Food Composition Analysis – the
basics
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Moisture and Total Solids
Ash
Protein Analysis
Vitamin Analysis
Lipid (Fat) Analysis
Carbohydrate Analysis
Secondary Metabolites and Nutraceuticals
From: Nielsen, “Food Analysis”, 3rd edition, Kluwer, 2003
AOAC International
• Established in 1884 by USDA as the Association
of Official Agricultural Chemists
• Now the Association of Official Analytical
Chemists (reflects membership)
• Includes microbiologists and food scientists
• Most of the accepted methods to analyze foods
have been developed and/or validated by AOAC
• Three methods validation programs, the AOAC®
Official Methods Program® Peer-Verified
Methods Program, and the AOAC®
Performance Tested Methods Program
Moisture content and total solids
• Necessary to know when computing nutritional
value
• May affect stability of dehydrated foods
• Moisture content may be specified in
compositional standards
– (i.e. cheddar cheese must be < 39% moisture)
• 3 forms of water in food products
– free water
– adsorbed – held tightly in cell walls or to proteins
– hydrates – some proteins or salts exist as hydrates
• Best method depends on primary form and may
be specified by AOAC guidelines
Methods to determine water
content/total solids
• Sample handling must be controlled to avoid inadvertent
moisture loss
• Oven drying – sample heated under specified conditions,
weight determined by difference
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convection, forced draft or vacuum ovens
sample may be steam-dried or air dried prior to oven drying
particle size & surface area affects rate of loss
high temp. (250oC) dries more completely, lessens time required,
but may cause decomposition, loss of volatiles
– carbohydrate decomposition also possible
• Vacuum ovens allow drying at a lower temp, shorter time
• Freeze-drying can prevent thermal decomposition
– requires sample be pre-frozen
Methods to determine water
content – non-oven
• Microwave analysis
• Infrared drying
• Distillation
– sample is co-distilled with high bp solvent
• Karl Fischer titration
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more accurate for low-moisture foods
2H2O + SO2 + I2  C5H2SO4 + 2HI
pyridine & methanol used to dissolve reagents
Unreacted iodine is measured visually or by potentiometry
• Electrical methods
• Refractometry
• Water activity – measure of vaporization
Ash
• Inorganic matter remaining after oxidation or
ignition of a sample
• Ash content = total mineral content
• Dry ashing converts most minerals (Fe, Se, Pb,
etc.) to oxides, sulfates, phosphates, chlorides &
silicates
• Wet ashing used for minerals where
volatilization is an issue
• Uses mixtures of HNO3, H2SO4/H2O2 and HClO4
to oxidize materials completely
• Fresh foods usually low in ash; high-ash foods
linked to digestive issues
Basic protein analysis
Nielsen, Ch. 9
• Food proteins are varied in structure and size (5 kDa –
1000 kDa or more)
• N is the distinguishing element and N content ranges
from 13 – 19% in proteins
• accurate analyses important particularly for enzymes
• other N-containing molecules (free aa’s, small peptides,
nucleic acids, alkaloids, amino sugars, some vitamins)
interfere
• proteins easily denatured by heat, acid, base, organics &
detergents
• most analyses based on determination of N, peptide
bonds, aromatic aa’s, uv-absorption, light-scattering and
binding dye molecules
Peptide backbone
R
R
O
H
H
H
N
C
H3 N
O
H
H
N
C
N
H
C
R
O
H
H 3C
CH 3
O
H
N
C
C
O
H3 C
HC
H
N
H
O
C
O
H
H2 C
OH
• R groups (aa side chains) may be hydrophilic (polar),
hydrophobic (nonpolar), aromatic, acidic or basic
• Some analyses (e.g. Kjeldahl method) require hydrolysis of
peptide linkages to liberate free amino acids (digestion)
• Some require that peptide linkages remain intact so that
peptide functional group can be detected (IR), metal chelation
• Some require intact protein structure which can interact with
a dye, producing a detectable endpoint
Kjeldahl method (AOAC)
• Sample is digested with H2SO4 and a metallic catalyst
(Hg, SeO2, or Cu)
• KMnO4 added to fully oxidize N to NH4SO4
• Base is added to release free NH3, which is then distilled
into boric acid solution
– (NH4)2SO4 + 2 NaOH  2 NH3 + Na2SO4 + 2 H2O
– NH3 + H3BO3  NH4+ + H2BO3– H2BO3- + H+  H3BO3
• Borate is titrated with std. HCl
– Moles HCl = moles NH3 = moles N in original sample
– avg. protein = 16% N, so %N x 6.25 = % protein
• Disadvantage: measures all N sources, slow
Dumas method (AOAC)
• Combustion of samples releases N2 gas
• N2 quantified by GC with TCD detection
Infrared spectroscopy methods (AOAC)
• Protein can be quantified using absorption bands in the IR
(2.5 – 15 um or 4000-600 cm-1)
• Amides have several bands in 1560-1695 cm-1 range
(C=O stretch, N-H bending)
• Near IR (700-2500 nm) instruments also used to detect N-H
deformation abs @ 2080-2220 nm (more on IR: Ch. 24)
• J. Agric. Food Chem. 2010, 58, 702–706 – NIR with PLS regression analysis to
predict protein content of bean seeds
Biuret and Lowry methods
• Under basic conditions, peptide bonds complex Cu2+ 
purple color
• Biuret reagent: CuSO4, NaOH, potassium sodium
tartrate (stabilizes Cu2+)
• Reagent mixed with sample at RT, abs @ 540 nm
measured, std against BSA
• Lowry method uses Biuret reagent plus Folin-Ciocalteu
reagent (phosphomolybdic/phosphotungstic acids) which
reacts with tyrosine & tryptophan  blue-green color
• Samples measured @ 650 nm
• Advantage: greater sensitivity and specificity
• Disadvantage: interference by sugars, lipids, phosphate
buffer, polyphenols
Bradford assay
• Uses Coomassie Brilliant Blue G-250 dye
• When protein solution is acidified below pI,
electrostatic interaction w/dye
• Changes red  blue upon binding protein
• lmax 465 nm  595 nm
• Abs read @ 595 nm
• Protein concentration
determined by comparison to std curve for BSA
Advantages: even more sensitive than Lowry
and no interference from sugars or polyphenols