Proximate analysis and fibre fraction

1. Proximate analysis
and it’s limitations
K.GURU MOHAN REDDY
TVM/2016-13
DEPARTMENT OF ANIMAL NUTRTION
COLLEGE OF VETERINARY SCIENCE, TIRUPATI
SRI VENKATESWARA VETERINARY UNIVERSITY

2. 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.

3. • In Weende’s system of analysis "soluble carbohydrate" was called
Nitrogen-free extract (NFE)
• These workers emphasized that carbohydrates could be grouped into:
(1) the starches and the sugars and
(2) a coarse fibrous fraction

4. • Later on an insoluble residue after boiling the fat free food sample
first with dilute acid and then with dilute alkali was obtained based on
the acid stomach digestion and the subsequent alkaline intestinal
digestion of consumed food.
• At the end of digestion, insoluble organic residue was denoted as
crude fibre.

5. feed or
biological
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

6. 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.

7. Feeds containing 10% moisture are called as AIR-DRIED feeds.
FEEDS MOISTURE%
Leafy succulent roughages 80%
Green grass 75%
THICK STEMMED CROPS LIKE
Maize & Jowar
70%
Hays 12-14%
Straws 10%
Concentrates like grains, brans, oil
cakes
10 %

8. 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.

9. • 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.

10. 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.

11. • 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 permits a correction to be made from
titrable acids in the distillate.
• 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*).

12. • For green fodders the samples should be taken from the field when
there is no dew on the fodders.
• The plants should be harvested little above the ground level (2-3cm)
and brought to the laboratory where the plants are cut into smaller
pieces (2-3cm size). It is better to have all the parts represented.
• About 1-2 kg sample may be prepared and 100g sample in a clean dry
pre-weighed tray may be taken and is kept in the oven for overnight
at 100°C.

13. General Methods of Drying
• 1.Low-temperature drying:
• Some laboratories adopt low temperature drying using vacuum drying
oven (Temp. 30°C, 16 mm mercury pressure).
• This type of drying will reduce losses of volatile labile compounds, losses
due to prolonged cnzymic activity are likely to be large.

14. • 2.High-temperature drying:
• Most of the laboratories adopt high temperature drying using oven
at 100°C.
• In this case there are losses of volatile or heat labile components.

15. • 3. Freeze drying:
• Where it is desirable that changes in chemical composition should be kept to a
minimum during drying, freeze drying has been employed, but this can not be
used to determine dry matter content as sample do not come to a standard end
point.
• It has been observed that more volatile organic compounds may also be removed
during the process.

16. Estimation of Dry Matter in Forages, Faeces and
Concentrates
• APPARTUS
• Aluminium moisture cup with lid,
• Hot air oven,
• Metal tongs,
• Asbestos sheet,
• Desiccator,
• Analytical balance,
• Weight box.

17. Procedure
• Weigh accurately about 5 g of the material in a empty aluminium
dish, having a diameter of at least 50 mm and a depth of about
20 mm, shake the dish until the contents arc evenly distributed.
• Place the dish in an air oven maintained at 100°C+ 2°C and dry for at
least 2 hours.
• Cool in a desiccator and weigh.

18. • Moisture, percent by weight =100× (w1- w2)
w1-W
• Where,
• W1= Weight in gram of dish with the material before drying.
• W2 = Weight in gram of the dish with the dried material.
• W = Weight in gram of the empty dish.
• DM= 100- Moisture per cent

19. 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.

20. Apparatus
• Dean and Stark assembly,
• Electrical heating mantles ,
• Liebig condensers.
• Reduction adapters.
• Round bottomed flasks (with ground glass joints - one litre capacity)

21. REAGENTS
• Ethanol,
• Phenolphthalein indicator,
• Sodium hydroxide 0.1 M.
• Toluene

22. Procedure
• Weigh 70-80 g of silage and molasses into the flask and add 400 ml of
toluene.
• Heat the moisture of silage and molasses with toluene and adjust the
controller until the toluene boils steadily.
• After 90 minutes, and subsequently at 15 minutes intervals, note the
volume of aqueous phase in the receiver.

23. • When two consecutive equal readings are obtained, discontinue the
heating, allow the receiver and its contents to cool to room
temperature and record the volume to the aqueous phase.
• Pipette 10 ml of the aqueous phase into a 100 ml conical flask.
• Add 40 ml ethanol, previously neutralized to phenolphthalein,
• Titrate with 0.1 M sodium hydroxide using phenolphthalein indicator.

24. Calculation
• Obtain the dry matter (DM) in the silage, form the expression
DM= (99.8 V ÷W) × ( 1-ft)/10
• Where ,
V = total volume of aqueous phase (ml).
W= weight of silage taken (grams).
t= titre (ml 0.1 M sodium hydroxide
f = a factor dependent on the constituent acids of the sample but
which for practical purposes may be taken as 0.0055

25. 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.
• Newly developed method helped in the recovery of 95-98% of added
distilled water in the molasses.

26. Procedure
• 10-15 g molasses is weighed in the distillation flask of Dean and Stark
assembly.
• 25 ml of xylol (AR) and 3 ml hydrochloric acid (AR) are also added.
• It is mixed completely by shaking and keep overnight in a moisture
free place .
• The contents are distilled on oil bath at 120°C temperature for 15-20
minutes only.

27. • The water in the receiver is allowed to separate from the xylol by
moving a spiral copper wire up and down in the condenser and
receiver occasionally, thus the layer of water is settled at the bottom
of receiver
• The receiver is immersed in water at about 27°C for at least 15
minutes or until xylol layer is clear, and then volume of water (actual
water and acid water) is deducted out of total volume recorded and
result is calculated.

28. calculations
• Moisture content, per cent by weight
= (V×D×100) ÷ W
Where ,
V = Actual volume of water (ml) (Volume of water observed
from the receiver-3)
D = Specific gravity of water at the temperature at which the
volume of water is read.
W = Weight in gram of the material taken for the test. In this
method, efficiency of recovery of added water is 95-98%.

29. Crude Protein
• According to Weende's food analysis scheme, nitrogenous fraction of
food may be divided into two portions protein and non-protein.
• Protein is made up of dispensible amino acids-glutamic acid, aspartic
acid, arginine, glycine, histidine, cystine, tyrosine;
• indispensable amino acids – lysine, tryptophan, leucine,
phenylallalanine, methionine, threonine, isoleucine, valine.

30. • Another-parts of non protein nitrogen (NPN) consists of urea nitrogen,
ammonia nitrogen, uric acid nitrogen, free amino acids, amines, etc.
• Overall portions-true protein and non protein nitrogen.
• True protein is estimated by Stutzer reagent method, (where protein is
precipitated with cupric hydroxide in alkali conditions)
• while non-protein nitrogen fractions, e.g. urea nitrogen, ammonia
nitrogen are estimated accurately by Conway diffusion technique.

31. • Generally, pure proteins yield 5.25-5.75 calories of GE/g .
• Crude protein is estimated by Kjeldahl digestion 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.

32. calculations
• Crude protein = N X 6.25, where N is nitrogen (%)
• Factor 6.25 is based on the fact, that protein contains 16% nitrogen

33. 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.

34. • Too high values may be encountered with silages, and sometimes oil cakes
due to oxidized fat or calcium soaps in faeces, which are not completely
extracted.
• Treatment with acid a head of the extraction of faeces, and when the feed-
stuffs are extracted with a better solvent (Chloroform-methanol, benzene-
methanol, ether-methanol, acetone or isopropyl alcohol), it gives better
results.

35. • Parts of the feed fat particularly polar lipids, which are integrated in the cell
structure, are not as easily extractable.
• Sometimes, the faeces of animals contain fatty acids bound to calcium and
may escape the assay in this way.
• A series of analyses conducted earlier, which reveal that the yield of
extractable fat is increased by pretreatments with hydrochloric acid (HCL).

36. • Generally, gross energy of crude fat is 9.40 kcal/g of feed.
• Generally, two methods of extraction of the fat from biological samples are
followed.
• In the first method, weight of extracted material is recorded after
evaporation the solvent and loss in weight of the moisture free sample
following its extraction is known as crude fat.
• In the second method, difference in the weight of oil flask is recorded,
which is denoted as crude fat. Second method gives more accurate results.

37. 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).

38. Effects of the Weende’s crude fibre procedure on
fat free food
Constituent Boiling with 1.25%
H2SO4
Subsequent boiling with
1.25% NaOH
Protein Partial extraction Complete extraction
Starches and Sugars Hydrolysis and extraction -
Cellulose Slight effects Slight effects
Hemi-cellulose Variable extraction Extensive but highly variable extraction
Lignin Slight effects Extensive but highly variable extraction

39. • 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.
• It has been experienced that Weende crude fibre may be a misleading
index of the overall digestibility of a feed, because crude fibre itself is at
least as highly digested as the soluble carbohydrate (nitrogen free extract).

40. • 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.
• Generally, Gross Energy yield from carbohydrate is 4.15 kcal/g.

41. • 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 + crude fat + crude fibre + total ash)

42. Main features of Weende's system of analysis
• Lignin is indigestible and dissolved into the nitrogen free extract (NFE)
by the use of sodium hydroxide.
• carbohydrate is divided into two fractions, i.e. highly-digestible (NFE)
less digestible (crude fibre) .
• The highly digestible fraction calculated by difference is supposed to
represent available carbohydrates.
• It is a very simple scheme of analysis which is worldwide accepted.

43. Limitations
Crude protein:
• It over estimates feed protein values because:
• Some nitrogen is inorganic (nitrates, ammonium salts & urea – which
is 46% nitrogen).
• Some nitrogen is organic but non – protein (nucleic acids).

44. Total ash
• It is not truly representative of mineral levels in either quantitative or
qualitative terms.
• This is true for sulphur & phosphorus either of which could be derived
from the organic or inorganic fractions.
• It is accurate for total Sodium (Na), Potassium (K) & Calcium (Ca).
• It is not correlated to availability to animals

45. Crude fiber
• It does not give a true estimate of the least digestible part of fiber in
many feedstuffs – particularly straw.
• During the boiling with acid and alkali, some solubilisation and thus
loss of lignin and hemicellulose occurs.

46. Ether extract
• The EE is supposed to contain only true fats i.e. esters of fatty acids
with glycerol.
• But in reality the crude fat also contain chlorophyll, sterols , waxes
and other plant constituents soluble in ether.
• Nitrogen Free Extract:
• It is not an accurate assessment because it is influenced by errors in all
the constituent analyses.

47. Fibre fractionations

48. 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
*Lignin, silica & some protein * organic acids, protein, pectin

49. • Cell contents- are soluble & digestible by enzymes secreted
in digestive tract of all animals.
• Cell wall contents- are insoluble & are digested by only
micro-organisms in the gastro-intestinal tract.

50. --- NDF ---NDS – have an average digestibility of about 98%
Acid detergent solubles
ADF (Acid detergent fibre) Hemicellulose
Cellulose ,Lignin , Silica
72% H2SO4
KMnO4 CELLULOSE dissolved
Method (pH 3.0) Lignin + minerals 550oc
Ash LIGNIN lost by ignition
Cellulose +minerals 550 oc Lignin lost by oxidation
Ash Cellulose lost by ignition

51. 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.

52. • 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.

53. • It under estimates the cell wall content of legumes.
• Legumes and other non-grass species contain relatively high
concentrations of pectic polysaccharides that are extracted by neutral
detergent & not included in their NDF fraction.
• Hence Neutral Detergent Fiber & cell wall are not synonyms.

54. 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 )

55. 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.

56. • 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.

57. Acid Detergent Insoluble Nitrogen
• Forage processing temparatures of over 500C tend to increase the
lignin yields with either of the above methods largely by the
production of artifact lignin via the non enzymic browning reaction.
• The nitrogen content of the ADF is considered to be a sensitive
measure of the extent of such damage and serves as a basis for
estimating artifact lignin.
• This is called acid detergent insoluble nitrogen (ADIN).
• CP of acid detergent insoluble nitrogen is ADICP.

58. 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.

59. THANK YOU