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FEED EVALUATION

PHYSICO-CHEMICAL
ANALYSIS
by
Margarita T. Arnaiz

OUTLINE

1. Introduction
2. Physical evaluation
3. Chemical evaluation
a. proximate analysis
moisture, crude protein, crude fat, crude fiber, ash, NFE

b. specific nutrient content


amino acids, fatty acids, vitamins and minerals

c. anti-nutrient contents
d. others
4. Exercise (problem solving)

Feed and ingredient evaluation

important to ensure effectiveness of feeds


to provide optimum growth of the animals
no single test will provide necessary data
for adequate feed evaluation
variety of tests methods are utilized to
provide information required to assess the
quality and nutritional value of feeds

Evaluation methods
Physical rely on the physical characteristics of
feedstuffs but little information on the nutritional value
Chemical characterize or define the chemical nutrients
present
Biological utilized live organisms, i.e., actual feeding
experiment; ascertains the true value of the feedstuff to
the animal; time-consuming, expensive, and requires
facilities for holding the animals
Microbiological use of microorganisms to evaluate
presence of nutrients;

Physical Evaluation

Use of sense
smell rancidity of oil
taste off flavors
visual extraneous matters; homogeneity of ingredients
touch wetness, dryness

Attractability how fast the animal is attracted


Bulk density wt in gram/liter
Water stability test-weight loss after it is placed
in water after a certain period

Chemical Evaluation

Proximate analysis
Developed by Wendee Experimental Station in
Germany over a 100 years ago
most generally used chemical scheme for describing
feedstuffs
general overview of the chemical composition feeds,
partitioning compounds with common chemical
properties

Proximate analysis

Moisture
Crude protein
Crude fat or ether extract
Crude fiber
Ash
Nitrogen free extract

Flow Diagram for Proximate analysis


Feed sample
Oven dry@ 105C

Moisture
Moisture free sample
Digestion
Distillation
Titration

Ether extraction

Crude fat, Cft


Incinerate@600C
Fat free residue

Crude protein, CP
Ash
Boil in acid, boil in alkali
Oven dry@105C, incinerate
@ 550C
100-(CP+Cft+Cfb+Ash)

NFE

Crude fiber, Cfb

Sample preparation

formulated feed
as received

Ground feed

grinder
% moisture determination

Oven dry @ 105 to


remove moisture

Other proximate components

Moisture

Loss in weight after oven drying at


105 - 135oC
High moisture content can lead to
growth of molds during storage
Moisture content should not be
more than 10% prior to storage

Moisture
analyzer

Reference Test method


-AOAC 930.15 (oven method)
-Moisture analyzer
Oven

Crude protein (CP)


Proteins are complex nitrogenous cmpds essential to life; source
of energy
Kjeldahl technique of analysis
-developed byJohan Kjeldahl (1883)
- discovered that proteins contain more or less 16% nitrogen
ReferenceTest method :AOAC 984.13
Principle: Sample is digested in H2SO4, using CuSO4 catalyst,
converting N to NH3, which is distilled and titrated
Steps
1.Digestion nitrogenous cmpds added with H2SO4 to break N bonds, forming NH4+
2: Distillation - NH4+is converted to NH3 (g) by NaOH, NH3(g) is distilled into acid soln
3.Titration Unreacted acid titrated with NaOH

Calculation: Nitrogen = (ccxN)acid-(ccxN)base x 14.0067


%Nitrogen =(Wt N/wt sample) x 100
%CP
= 6.25 X Nitrogen

Crude protein analysis by Kjeltec method


(automatic distillation and titration)

Spl, 0.1g

Digested sample

Catalyst,
CuSO4+K2SO4

To Kjeltec apparatus

H2SO4,conc
6 mL

Digestion at 420oC

3.1.NaOH added,
3.2.NH3(g) directed towards vessel
with boric acid + indicators
3.3.Titration with std acid (HCl) with
end point electrode sensor
3.4. automatic calculation of %Cp

1.Insert tube with digested


spl
2.Input weight
3. Automatic distillation
and titration
4. Read out % CP
(Factor 6.25)

Conversion factors for some protein sources


Origin

Per cent N in protein,


%

Conversion Factor

Oil seed
proteins
Cereal
proteins
Plant leaf
Animal or fish

18.5

5.4

17

5.9

15
16

6.6
6.25

Crude fat/ether extract


Material extractable with organic
solvent, e.g. ether
Extractable matters incl lipids, fat
soluble vitamins (A,D&E), some cmpds
with little nutritional value to animals
(chlorophyll, volatile oils, resins
pigments, waxes)
Soxhlet method of analysis
ReferenceTest method: AOAC 991.36
(Using SOXTEC 2055)

Soxhlet apparatus (conventional)

Steps;
1. boiling in solvent (petroleum ether)
2. Draining of solvent
3. Recovery of solvent
4. Drying of cups with fat residue at 105oC
Calculation :
Soxtec 2055 (automated)
%Fat = [(Wt cup with fat residue-Wt cup) /Wt sample]x100

Crude fiber

Refers to the residue of a feed that is


insoluble after successive boiling in acid and
base
Generally regarded as the indigestible
portion of carbohydrate having no nutritional
value
Method of determination simulates the
digestive tract, acidic in the stomach and
alkaline in the small intestine
Reference Test Method : AOAC 978.10
using Fibertec 1020
Steps:
1. Defatted sample in fritted glass crucible
boiled with acid, filtered & wash with water
2. Boiling with alkali, filtered & wash with water
3. Drying at 105oC
4. Ashing at 500oC

Fibertec

Calculation: % Cfb = [(Wt at 105oC Wt at 500oC)/ initial Wt sample] x100

Ash
Inorganic residue
obtained by burning off
organic matter
Residue after burning
allows for mineral analysis
Reference Test Method:
AOAC 942.05
Sample is burned in
furnace for 2 hours at
600oC. The loss in weight
is the amount of ash.
Furnace

Nitrogen-free extract (NFE)

Represents supposedly the soluble


carbohydrate of the feeds, but may also
contain hemicellulose and lignin
Determined by mathematical calculation

% NFE = 100 (%Cft + % CP + % Cfb+%Ash)

Summary of proximate analysis


Component

Reference Test Method

Determination

Moisture

a)AOAC 930.15: Moisture in Animal Feed


b) Moisture analyzer (Mettler Toledo MJ33)

Loss in weight after drying x 100


a)Loss in weight after drying x 100
b) direct reading

Crude fat/ether
extract

AOAC 991.36: Fat (Crude) in Meat and Meat


Products
(by SOXTEC 2055)

(Wt fat residue*/wt of sample)x 100

Crude fiber

AOAC 978.10: Fiber (Crude) in animal Feed


and Pet Food
(By FIBERTEC M6 1020)

[(Wt at 105-wt at 500)*/initial weight] x


100
*with blank correction

Crude protein

(by KJELTEC 2300)) AOAC 984.13: Protein


(Crude) in Animal and Pet Food

% N x 6.25)

Ash

AOAC 942.05: Ash of Animal Feed

Loss in weight after ashing x 100

NFE

By difference

100-(CP+Cfb+Cft+Ash)

*with blank correction

Energy content

Indirectly from the


estimated composition (i.e.
protein, lipid, &
carbohydrates) by
multiplying the energy
conversion factor for each
nutrient and summing them

www.techknow.org.uk

Can be measured directly


using the bomb calorimeter

Bomb calorimeter

Specific Nutrient contents

Amino acids
Fatty acids
Vitamins
Minerals

Anti-nutrient contents
Endogenous (urease,trypsin inhibitors, etc)
Exogenous ( aflatoxins, pesticides, heavy metals, etc)
Feed additives
Antibiotics, antioxidants ( ex. Ethoxyquin)

Amino acids
Amino acids (AA) can be
analyzed using a dedicated
amino acid analyzer or high
performance liquid
chromatograph (HPLC)
Purified proteins are hydrolyzed
and injected in HPLC with
fluorescence detector and cation exchange resin column.
Mobile phase with varying ionic
strength and pH carries the
sample across the column
where separation of different AA
takes place. Identification and
quantification is effected by
calibration of an AA standard
solution

HPLC

Fatty acid
Fatty acids serve as source of
energy for many physiological
functions in organisms
Can be analyzed using gas
chromatograph (GC) with flame
ionization detector
Lipid/fat are extracted from the
sample using organic solvents
(chloroform-methanol),
saponified and formed into
esters prior to injection in the
GC. The sample upon injection
is carried by an inert gas (e.g.
He) across a capillary column
where separation takes place.
Identification and profiling is
aided by a mixed fatty acid
standard.

GC

Vitamins
Variety of nutrients which
do not include proteins,
lipids, and carbohydrates
Required in small
amounts for normal
growth
Can be water soluble
( thiamin, riboflavin,
ascorbic acid, etc)
Some vitamins can be
analyzed using HPLC

Minerals
Inorganic components
which can be analyzed
from the ash (Ca, P, Fe,
etc)
Can be analyzed using
atomic absorption
spectrophotometer
(AAS), inductively
coupled plasma (ICP),
and also by colorimetric
methods

Anti-nutrients determination

Substances that can affect the health and


normal performance of the animal, either
toxic or inhibit metabolic reactions
Trypsin inhibitors - spectrophotometry
Aflatoxin - chromatography
Pesticides Gas Chromatography
Heavy metals - AAS

Feed additives
Feed components that can affect food safety of
aquaculture products
Antibiotics
- added to feeds to control diseases (treatment and
prophylaxis) or as growth enhancer
Anti-oxidants
- added to feeds to prevent their deterioration, especially
oxidation of fats (prevention of rancidity), ex. Ethoxyquin
Mostly analyzed and widely accepted method of analysis
is by the use of HPLC

Sample preparation, extraction and HPLC analysis for antibiotics


Sample
Homogenize

Shake & centrifuge

Solvent removal

Filter
( 0.22m membrane filter)
Inject in HPLC

Analysis & quantification

Exercises for proximate analysis


Moisture

Wt of dish

Wt initial

Wt Final

Trial 1

22.0879

24.3992

24.3435

Trial 2

24.8102

26.9212

26.7729

Ash

Wt crucible

Wt Spl

Wt crucible+ash

Trial 1

19.3038

2.0211

19.9151

Trial 2

20.6886

2.0121

21.2946

CP

Wt sample

%N

Trial 1

0.1019

8.857

Trial 2

0.1063

8.8684

CFt

Wt cup

Wt sample

Wt cup +fat

Trial 1

22.397

1.0009

22.4229*

Trial 2

22.3373

1.0422

25.3678*

Cfb

Wt crucible @
105

Wt crucible @500

Net wt of Cfb

Trial 1

29.517

29.49

0.0188*

Trial 2

27.0086

27.0086

0.0139*

%Moisture

%Ash

%CP

%Cft

% Cfb

With blank correction

Problem: Estimate the nitrogen free extract (NFE) of the sample

Average

THANK YOU

Exercises for proximate analysis


Moisture

Wt of dish

Wt initial

Wt Final

%Moisture

Average(%)

Trial 1

22.0879

24.3992

24.3435

11.31

11.32

Trial 2

24.8102

26.9212

26.7729

11.33

Ash

Wt crucible

Wt Spl

Wt crucible+ash

%Ash

Trial 1

19.3038

2.0211

19.9151

30.25

Trial 2

20.6886

2.0121

21.2946

30.12

CP

Wt sample

%N

%CP

Trial 1

0.1019

8.857

55.36

Trial 2

0.1063

8.8684

55.43

CFt

Wt cup

Wt sample

Wt cup +fat

%Cft

Trial 1

22.397

1.0009

22.4229*

2.59

Trial 2

22.3373

1.0422

25.3678*

2.93

Cfb

Wt crucible @
105

Wt crucible @500

Net wt of Cfb

% Cfb

Trial 1

29.517

29.49

0.0188*

1.88

Trial 2

27.0086

27.0086

0.0139*

1.33

30.18

55.39

2.76

1.61

With blank correction

Problem: Estimate the nitrogen free extract (NFE) of the sample (10.O6%)

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