 Moisture percent orị

Dry mater %ị ẳ

c aị


b aị


a ẳ empty weight (g) of aluminium tray or glass Petri dish

b ¼ weight (g) of tray or Petri dish with sample before oven drying



Nutritional Evaluation of Forages

c ¼ weight (g) of tray or Petri dish with sample after oven drying


1. In case of long fodders, they may be cut into small pieces for convenience of

handling with least disturbance to its structure.

2. Keep half open the cover of the ventilator on the top of oven, especially in case

of samples of high moisture, to avoid water accumulation inside the oven due to


3. If extraneous moisture is present in case of green forages, care should be taken to

overcome its loss during handling to assess actual dry matter truly representing

the situation of bulk or lot from which it is drawn.


Dry Matter in Laboratory Samples

In order to get uniform idea about the chemical composition, it is customary to

express it on moisture-free basis which can be estimated in ground and stored bulk

samples preserved after DM estimation.






Moisture cup (aluminium) with lid (2 cm depth and 5 cm diameter)

Hot air oven

Chinametric balance



1. Dry a clean moisture cup with lid in a hot air oven at 100 Ỉ 2 C for 10–15 min

and cool in a desiccator and not the empty weight.

2. Spread uniformly the ground sample to be analysed on a sheet of white paper for

sufficient time to attain room temperature.

3. Transfer representative sample of suitable quantity with the help of spatula from

different places into the moisture cup and note the weight with lid.

4. Dry the sample in hot air oven at 100 Ỉ 2 C for 8–12 h with partially opened lid.

5. Cool in a desiccator with closed lid and weigh.

6. The process of drying, cooling and weighing is repeated till the difference

between two successive weighings is less than 1 mg. The moisture-free sample

is retained in desiccator for the estimation of ether extract.


Moisture %ị ẳ

b aÞ À ðc À aÞ


ðb À aÞ

Dry matter ð%Þ ¼ 100 À Moisture percent ðorÞ

Dry mater ð%Þ ¼

ðc À aị


b aị


Estimation of Dry Matter (DM)



a ẳ empty weight (g) of moisture cup with lid

b ¼ weight (g) of moisture cup with lid and sample before oven drying

c ¼ weight (g) of moisture cup with lid and sample after oven drying


DM in Silage, Haylage and Molasses

Oven drying of materials having heat labile compounds may underestimate the

moisture content. Alternatively, the moisture in such feedstuffs can be estimated by

toluene distillation method.


The actual volume of water is measured by distillation in the presence of excess

toluene after correcting the volume for total acidity that is occupied by volatile acids.

Equipments and Glassware

1. Toluene distillation set with condenser

2. Graduated flask

3. Pipette







Ethanol (80% alcoholic ammonia solution neutralized to phenolphthalein)

0.1 N NaOH

Phenolphthalein indicator






Add 400 mL of toluene to the distillation flask containing 70–80 g of sample.

Distil the contents for 6–8 h at the rate of 2–3 drops per second.

Stop distillation after observing two equal consecutive volumes at 15 min interval.

Wash the condenser with toluene to remove traces of water present in it and

continue distillation for another 15 min. Keep the graduated receiver flask in

water bath at 20 C for 20 min and note the volume of water at room temperature.

5. Transfer the aqueous layer to 100-mL volumetric flask and make up the volume

with CO2-free distilled water.

6. Add 40 mL neutralized ethanol to 10 mL of diluted distillate and titrate against

0.1 N NaOH in the presence of phenolphthalein.


DM ẳ 100


t f =10ị




Nutritional Evaluation of Forages










volume (mL) of aqueous phase

weight (g) of sample

volume (mL) of 0.1 N NaOH utilized

factor depending on the constituents of acids of the sample (normally 0.00555

is taken for all practical purposes)

Note: No acid correction is needed in case of molasses.


Crude Protein (CP)


The Kjeldahl Nitrogen method is the most frequently used procedure for measuring

nitrogen and in turn the protein content in biological materials. In this method, the

amino (–NH2) nitrogen is oxidized by sulphuric acid in the presence of catalyst of

(NH4)2SO4. The ammonium ion is converted to NH3 by NaOH and collected by

distillation. The NH3 is then quantitatively titrated against standard acid (HCl or

H2SO4) of known strength and nitrogen in the sample is computed. The crude

protein is obtained by multiplying the nitrogen content with factor 6.25 (16%

nitrogen in protein for most of the feeds in general).

Equipment and Glassware







Digestion bench placed in digestion chamber

Distillation unit (micro-Kjeldahl distillation apparatus)

Kjeldahl flask (500–800 mL)


Conical flask

Volumetric flask


1. Commercial sulphuric acid

2. Digestion mixture [1 part copper sulphate (acts as catalyst) : 10 parts sodium or

potassium sulphate (raises boiling point)]

3. 40% Sodium hydroxide

4. Tashiro’s indicator

5. N/7 H2SO4


The method of estimation of nitrogen/crude protein encompasses digestion,

distillation and titration.


Crude Protein (CP)



1. Transfer exactly weighed suitable quantity (as described under sampling) of

sample into Kjeldahl flask.

2. Add 20–50 mL commercial sulphuric acid depending upon the type of sample.

3. Add 5–10 g of digestion mixture.

4. Boil the contents for 2–3 h on a digestion bench placed in a digestion chamber till the

solution is clear without leaving any undigested black particles. Adhering materials

inside walls of the flask needs one or two washings in between after cooling.

To avoid bumping, a few glass beeds may be placed inside the Kjeldahl flask.

5. Transfer the digested material present after cooling by dissolving with nitrogenfree tap water into volumetric flask followed by 5–6 repeated washings. Make up

the final volume after cooling.

6. Similarly run a blank without sample.


1. Place a conical flask containing 10 mL of Tashiro’s indicator at the end of

condenser of micro-Kjeldahl distillation apparatus. Care should be taken to see

the tip of the condenser is completely dipped inside the indicator to avoid escape

of released ammonia during distillation.

2. Pipette out 5–10 mL of aliquot of digested sample from the volumetric flask into

the distillation unit.

3. Add 10–20 mL of 40% NaOH sufficient to make the contents alkaline (i.e. till

the contents turn blue or black). Wash with a small quantity of distilled water

and close the receiving end immediately with a pinch cock. Seal the funnel with

a little amount of distilled water to avoid escape of ammonia.

4. Steam distil the contents of the distillation unit by boiling the water in a round

bottom flask (avoid bumping by addition of glass beads) connected to the

distillation unit.

5. Collect around 30–50 mL distillate (or at least 2 times the quantity of indicator

taken) to ensure all nitrogen in the form of ammonia is distilled. Red colour turns

to green.

6. Remove the conical flask with distillate after washing the tip of the condenser

with a few millilitres of distilled water.

7. Wash the distillation unit 2 or 3 times with distilled water with the help of back

suction developed by vacuum due to displacement of boiling flask from the

heater to make the apparatus ready for distillation of next sample.

Note: In place of Tashiro’s indicator, standard N/7 H2SO4 may also be taken with

methyl red indicator in the receiver flask (conical flask). In which case, the distillate is

back titrated against N/7 NaOH to know the actual volume of N/7 H2SO4 consumed.


1. Titrate the distillate in the conical flask against standard N/7 H2SO4 solution

taken in a burette till the red colour just reappears.

2. Note the volume of N/7 H2SO4 consumed.

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40 Estimation of Indoleactic Acid (Knegt and Bruinsma 1973)

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