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PHYSICO CHEMICAL
ANALYSIS
FEED EVALUATION
by
Margarita T. Arnaiz
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OUTLINE
1. Introduction2. Physical evaluation
3. Chemical evaluation
a. proximate analysismoisture, 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)
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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
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Evaluation methods
Physicalrely on the physical characteristics of
feedstuffs but little information on the nutritional value
Chemicalcharacterize or define the chemical nutrients
present
Biologicalutilized 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
Microbiologicaluse of microorganisms to evaluate
presence of nutrients;
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Physical Evaluation
Use of sensesmellrancidity of oil
tasteoff flavors
visualextraneous matters; homogeneity of ingredients
touchwetness, dryness
Attractabilityhow fast the animal is attracted
Bulk densitywt in gram/liter
Water stability test-weight loss after it is placedin water after a certain period
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Chemical Evaluation
Proximate analysis Developed by Wendee Experimental Station in
Germany over a 100 years ago
most generally used chemical scheme for describingfeedstuffs
general overview of the chemical composition feeds,
partitioning compounds with common chemical
properties
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Proximate analysis
Moisture
Crude protein
Crude fat or ether extract Crude fiber
Ash
Nitrogen free extract
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Flow Diagram for Proximate analysis
Feed sample
Oven dry@ 105C
Moisture
Moisture free sample
Incinerate@600C
Ash
Crude protein, CP
100-(CP+Cft+Cfb+Ash) NFE Crude fiber, Cfb
Boil in acid, boil in alkali
Oven dry@105C, incinerate
@ 550C
Fat free residue
DigestionDistillation
TitrationCrude fat, Cft
Ether extraction
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Sample preparation
grinder
formulated feed
as receivedGround feed
% moisture determination
Other proximate components
Oven dry @ 105 to
remove moisture
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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
Reference Test method
-AOAC 930.15 (oven method)
-Moisture analyzer
Moisture
analyzer
Oven
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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 CuSO4catalyst,
converting N to NH3, which is distilled and titrated
Steps
1.Digestionnitrogenous 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 soln3.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
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Crude protein analysis by Kjeltec method
(automatic distillation and titration)
Digestion at 420oC
Catalyst,
CuSO4+K2SO4
H2SO4,conc
6 mL
Spl, 0.1g Digested sample
1.Insert tube with digestedspl
2.Input weight
3. Automatic distillation
and titration
4. Read out % CP
(Factor 6.25)
To Kjeltec apparatus
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
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Conversion factors for some protein sources
Origin Per cent N in protein,
%
Conversion Factor
Oil seed
proteins
18.5 5.4
Cereal
proteins
17 5.9
Plant leaf 15 6.6Animal or fish 16 6.25
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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)
Soxtec 2055 (automated)
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 :
%Fat = [(Wt cup with fat residue-Wt cup) /Wt sample]x100
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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 500oCFibertec
Calculation: % Cfb = [(Wt at 105oCWt at 500oC)/ initial Wt sample] x100
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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
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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)
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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 100b) 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
*with blank correction
Crude fiber AOAC 978.10: Fiber (Crude) in animal Feedand Pet Food
(By FIBERTEC M6 1020)
[(Wt at 105-wt at 500)*/initial weight] x100
*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)
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Energy content
Can be measured directly
using the bomb calorimeter
Indirectly from theestimated composition (i.e.
protein, lipid, &
carbohydrates) by
multiplying the energy
conversion factor for eachnutrient and summing them
Bomb calorimeter
www.techknow.org.
uk
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Specific Nutrient contents
Amino acids
Fatty acids
Vitamins
Minerals
Endogenous (urease,trypsin inhibitors, etc)
Exogenous ( aflatoxins, pesticides, heavy metals, etc)
Feed additives
Antibiotics, antioxidants ( ex. Ethoxyquin)
Anti-nutrient contents
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Amino acids
Amino acids (AA) can be
analyzed using a dedicatedamino acid analyzer or high
performance liquid
chromatograph (HPLC)
Purified proteins are hydrolyzed
and injected in HPLC withfluorescence detector and cat-
ion exchange resin column.
Mobile phase with varying ionic
strength and pH carries the
sample across the columnwhere separation of different AA
takes place. Identification and
quantification is effected by
calibration of an AA standard
solution
HPLC
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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
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Vitamins
Variety of nutrients whichdo 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
Inorganic components
which can be analyzed
from the ash (Ca, P, Fe,
etc)
Can be analyzed using
atomic absorptionspectrophotometer
(AAS), inductively
coupled plasma (ICP),
and also by colorimetricmethods
Minerals
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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
PesticidesGas Chromatography
Heavy metals - AAS
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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
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Shake & centrifuge
Solvent removal
Filter
( 0.22m membrane filter)
Inject in HPLC
Homogenize
Sample preparation, extraction and HPLC analysis for antibiotics
Analysis & quantification
Sample
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Exercises for proximate analysis
Moisture Wt of dish Wt initial Wt Final %Moisture Average
Trial 1 22.0879 24.3992 24.3435
Trial 2 24.8102 26.9212 26.7729
Ash Wt crucible Wt Spl Wt crucible+ash %Ash
Trial 1 19.3038 2.0211 19.9151
Trial 2 20.6886 2.0121 21.2946
CP Wt sample % N %CP
Trial 1 0.1019 8.857
Trial 2 0.1063 8.8684
CFt Wt cup Wt sample Wt cup +fat %Cft
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 % Cfb
Trial 1 29.517 29.49 0.0188*
Trial 2 27.0086 27.0086 0.0139*
With blank correction
Problem: Estimate the nitrogen free extract (NFE) of the sample
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THANK YOU
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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 30.18
Trial 2 20.6886 2.0121 21.2946 30.12
CP Wt sample % N %CP
Trial 1 0.1019 8.857 55.36 55.39
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 2.76
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 1.61
Trial 2 27.0086 27.0086 0.0139* 1.33
With blank correction
Problem: Estimate the nitrogen free extract (NFE) of the sample (10 O6%)