Analysis of Bulk Food Components (Proximate Analysis)

Bibliography
Kirk,RS & Sawyer,R (1991) Pearsons composition & analysis of Foods, 9th edn, Longman, ISBN 0582409101 Nielsen S.S. (2003) Food analysis 3rd ed. Kluwer Academic/Plenum Publishers, 2003. ISBN: 0306474956 Coultate,TP (2009) Food The chemistry of its components, 5th edn, Royal Society of Chemistry, ISBN 9780854041114 Near IR spectroscopy: http://www.spectroscopyeurope.com/NIR_18_3.pdf

Definition of Proximate Analysis

Analysis of foods for nitrogen (for protein), ether extract (for fat), crude fibre and ash (mineral salts), together with soluble carbohydrate calculated by subtracting these values from the total (carbohydrate by difference).

Why Analyse Food?

Legislation e.g. fat in chocolate, additives Nutrition - a) macronutrients; b) micronutrients Nutritional labeling eg fat, protein Toxicology (a) Natural toxins e.g. alkaloids (b) fungal toxin (mycotoxin) (c) contaminant Control of Trade Food manufacture & Quality

Accuracy and Precision

Accuracy - closeness of observed result to true result Precision - Agreement between replicate determinations

Accurate Precise (no bias) Reproducible

Inaccurate Precise or imprecise? (reproducible) Bias or no bias

Composition of Foods
Water Protein Fat Carbohydrates (soluble) Dietary fibre (non-starch polysaccharides plus lignin) Minor components eg vitamins, pigments, organic acids, flavonoids etc.

Strategy for Food Analysis

Obtain representative sample http://www.encyclopedia.com/video/0j8DpLjI meA-proximate-analysis-samplepreparation.aspx Pretreatment e.g. heating to inactivate enzymes, drying etc Extraction/ matrix reduction/ particle size reduction often required Separation/ purification Identification (minimise errors at all stages)

WATER IN FOOD
1.FREE WATER- water that is free to act as a solvent for molecular solutions or colloids 2.ADSORBED WATER - Water is adsorbed onto internal or external surfaces of solids (hydrogen bonding) 3.CONSTITUTIONAL WATER- Present in chemical combination as water of hydration NB - Boiling point 3>2>1

Moisture Content of Food

FRUIT Apples 79-91% Oranges 83-89 VEGETABLES Carrots 83-91 Tomatoes 91-97 MILK PRODUCTS Milk 87-90 Cheese (hard) ~37 MEAT PRODUCTS Beef cuts 55-70 % Ham 53 FISH Cod 83 Salmon 63 DRIED FOODS Soup Powder 7 Cocoa 5

Determination of waterOven Drying

Sample preparation- grinding, dispersion Select weight of sample and container size. Distribution of sample important Drying conditions: 70-130C / 25-760 mm mercury pressure Dry for Constant time or to constant weight Air stream if infrared heating used

Problems in Oven Drying

Mainly free water Reactions of carbohydrates may produce water Loss of other volatiles Oxidation of unsaturated fatty acids, phenols in some foods Practical problems:- case hardening, reabsorption of moisture during weighing

Karl-Fischer Titration to Determine Water Content

Titration between food containing water extracted into, or dissolved in, methanol and Karl Fischer reagent ( iodine, sulfur dioxide, pyridine in methanol) Determine end point of titration by electrical conductivity I2 + SO2 + H2O + CH3OH + 3 C5H5N = 2C5H5N.HI + C5H5NH. SO4.CH3

Dean and Stark Volumetric method for water

Collected water Solvent (toluene) food Heat

Lipid Analysis
All lipids are soluble in non-polar organic solvents and have low solubility in water Lipid class mainly comprises edible fats, which contain triglycerides (>90%), diglycerides, monoglycerides, phospholipids (<2%), sterols (<1%), tocopherols (~200ppm), free fatty acids

Fat content of foods

Butter 80% Margarine 80% Reduced / Low-fat spreads 20-60% Chocolate 30% Milk 3 - 4%

Soxhlet extractor for lipids in solid foods

Water in

Water out food

solvent

http://www.rsc-teacherfellows.net/labTechniques/SoxhletExtractionAnimation.htm

Fat extraction (contd)

Soxtec method: http://www.encyclopedia.com/video/ylyjm8 iY23Q-proximate-analysis-percentfat.aspx Treat food with acid or alkali to release lipid from interfering components eg Rose-Gottlieb method Treat food with conc. ammonia. Then extract fat with solvents (eg milk products)

Gerber method for fat in milk products

Volumetric method Treat milk in milk butyrometer with Macdonalds reagent ( emulsifier, butanol, methylated spirits solution) or concentrated sulfuric acid. Heat at 65C for 5 min. Centrifuge. Heat at 65C for 5 min. DIRECT READING IN % FAT ON CALIBRATED SCALE

Milk butyrometer for the Gerber method

Add milk, Macdonalds reagent/ sulfuric acid

fat

Aqueous phase of milk, Macdonalds reagent

stopper

Johan Kjeldahl

Development of Kjeldahl method

Johan Kjeldahl
employed by Carlsberg brewery to develop assay for grain protein

less protein in grain = more beer

published 1883

Protein Content of Food

Flour milk cheese (swiss) eggs orange juice beef (roast)

% Dry wt 11.9 26.9 45.1 49.3 6.3 62.5

% Fresh 10.5 3.5 27.5 12.8 0.8 25.0

KJELDAHL PROTEIN ANALYSIS

Weigh food; add conc.sulphuric acid, selenium and potassium sulphate; heat under reflux until sample is fully dissolved Dilute to volume. Introduce into distillation apparatus. Add sodium hydroxide Distill and collect in boric acid solution Add screened methyl red indicator and titrate with sulphuric acid till grey colour

Kjeldahl distillation unit

Sodium hydroxide

Food digest

Boric acid

Chemical Changes in the Kjeldahl Analysis of Protein

Digestion Protein N + H2 SO4 ------> (NH4)2 SO4 Distillation (NH4)2 SO4 + 2NaOH -----> NH3 + Na2 SO4 NH3 + H3 BO3 --------> NH4 .H2 BO3 Titration (screened methyl red) 2NH4 .H2 BO3 + H2 SO4 -----> (NH4)2 SO4 + 2H3 BO3

Nitrogen to Protein Conversion factors

Wheat, barley, oats 5.83 Rice 5.95 Soybean 5.71 Milk 6.38 Meat, eggs 6.25

Other methods of Nitrogen determination

Dumas-Pregl Burn sample to CO2, H2O and nitrogen oxides (NO, N2O, NO2) at 1100oC. Reduce nitrogen oxides to nitrogen by heating with copper (at 600oC); remove CO2 and H2O, and determine nitrogen by gas chromatography http://www.encyclopedia.com/video/K87F Qf_yYYE-proximate-analysis-percentprotein.aspx

Carbohydrate analysis
Sugars - sweet, crystalline, water-soluble e.g.glucose, sucrose, lactose, fructose Polysaccharides - polymers which contain between 10 and several thousand sugar units eg starch Polysaccharides other than starch are non-digestible so are classified as dietary fibre (together with lignin)

Reducing sugars
Molecules with free aldehyde or ketone group are reducing sugars e.g. glucose fructose, lactose Sucrose is not a reducing sugar, since it does not contain a free aldehyde or ketone group; sucrose can readily be hydrolysed to glucose + fructose (acid catalysed)

Lane and Eynon method for Reducing Sugars

Reagent: Fehlings solution - alkaline copper tartrate Principle: a volumetric procedure based on a titration in which reducing sugars reduce Cu(II) to Cu(I). A redox indicator (methylene blue) is used. This is blue in presence of Cu(II), and becomes when all Cu (II) is reduced.

Lane and Eynon method for Reducing Sugars (II)

Procedure - heat Fehlings solution to boiling in a beaker. Add methylene blue indicator. Add sugar solution from a burette keeping sample boiling until blue colour disappears leaving an orange-red liquid (NB standardise the Fehlings reagent with a known sugar solution)

Dietary fibre
Definition: Polysaccharides (and other molecules e.g. lignin) that are not hydrolysed by the endogenous secretions of the mammalian digestive tract. Components include cellulose, hemicellulose, lignin and pectins from the walls of cells; resistant starch; and several other compounds

Sources of dietary fibre

vegetables, wheat and most other grains. Foods rich in soluble fibre include fruits, oats, barley and beans.

Analysis of Dietary Fibre

Methods can be classified as: Nonenzymatic-gravimetric Enzymatic-gravimetric Enzymatic-chemical methods, which include

Dietary fibre classified according to solubility in water

Total Dietary Fibre (TDF) - sum of soluble and insoluble polysaccharides and lignin Insoluble dietary fibre (IDF) which includes celluloses, some hemicelluloses and lignin; Soluble dietary fibre (SDF) which includes -glucans, pectins, gums, mucilages and some hemicelluloses.

Dietary fibre in foods

Product AOAC method Englyst method (g/100g)* (NSP) (g/100g) Apples (with skin) 2 1.6 Bananas 1.9 1.1 Carrots (boiled) 3.1 2.5 Baked beans 4.2 3.7 White bread 2 1.5 Brown bread 4.5 3.5 Wholemeal bread 7.4 5.8 *non-starch polysaccharides + resistant starch + lignin

AOAC method
gelatinize with a thermo-stable -amylase treat with protease and amyloglucosidase (to remove protein and starch) separate SDF and IDF by filtration Dry residue after filtration (IDF) and correct for protein and ash content. Weigh filtrate, containing SDF, precipitated with ethanol, recovered by filtration and dried. TDF = SDF + IDF.

Englyst method
isolation of DF with -amylase, followed by treatment with a mixture of pancreatin and pullulanase. Hydrolysis with sulfuric acid at 100C/ 2 h. Add NaOH and 3,5-dinitrosalicylate to develop colour; 100C for 10 min . absorbance was measured at 530 nm Prepare calibration plot with standard glucose samples.

New methods
Near infrared spectoscopy for fat, protein, carbohydrate and water but needs calibrating for each food. Nuclear Magnetic Resonance for fat HPLC for carbohydrates

Near IR spectra of soybean powder and defatted soybean

 Uses radiation in range 12500 - 4000 cm-1 Use for analysis of protein, fat, water, starch, sugars, fibre, alcohol etc Used widely by food industry for quality control eg grains (wheat, rice), soybeans, meat, wine near IR peaks are broad, overlapping peaks Calibrate method using standards with measured parameter determined by another method Use multiple linear regression to relate concentration to absorbance values at several wavelengths.

Near Infrared analysis