Principle of various techniques in estimating chemical and biochemical constituents in feeds

and fodder

K.GURU MOHAN REDDY

TVM/2016-13

DEPARTMENT OF ANIMAL NUTRTION

COLLEGE OF VETERINARY SCIENCE, TIRUPATI

SRI VENKATESWARA VETERINARY UNIVERSITY

Weende's System of Analysis

• A team of scientists under the leadership of Henneberg and

Stohmann developed proximate principles analysis system in the

sixties of last century at Weende Experimental Station.

• Main components of different fractions in the proximate analysis of

foods are moisture, ash, crude protein, ether extract, crude fiber,

and nitrogen free extract.

Feed sample

Dry matter

Inorganic matter(ash)

residue

ash is treated with excess conc. HCL and extracted

Acid soluble ash

(used for minerals

estimation)

Acid insoluble ash

organic matter

(loss in weight due to ashing in furnace)

crude protein

(N*6.25)

Ether Extract

( portion soluble in

petroleum ether)Carbohydrates

NFE

(Soluble sugars )CF

(the residue after EE is boiled with weak acid & weak alkali)

Dried residue left is ignited The difference in wt. is CRUDE FIBER

Moisture

Moisture (water)

• Water is an important constituent of all plant and animal tissues.

• It is determined by drying a feed sample in a hot air oven at 1000C for

a specified length of time.

• The loss of wt. is the moisture content of sample.

• The residue left after the removal of moisture is Dry matter content of

the sample.

Significance of moisture content of feeds

• As moisture content of feed varies , its proximate composition will also

varies and consequently its nutritive value varies.

• Moisture content of feeds is also significant in calculating the cost per

unit weight of feeds.

• Useful in the Classification of feed into succulent & non-succulent

feeds.

• As the moisture level increases dry matter level decreases. While

purchasing of feed , the dry matter content is taken into consideration rather

than gross weight of the feed.

• Moisture content is significant in the storage feeds.

• In general feeds with more than 11% moisture get mouldy & spoiled.

• Moisture levels determine the keeping quality of hay & losses in making.

Dry Matter

• Dry matter is generally defined as the constant weight ,a sample

attains when heated at 100°C (occasionally I05°C)

• Dry matter is calculated by weighing after drying to constant weight in

an oven at 100-105°C.

• Generally, molasses, milk or feeds with a high moisture content is

preferably dried by, increasing from 50°C to 105°C after 24 hours.

• Hay, grasses and silages are usually predried at 60-70°C and roots in small

cut samples at 70-80°C while starchy feeds can stand 120-140°C.

• Silage suffer losses of volatile components in oven drying.

• The method of toluene distillation is used for silage moisture estimation.

• Moisture in molasses may be estimated by "Dairy search" method based

on release of bound water by treatment with hydrochloric acid and then

extraction it with xylol (Krishna et al., 1972*).

Toluene distillation

• Determination of dry matter in silage by toluene distillation .

• The dry matter in silage is calculated from the volume of water

removed by distillation in the presence of toluene.

• A titrimetric determination of the total acidity of the distillate is used

to correct the measured volume of the distillate for the volume

occupied by volatile acids.

Dairy search method

• "Dairy search" method for moisture determination in molasses.

• A simple method was developed at NDRI, Karnal, to estimate

moisture in molasses by disintegrating the carbohydrate moiety with

the help of hydrochloric acid and completely extracting thereby

bound water with the help of xylol.

Crude protein

• Crude protein is estimated by Kjeldahl procedure, where organic

matter is digested by heating in concentrated sulfuric acid.

• From the N-containing organic molecules ammonium sulfate is

formed.

• The amount of ammonia is estimated by distillation, and then titrated

against standard acid solution.

• Crude protein = N X 6.25, where N is nitrogen (%)

Crude Fat

• Crude fat is also known as Ether Extract.

• This is a combination of simple fat- fatty acid esters, compound fat,

neutral fat, sterols-Pseudo fat (Vitamins A, D2, D3,E, K) and carotene.

• Fat is estimated by extraction with petroleum ether (boiling point 40-

60°C) by a Soxhlet extractor.

Carbohydrate

• Carbohydrate portion of biological material is made up of two parts-nitrogen free

extract and crude fibre.

• Nitrogen free extract is also known as soluble carbohydrates, which consist of

- Water,

- Soluble vitamins,

- Monosaccharides (simple pentose or hexose sugars),

- Oligosaccharides (compound sugars) Polysaccharides (starches).

• Insoluble carbohydrate (crude fibre) is mainly polysaccharides consisting of

hemi-cellulose and cellulose.

• Actually, residue of a feed that, is insoluble after successive boiling with

dilute acid and dilute alkali is known as a crude fibre and contains besides

cellulose a part of nitrogen also.

• Crude fibre gives an indication of bulkiness of a feed.

• As per Weende's methods, some of lignin, pentosans and part of the

cellulose are dissolved and included in calculation of NFE.

• Ash

• The residue from burning any biological material in furnace at 550°C is

called ash

• Organic Matter

• This is calculated by deducting the ash per cent from dry matter.

• Nitrogen Free Extract (NFE)

This is calculated as given below:

• NFE=100 - (Moisture + Crude Protein + Ether Extract + Crude Fibre + Total Ash)

Vansoest Method –Partioning of Forage Fiber

FEED SAMPLE

Boiled with Neutral detergent at pH 7

NDF ( Neutral detergent fiber) NDS (Neutral detergent solubles)

---plant cell wall contents ---cell contents and pectin

*Hemicellulose *SOLUBLE CARBOHYDRATES

*Cellulose *Starch

*Ligni,silica & some protein * organic acids,protein,pectin

--- NDF ---NDS – have an average digestibility of about 98%

Acid detergent solubles

ADF (Acid detergent fibre) Hemicellulose

Cellulose ,Lignin , Silica

72% H2SO4

KMnO4 method

(pH 3.0) Lignin + minerals 550oc Cellulose dissolved

Silica LIGNIN lost by ignition

Cellulose +minerals Lignin lost by oxidation

550oC

Silica Cellulose lost by ignition

Neutral Detergent Fiber

• The method utilizes detergents which complex with protein to render

it soluble & utilizes a chelating agent (EDTA) to remove heavy metal &

alkaline earth contamination.

• This procedure involves the separation of feed dry matter into two

fractions –high digestible & low digestible by boiling a 0.5 -1 g sample

of feed in a NDS solution(3% sodium lauryl sulfate buffered to a pH 7)

for one hour & filtering.

• NDF as determined by Van Soest procedure is considerably higher

lignin & hemicellulose are included in the NDF fraction.

• Crude protein content of NDF is neutral detergent insoluble CP

(NDICP).

• NDF can be equated with the cell wall content of grass & cereals.

• If preceded by a starch extraction , it can be equated with the cellwall

content of many other feed ingredients.

Acid Detergent Fibre

• This procedure is used for the purpose of determining the lignin in a

forage sample.

• In this method the acid detergent fibre procedure is used as a

preparatory step.

• This involves boiling of a 1 g sample of feed in an acid detergent

solution for one hour and filtering . The insoluble makes up A.D.F.

• NDF- ADF= hemicelluolse (+ limited amount of protein )

Acid Detergent Lignin & Permanganate Lignin

• In order to determine the amount of lignin present, the ADF is then

digested in 72% H2SO4 at 15OC for 3 hrs and filtered .

• The residues remaining after washing and drying is weighed and

ashed.

• The ash remaining approximates the silica present, while the loss in

weight during ashing approximates the lignin and is referred to as

acic detergent lignin (ADL) or more specifically as acid insoluble

lignin.

• An alternative method for determining lignin which has advantages

for certain materials involves the oxidation of the lignin of ADF with

an excess of acetic acid-buffered KMnO4 solution.

• Lignin so determined is referred to as permanganate lignin.

• A variation of this method may be used to allow for the cutin present

in many seed hulls , which otherwise would be measured as lignin.

Determination of True protein and NPN

• The insoluble protein left by precipitation with a suitable precipitation

agent after filtering NPN is termed as true protein.

• The NPN is calculated by difference between the total crude protein

nitrogen and the value of the precipitated true protein nitrogen.

• The various precipitating agents commonly used are tungstic acid,

trichloroacetic acid (TCA) , copper hydroxide , zinc-barium hydroxide

etc,.

Determination of Free Fatty Acids

Principle

• Free fatty acids are extracted from lipids of feed sample by using Dolls

extraction mixture ( 40:10:1, heptane : isopropanol : acetic acid).

• The free fatty acids form a complex with cupric ions when mixed with

copper reagent.

• The coloured complex formed with copper is soluble in chloroform

and diethyl di thio carbamate is used as colour developer.

Mineral Estimation- Thalapatra method

Estimation of Calcium

Principle:

• When a solution containing calcium is treated with ammonium

oxalate all the calcium present is precipitated as calcium oxalate.

• The precipitate on treatment with sulphuric acid dissolves forming

calcium sulphate liberating free oxalic acid.

• This oxalic acid is quantitatively estimated by titration against

standard N/10 KMnO4 solution to arrive at the calcium content

present in the given solution.

• 1ml of N/10 KMnO4 solution = 2 mg of calcium

Estimation of Phosphorous

Principle

• Phosphorous present in the ash solution is precipitated as ammonium phospo molybdate.

• The precipitate is washed till free from acid and dissolved in a known excess of standard N/10 NaOH.

• The excess alkali is back titrated against standard N/10 HCl to arrive at the exact quantity of standard N/10 NaOH required.

• 1 ml standard N/10 NaOH = 0.1347 mg of P

Estimation of Magnesium

Principle

• Calcium is removed by precipitation as oxalates.

• Then Magnesium from Calcium filtrate is precipitated in an alkaline

medium as Magnesium ammonium phosphate by disodium

phosphate.

• The precipitate is washed with ammonical water to remove free

chlorides, then dried, ignited in a tarred crucible and weighed as

magnesium pyrophosphate

• MgCl2 + Na2HPO4+ NH4OH ……….. MgNH4PO4 + 2 NaCl + H2O

• 2 MgNH4PO4 on heat gives MgP2O7 + 2 NH3 + H2O

Estimation of Minerals – Atomic Absorption Spectrophotometry

• First of all a suitable quantity of sample material is oxidized with a

mixture of acids like perchloric acid, sulphuric acid and nitric acid in

the ratio of 0.5: 1: 4 and the material is brought into solution with

triple-distilled water.

• The test material (acid digest) in solution is delivered through a

nebulizer into a spray chamber.

Sample preparation-Wet oxidation (digestion)

• It is a procedure for oxidizing organic substances by using acids and

oxidizing agents.

• Minerals are solubilized without volatilization.

• The combustion of samples of feeds, fodders, feces, urine, blood and

water is carried out by triple acid digestion method.

Procedure for feeds and fodders

• Take 1-2 g material powdered in glass pestle and mortar.

• Weigh accurately and transfer into a 100 ml Kjeldahl flask.

• Moisten the material with 2 ml water; add 2 ml of Sulphuric acid and

2 glass beads.

• Heat the mixture gently over an electrical micro-digestion bench till a

homogenous dark brown liquid is formed

• After cooling the flasks, add 0.5 ml per chloric acid and 5 ml Nitric acid and

heat till the dark brown color become faint.

• Cool the flask, again add 2.5 ml Nitric acid and heat again till the material

changes to yellow or color less mass.

• At this stage add 5 drops of per chloric acid to flask to ensure the removal

of last traces of organic matter.

• Heat again until thick white fumes of per chloric acid no longer come out.

• Add little water to the flask, while still warm, to keep the solid salts as

solution.

Flame Photometer

Definition:

A photoelectric flame photometer is an instrument used in inorganicchemical analysis to determine the concentration of certain metal ionslike sodium, potassium, calcium and lithium.

Principle:

When an alkali metal salt is drawn into a non-luminous flame, it willionize, absorb energy from the flame and then emit light of acharacteristic wavelength as the excited atom decay to the unexcitedground state.

ELEMENT EMISSION

WAVELENGTH(nm)

FLAME COLOUR

Sodium(Na) 589 Yellow

Potassium(K) 766 Violet

Barium(Ba) 554 Lime green

Calcium(Ca) 662 Orange

Lithium(Li) 670 Red

Colorimetric Method of Trace Element Analysis

• Colorimetric analysis is a method of determining the concentration of

a chemical element or chemical compound in a solution with the aid

of a color reagent.

• It is applicable to both organic compounds and inorganic compounds

and may be used with or without an enzymatic stage.

Steps in Estimation of Trace Elements

• Production of an acidic solution of inorganic elements in the biological

sample after removal of organic matter by dry ashing or wet oxidation.

• Removal of interfering substances.

• Determination of selected elements, by using colorimetry.

• Results of trace elements determination are expressed as parts per million

(ppm), mg/kg or µg / g dry matter.

Estimation of Copper

• Callan and Henderson (1929) discovered that when di ethyl di thio

carbamate is added to solution of copper, a golden brown color is

produced.

• Mc Far lance (1932) found that colored copper salt could be rapidly

and quantitatively extracted from aqueous solution by amyl alcohol

and that the color was intensified in the organic solvent.

• The color complex is stable for at least two hours and the pH of

solution has little effect on the color intensity b/w pH 5.9 and 9.2.

• Iron gives a brown color with the reagent and is the only substance in

biological ash which is known to interfere significantly.

• However, when sodium pyro phosphate is added, iron pyrophosphate

is formed and this complex does not react with carbamate, whereas

the reactivity of copper is unaffected.

• In this micro estimation method of copper, amyl alcohol is used

because ionization of copper salt is depressed in the organic solvent,

this intensifying the color and so giving the reaction greater

sensitivity.

Estimation of Cobalt

Principle:

• Cobalt is extracted ash solution with dithizone, cobalt forms a red

complex with sodium 1-nitroso-3-naphthol-3, 6-disulphonate, nitro so

–R salt, which is stable in boiling nitric acid.

• Three moles of reagent combines with one mole cobalt.

Estimation of Manganese

Principle

• The colorimetric determination of manganese by oxidation to

permanganate in acid solution is both sensitive and specific. Per

iodate oxidases manganous salts converted smoothly to

permanganate. The reaction is as follows:

• 2 Mn++ + 5 IO4- + 3 H2O = 2MnO4

- + 5IO3- + 6H+

Estimation of Vitamins

Estimation of Vitamin A

Principle:

• When antimony trichloride reacts with Vitamin A in chloroform

solution a blue color develops. Vitamin A is estimated from

colorimeter readings by use of a standard curve.

Estimation of D2 and D3

Principle:

• By proper control of the composition of an antimony trichloride reagent, it can be

shown that saturated sterols do not react there with as chromogens but

unsaturated steroids do.

• Sterols with one double bond in ring B give a yellow colour with absorption

maximum at 500 nm sloping from the violet towards the red.

• Sterols with two double bonds in ring B shows a maximum at 510 to 515 nm.

• While vit-D2 and D3 have the very sharp and high maximum absorption at 500nm.

Estimation of thiamine

Principle

• Alkaline potassium ferricyanide oxidizes thiamin to thiochrome which

is a fluorescent compound.

• The thiochrome is extracted in isobutyl alcohol and measured in a

fluorimeter.

Estimation of riboflavin

Principle

• Riboflavin fluoresces at wavelength 440 to 500 nm.

• The intensity of fluoresence is proportional the concentration of

riboflavin in dilute solution.

• The riboflavin is measured in terms of the difference in fluorescence

before and after chemical reduction

Estimation of Pyridoxine (B6)

Principle:

• The tissue is extracted and hydrolysed by acid and enzyme action.

• The proteins are removed by tungstic acid precipitation and the

pyridoxine adsorbed on super filtrol at pH 3.

• The pyridoxine is selectively eluted by alkaline alcohol and converted

into an azo-dye which is estimated colorimetrically.

Estimation of choline

Principle:

• The dry material is exhaustively extracted with methanol. The dissolved

material is hydrolysed by aqueous Ba(OH)2 .

• The choline is precipitated from the neutralized saponification mixture by

Reinecke salt, separated, dissolved in acetone and compared in a

colorimeter.

• This method has been applied satisfactorily on dried yeast, dried liver

extract, peanut meal and fresh liver.

Estimation of Vitamin C

Principle:

• Amount of Ascorbic Acid can be determined by standard solution of

iodine.

• The iodine is reduced by the ascorbic acid to form iodide.

• Ascorbic acid converted to dehydro ascorbic acid and two protons

two electrons.

I2 + 2e- ………. 2 I-

Estimation of Anti-nutritional Factors

Definition

• The anti-nutritional factors (ANFs) may be defined as those substances

generated in natural feed stuffs by the normal metabolism of species and by

different mechanisms (e.g., inactivation of some nutrients, diminution of the

digestive process or metabolic utilization of feed) which exert effects contrary to

optimum nutrition.

Estimation of total phenolic compounds

Principle

• Phenolic compounds , when reacts with Folin Ciocalteu reagent at

alkaline pH gives absorbance maximum at 725 nm.

• Estimation of Simple Phenolic Compounds (Non – Tannin Phenolics)

• Principle

• Polyvinyl poly pyrrolidone (PVPP) has the property to bind tannins but

not the simple phenolic.

• Thus, tannins and extract containing tannins as well as phenolic

compound can be removed by precipitation with PVPP and filtrate

can be used for the estimation f non tannin phenolic compounds.

Estimation of pure tannins compounds

Principle

• Pure tannins can be estimated by the difference method I.e., by

subtracting non tannin phenolic compound from the amount of total

phenolic present in a sample.

Estimation of condensed tannins

Principle

• Condensed reacts with ferric reagent in the presence of Butanol-HCL.

The color is persistent and absorbance maximum is at 550nm.

Estimation of Total Tannins (volumetric method)

Principle

• Tannins can be estimated as quercitannic acid by titrating against

potassium permanganate in the presence of indigo carmine

Estimation of Mimosine

Principle

• Mimosine is extractable in dilute HCl and it gives color reaction with

FeCl3reagent, which can be measured at 535 nm.

Estimation of Non- Starch Polysaccharide

• Non- structural carbohydrates (NSC) minus starch and sugars equalsNSP.

• Starch and sugars can be measured directly.

• The net fraction can reasonably calculated by difference using one oftwo formulae:

• NSC = 100 – ( NDF + PROTEIN + FAT + ASH )

• NSC = 100 –{( NDF –NDF Protein) + protein + fat + ash}

• NSP= NSC- (sugars + starch )

• The NSP do not generally include native hemicellulose and celluloses

that are ordinarily a part of lignified cell wall matrix, which recovers

hemicellulose and cellulose.

• So determination of organic matter, starch, protein, fat, NDF and

lignin will help to estimate NSP indirectly

Determination of starch

Principle:

• Powdered feed sample is treated with alcohol to solubilize free

sugars, lipids, most pigments and circular waxes.

• The residue rich in starch is solubilized with per chloric acid and the

extract is treated with anthrone- sulphuric acid to determine glucose.

Determination of Free Sugars

• The alcohol soluble portion containing free sugars includes Hexoses,Pentoses and Uronic Acid which are to be determined separately.

Measurement of monosaccharide’s (Hexoses)

Reagents

• Anthrone / thiourea reagent: 340ml H2O +660 ml H2SO4 (conc.) + 10 gThiourea + 0.5 g Anthrone. Warm the mix to dissolve at 80-90oC. Cooland store at 0-4Oc.

• Standard glucose (10-40 µg/ml)

Measurement of Pentoses

Reagents

• Orcinol 10% solution in 95% ethanol.

• Ferric chloride- Hydro chloride acid reagent: 0.1% (w/v) anhydrous

ferric chloride (FeCl3) in conc.HCl.

• Working reagent = 1 part orcinol solution + 2 parts FeCl3-HCl reagent

Measurement of Uronic acid

Reagents

• 0.025M sodium tetra borate deca hydrate in conc. H2SO4

• 0.125% carbazole in ethanol

• Standard stock = 0.1% (w/v) glucoronate in saturated benzoic acid.

• Working standard : 10-40µg/ml

REFERENCES

• LIVESTOCK NUTRITIONAL ANALYTICAL TECHNIQUES-GOPAL KRISHNA

• MODREN ANALYTICAL TECHNIQUES USED IN ANIMAL NUTRITION

RESEARCH

• PRINICIPLES OF ANIMAL NUTRITION AND FEED TECHNOLOGY-

D.V.REDDY.

• ANIMAL NUTRITION SCIENCE-GORDON Md.DRYDEN.

• BASICS OF ANIMAL NUTRITION –JASPHAL SINGH & HERNDAL.