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Food Composition Analysis – the basics Moisture and Total Solids Ash Protein Analysis Vitamin Analysis Lipid (Fat) Analysis Carbohydrate Analysis Secondary.

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Presentation on theme: "Food Composition Analysis – the basics Moisture and Total Solids Ash Protein Analysis Vitamin Analysis Lipid (Fat) Analysis Carbohydrate Analysis Secondary."— Presentation transcript:

1 Food Composition Analysis – the basics Moisture and Total Solids Ash Protein Analysis Vitamin Analysis Lipid (Fat) Analysis Carbohydrate Analysis Secondary Metabolites and Nutraceuticals From: Nielsen, “Food Analysis”, 3 rd edition, Kluwer, 2003

2 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

3 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

4 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 –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. (250 o C) 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

5 Microwave analysis Infrared drying Distillation –sample is co-distilled with high bp solvent Karl Fischer titration –more accurate for low-moisture foods –2H 2 O + SO 2 + I 2  C 5 H 2 SO 4 + 2HI –pyridine & methanol used to dissolve reagents –Unreacted iodine is measured visually or by potentiometry Electrical methods Refractometry Water activity – measure of vaporization Methods to determine water content – non-oven

6 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 HNO 3, H 2 SO 4 /H 2 O 2 and HClO 4 to oxidize materials completely Fresh foods usually low in ash; high-ash foods linked to digestive issues

7 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

8 Peptide backbone 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

9 Kjeldahl method (AOAC) Sample is digested with H 2 SO 4 and a metallic catalyst (Hg, SeO 2, or Cu) KMnO 4 added to fully oxidize N to NH 4 SO 4 Base is added to release free NH 3, which is then distilled into boric acid solution –(NH 4 ) 2 SO 4 + 2 NaOH  2 NH 3 + Na 2 SO 4 + 2 H 2 O –NH 3 + H 3 BO 3  NH 4 + + H 2 BO 3 - –H 2 BO 3 - + H+  H 3 BO 3 Borate is titrated with std. HCl –Moles HCl = moles NH 3 = moles N in original sample –avg. protein = 16% N, so %N x 6.25 = % protein Disadvantage: measures all N sources, slow

10 Dumas method (AOAC) Combustion of samples releases N 2 gas N 2 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 (Ch. 24)

11 Biuret and Lowry methods Under basic conditions, peptide bonds complex Cu 2+  purple color Biuret reagent: CuSO 4, NaOH, potassium sodium tartrate (stabilizes Cu 2+ ) 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

12 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 max 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

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