Food Analysis: Lipids Lecture PDF

Food Analysis: Lipids Saji George Associate Professor, Department of Food Science and Agricultural Chemistry, Macdonald-Stewart Building, Room-1039 (MS1-039) Tel: 514-398-7920, Fax: 514-398-7990 Email: [email protected] Web: http...

Food Analysis: Lipids Saji George Associate Professor, Department of Food Science and Agricultural Chemistry, Macdonald-Stewart Building, Room-1039 (MS1-039) Tel: 514-398-7920, Fax: 514-398-7990 Email: [email protected] Web: http://safe-nano.lab.mcgill.ca/ SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Content Lipid ▪ Relevance ▪ Principle ▪ Methods SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Lipids: Definition and Relevance Lipid: soluble in organic solvents but insoluble or sparingly soluble in water FDA definition: total fat is the sum of fatty acids from C4 to C24, calculated as triglycerides. Oils: triacylglycerols that are liquid at room temperature (olive oil) Fats: triacylglycerols that are solid at room temperature SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Lipids: Definition and Relevance General classifications 1. Simple lipids: ester of fatty acids with alcohol (i.e., fats, waxes) 2. Compound lipids: compounds containing groups in addition to an ester of a fatty acid with an alcohol (i.e., phospholipids, cerebrosides, and sphingolipids) 3. Derived lipids: substances derived from neutral lipids or compound lipids (e.g., fatty acids, long- chain alcohols, sterols, fat-soluble vitamins, and hydrocarbons) SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Lipids: Definition and Relevance Why is it important to measure Fat content in food materials? - Nutritional evaluation - Health / diet restrictions - Food quality (mouth feel texture) - Economic consideration SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Relevance of fat determination Levels of Fat content in the food products : Product Fat % (fresh) Fat % (dry) Milk 3.9 30 Bread 0.8 – 3.3 1.2 – 5.1 Avocados 26.4 76.4 Almonds 54.1 56.8 Potatoes 0.1 0.5 Apple 0.4 2.5 Asparagus 0.2 2.9 SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Principle of Fat Analysis The insolubility of lipids in water makes possible their separation form proteins and carbohydrates in foods. Lipids have a wide range of relative hydrophobicity depending on their molecular constituents. Lipids in Foods : Triglycerides (TG), Free fatty acids (FFA), Phospholipids, monoglycerides (MG), diglycerides (DG), sterols, complex lipids and waxes. SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Principle of Fat Analysis Crude fat represents mainly non polar lipids, Triglycerides (TG) because of using non polar organic solvents. Crude fat is extracted by dry extraction techniques (Soxhelt method and Goldfish method). Total lipids represent non polar and polar lipids, e.g. Fat content of milk TG (non polar) + phospholipids (polar lipids). Total lipids are extracted by wet extraction techniques (Babcock Method, Gerber Method, Majonnier Method and Bligh & Dyer method). SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Determination of Fat Content Fat Extraction (Nature of the sample) Dry Extraction (Crude fat) Wet Extraction (Total Lipids) Soxhelt Goldfish Babcock Majonnier Bligh&Dyer (Intermittent method) (Continues method) Fat Extraction (type of chemicals) Solvent Extraction Non solvent Extraction Soxhelt Goldfish Majonnier Bligh&Dyer Babcock Gerber (Intermittent method) (Continues method) (Discontinues method) SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Fat Analysis: methods Fat Extraction Dry Extraction (Crude fat) Wet Extraction (Total Lipids) Soxhelt Goldfish Babcock Majonnier Bligh&Dyer (Intermittent method) (Continues method) SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Determination of Crude Fat There are three basic ways of carrying out the Dry Extraction: - Discontinuous Extraction with a high ratio of organic solvent to sample. - Intermittent Extraction in which solvent is in contact with the sample intermittently, e.g. Soxhlet. - Continuous Extraction in which solvent is always in contact with the sample for a given period of time, e.g. Goldfish. SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Determination of Crude Fat ✓ Crude Fat refers to the residue obtained after removal of the non polar organic solvent used to extract a dried sample. ✓ The extraction of food material by a non polar solvent will give an estimate of “Crude Fat”, but not of “Total Lipids”. ✓ The major component of lipids in food materials is triglycerides. Crude Fat measures mainly the triglycerides which in many foods represent more than 95% of total lipids. SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Determination of Crude Fat Dry techniques (Soxhelt method and Goldfish method) are used to measure crude fat. Dry food sample Extraction solution of fat in the solvent non polar organic solvent Evaporation of organic solvent Residue (Crude Fat) The non polar organic solvent only extracts non polar materials (mainly TGs). SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Properties of Solvents used Ideal solvent properties, Should have a high solvent power for lipids and low or no solvent power for proteins, amino acids, and carbohydrates Should evaporate readily and leave no residue Should have a relatively low boiling point Should not leave behind any residue Should be nonflammable and nontoxic Should penetrate sample particles readily Should be nonhygroscopic Should be cost effective SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Determination of Crude Fat Extracting Solvents for crude fat determination. Most non polar organic solvents are used; Examples: 1- Hydrocarbons (e. g. Hexane, heptane and pentane) Petroleum ether (Combination of pentane and hexane, Most commonly used, low boiling point, volatile, nonpolar) 2- Diethyl ether (More polar than petroleum ether); it absorbs O2 and forms peroxides which in presence of heat could explode (safety concern). 3- n-Butanol is more polar than the other mentioned solvents. SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Determination of Crude Fat Pentane (C5)/ bp360C Hexane (C6)/ bp690C Heptane (C7)/ bp980C n-Butanol (C4-OH)/ Diethyl ether (C2H5-O-C2H5)/ bp 350C bp 1180C SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Determination of Crude Fat Petroleum Ether. ✓ It is the low boiling point fraction (bp 35-380C) of petroleum. ✓ It contains mainly hexanes and pentanes (not a single chemistry) ✓ It is more hydrophobic than diethyl ether and therefore selective for more hydrophobic lipids. SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Sample preparation Avoid lipid oxidation by, Using inert atmosphere (with nitrogen) Preparing sample under low temperature Avoiding excessive light General steps in sample preparation ▪ Removal of water: To enhance penetration of non-polar solvents ▪ Reduction of particle size: Grinding (of nuts) enhances surface area ▪ Separation of the lipid from bound proteins and/or carbohydrates through the use of alkaline or acid hydrolysis SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Sample preparation Meat Hydrolysis by HCl Solvent extraction for crude Sample fat Lipids in foods such as dairy, bread, flour, and animal products is bound to proteins and carbohydrates, and direct extraction with nonpolar solvents is inefficient. Acid hydrolysis, enable the penetration of solvent into the sample. break both covalently and ionically bound lipids into easily extractable lipid forms. SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Determination of Crude Fat Intermittent Extraction- semi-continuous solvent extraction Example: Soxhlet Extraction. Using soxhlet instrument Consist of a flask (2) for holding the solvent (1), a section containing the siphoning unit (Soxhelt flask) (6,7), paper thimble for holding food (4), and a condenser (9). Examples: AOAC Method 920.39C for Cereal Fat; AOAC Method 960.39 for Meat Fat. SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY Determination of Crude Fat Soxhelt flask Paper Thimble SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Determination of Crude Fat Soxhlet Extraction Steps 1. The sample is placed in filter paper thimble. 2. The solvent builds up in the extraction chamber. 3. The solvent accumulates in the extraction chamber (soxhlet flask) for 5- 10 mins that facilitate the extraction of crude fat from the sample. 4. The solvent + crude fat siphons back to the boiling flask. 5. Dismantle the unit, place the bottom flask containing solvent and fat on a heating mantle and evaporate the solvent to obtain crude fat 6. Measure the crude fat (weight or volume) left in the flask. SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Determination of Crude Fat The determination of Crude Fat is simple, but there are some variables should be considered: - Sample preparation (dried sample) - Choice of solvent (i.e. petroleum ether and diethyl ether) - Extraction (temperature, time and particle size) SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Determination of Crude Fat Continuous Extraction; Goldfish Method It is based on a closed loop of solvent evaporating, being condensed. The procedure involves placing the sample between the boiling solvent and a cold surface in a clean thimble. The solvent from a boiling flask (Beaker) continuously flows over the sample placed in the ceramic thimble. As boiling continuous, the solvent vaporizes, condenses on the cold surfaces and washes back through the samples to the boiling flask. After the complete extraction (6 hr or more), the condenser part of the instrument is disconnected and the solvent is evaporated. The residue left is weighed to determine total fat Limitations: Channeling- formation of a solvent channel in the sample, and fire hazard SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY The solvent condenses and flows over the sample Condenser Thimble Sample tube The solvent Boiling flask evaporates Goldfish SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY Water control valve Condenser Beaker Heater Heat control switch Elevation control Knob SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Determination of Total lipids The most important Wet Extraction methods are: - Babcock Method and Gerber Method (Non solvent wet extraction) - Majonnier Method (Solvent wet extraction) - Bligh and Dyer Method (solvent wet extraction) SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Determination of Crude Fat Discontinuous Extraction: Simplest version of extraction There is no continuous flow of solvent. The sample is extracted with a fixed volume of solvent. E.g: Majonnier, and Bligh and Dyer Method Disadvantages: Needs large amount of solvent (1 sample: 10 solvent). Low efficiency (As solvent extract fat, its ability to extract fat decreases) SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY Babcock Method (Non solvent wet extraction) Measures total lipids Samples contains water (e.g. Milk and dairy products) It is still recognized as a reference method for milk fat. The principle: Heat Wet sample + H2SO4 Centrifugation Fat coalesces/ (blob) of fat measured Volumetrically by the graduated portion of the Babcock bottle. H2SO4 digests protein to liberate the fat, generates heat and releases the fat. Total fat is measured volumetrically It is not applicable to products containing chocolate or added sugar without modification because of charring of chocolate and sugar by sulfuric acid. AOAC Method 989.04 and 989.10 for fat in milk SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY Gerber Method (Non solvent wet extraction) It is similar to Babcock method, Besides H2SO4, isoamyl alcohol is added to prevents the charring of sugar. Help releasing lipid from organic materials (carbohydrates) (AOAC Method 2000.18) SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY Mojonnier Method (Base or Alkaline Method): Can be used for both liquid and solid samples Milk, Dairy products, fish and meat) Principle 1 NH OH 2 Milk 4 Petroleum Ether Centrifugation Ethanol Diethyl ether Decant ether solution into previously weighed dish Mojonnier bottle Evaporation Mojonnier fat dish SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY Mojonnier method requires 4 reagents: 1- NH4OH dissolves protein of casein micelle membrane and fat globule membrane to separate any bond lipids from proteins; if the sample is sour, it neutralizes the acidic sample. 2- Ethanol prevents milk from becoming viscous (prevents possible gel formation). 3- Diethyl ether and Petroleum ether solubilize and extract fat; using Petroleum ether with Diethyl ether to prevent absorption of water by diethyl ether and dissolves more nonpolar lipids. SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY Bligh and Dyer Method (solvent wet extraction) Initially used for fish and sea foods and now for meat and meat products The principle: Extraction of lipids with a mixture of Chloroform: Methanol: Water CHCl3: CH3OH: H2O Method Food samples are mixed/homogenized in a chloroform-methanol solution and the homogenized mixture is filtered into a collection tube. A 0.88 % potassium chloride aqueous solution is added to the chloroform- methanol mixture containing the extracted fats to form two phases: the aqueous phase (top) and the chloroform phase containing the lipid (bottom). The phases are further separated in a separatory funnel or by centrifugation. Evaporation of the chloroform, to quantify total fat by weight. SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY Sample Extraction fat by monophasic (extraction solvent) Aqueous phase contains non fat Add CHCl3 or materials water to change Separation by separatory to biphasic state ✚ funnel CHCl3 contains fat Removing CHCl3 SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY Instrumental Methods 1- Infrared Method: fat can absorb IR energy at a wavelength 3.73μm; The more the energy absorption, the higher is the fat content of the sample. 2- Specific Gravity (Foss-Let Method): Fat is determined as a function of the specific gravity of a sample solvent extract. 3- Nuclear Magnetic Resonance (NMR). SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY  Recap Fat Analysis ▪ Relevance ▪ Classification ▪ Different methods of fat determination Dry extraction and wet extraction SAJI GEORGE @ S.A.F.E NANO, MCGILL UNIVERSITY