Lipid Analysis in Foods

• Lesson discusses lipid analysis, covering analytical methods for lipid quantification in foods, and focusing on solvent and non-solvent extraction techniques.

Lipids

• Major constituents of foods
• Important energy source
• Provides essential nutrients
• Excessive intake of certain lipids can have negative health effects
• Play a major role in determining overall physical characteristics, such as flavor, texture, mouthfeel, and appearance

Important Lipid Properties

• Total lipid concentration
• Type of lipids present
• Physicochemical properties i.e crystallization, melting point, smoke point, rheology, density, and color
• Structural organization of lipids within a food

Lipid Properties

• Soluble in organic solvents (ether, hexane, chloroform)
• Insoluble in water
• Includes triacylglycerols (TAGs), diacylglycerols, monoacylglycerols, free fatty acids, phospholipids, sterols, caretonoids, and vitamins A and D
• Lipid fraction of fatty foods are complex mixtures of molecules
• TAGs are the major component of most foods, typically making up 95–99% of total lipids present
• TAGs are esters of three fatty acids and a glycerol backbone
• Fatty acids in foods vary in chain length, degree of unsaturation, and position on the glycerol molecule

Lipid Analysis Considerations

• Water insolubility of lipids serves as the basis for their separation
• Extraction solvent selection is important
  • Glycolipids are mostly soluble in alcohols
  • Triacylglycerols are soluble in hexane and petroleum ether
• Necessary to free and solubilize these lipids in the organic solvent for successful extraction

Sample Preparation

• Depends on the type of food
• Depends on the nature of the lipid component i.e volatility, susceptibility to oxidation, physical state
• Depends on the type of analytical procedure used: solvent extraction, non-solvent extraction, or instrumental
• Should minimize any changes in the properties of the lipid fraction
• Preserve the sample by using a nitrogen atmosphere, cold temperature, low light, or adding antioxidants.
• Control temperature and handling of the sample if solid fat content or lipid crystal structure is important

Solvent Extraction Methods

• Commonly isolates lipids from foods to determine the total lipid content
• Proper sample preparation and preservation is necessary to ensure validity
• The sample should be pre-dried and ground
• Hydrolyzed with acid if it contains bound or complex lipids like lipoproteins and glycolipids
• A solvent should have high solvent power for lipids
• A solvent should have low or no solvent power for proteins, amino acids, and carbohydrates
• A solvent should evaporate readily, leave no residue, have a relatively low boiling point
• A solvent should be nonflammable and nontoxic in both liquid and vapor form
• Ethyl ether and petroleum ether are the most commonly used solvents

Continuous Solvent Extraction - Goldfish Method

• The solvent from a boiling flask flows continuously over the sample placed inside a ceramic thimble
• Lipid content is measured by weight loss from the sample and reported as crude fat
• Relatively faster but only process one sample at a time per set-up

Semi-Continuous Solvent Extraction - Soxhlet Method

• A sample is dried, ground into small particles, and placed in a porous thimble
• The thimble is placed in an extraction chamber suspended above a flask containing the solvent
• The flask is heated and the solvent evaporates into a liquid that drops into the extraction chamber containing the sample
• Eventually, the solvent builds up in the extraction chamber and completely surrounds the sample
• The chamber is designed to overflow exceeding a certain level which falls back down into the boiling flask
• As the solvent passes through the sample, it extracts the lipids, and carries them into the flask, the lipids then remain in the flask because of their lower volatility
• At the end of the extraction process, the flask containing the solvent and lipid is removed, the solvent is evaporated, and the mass of the remaining lipid is measured
• Requires more time than the continuous method but can analyze several samples simultaneously

Discontinuous Solvent Extraction - Mojonnier Method

• Applicable to liquid and solid samples
• Commonly used for determining fat of dairy products
• Sample is digested first with NH4OH with added EtOH to prevent possible gel formation
• The fat is then extracted with a mixture of ethyl ether and petroleum ether inside of a Mojonnier flask
  • ethyl ether extracts the polar lipids
  • petroleum ether extracts the non-polar lipids

Non-Solvent Extraction Methods

• Methods that do not rely on organic solvents but use other chemicals to separate the lipid from the rest of the food
• Commonly used for the determination of milk fat

Non-Solvent Extraction - Babcock Method

• H2SO4 is added to a known amount of milk
• The acid digests the CHON which generates heat and breaks down the fat globule membrane which releases the fat
• The addition of hot water and centrifugation separates the fat for quantification
• This method does not measure complex lipids and phospholipids since they are located in the aqueous phase.

Non-Solvent Extraction- Gerber Method

• Similar to the Babcock method but uses H2SO4 and isoamyl alcohol in a butyrometer with a graduated portion
  • Acid digests the CHON, carbs, and releases the fat
  • Maintains liquid state with generated heat
  • Isoamyl alcohol prevents charring of the sugars from heat/H2SO4
• This method does not measure phospholipids

Non-Solvent Extraction - Detergent Method

• A sample is mixed with a combination of surfactants, then separates in a Babcock bottle
• Surfactants break down the fat emulsion droplets in the milk releasing the fat
• The sample is centrifuged so the fat moves to the graduated neck of the bottle, used to determine its concentration
• Addresses the use of reagents like H2SO4, as in the Babcock method

Determination of Lipid Composition - Iodine Value

• Measures the average degree of unsaturation of a lipid
  • the higher the IV, the greater the # of C=C
• The lipid is weighed and dissolved in a suitable organic solvent, to which a known excess of iodine chloride is added
• Some of the ICl reacts with the double bonds in the unsaturated lipids, while the rest remains
• Iodine value is expressed as the grams of iodine absorbed per 100g of lipid

Determination of Lipid Composition - Saponification Number

• Measures the average MW of the TAG in a sample
• Saponification is the process of breaking down a neutral fat into glycerol and fatty acids by treatment with alkali: TAG + 3 KOH = Glycerol + 3 FA salts of K
• SN is defined as the mg of KOH required to saponify one gram of fat
  • A high SN indicates a shorter fatty acid chain and a lower MW

Determination of Lipid Composition - Acid Value

• Measures the amount of free acids present in a given amount of fat
• Uses a titrimetric method with the extraction of the lipid from a sample which is then dissolved in an EtOH solution with an indicator
  • This solution is then titrated with alkali (KOH) until a pinkish color appears
• AV is defined as the mg of KOH necessary to neutralize the fatty acids present in 1g of lipid
• AV may be overestimated if other acid components are present
• A good measure of the breakdown of triacylglycerols into free fatty acids, which has an adverse effect on the quality of many lipids.

Characterization of Physicochemical Properties

• Lipids are used in foods due to their characteristic physicochemical properties i.e mouthfeel, flavor, texture, appearance, frying.
• Important for food scientists to have analytical techniques that characterize their physicochemical properties: solid fat content, melting point, cloud point, smoke point, flash point, and fire point

Solid Fat Content

• SFC influences the spreadability, firmness, mouthfeel, processing and stability of foods
• Defined as the % of the total lipid that is solid at a particular temperature

Melting Point

• Each different TAG has its own unique MP, therefore they melt over a wide range of temperatures
• Clear Point is the temperature at which the fat completely melts and becomes transparent in a capillary tube
• Slip Point is the temperature at which the fat just starts to move downwards due to its weight in a capillary tube
• Wiley melting point is the temperature at which a disc of fat suspended in an alcohol-water mixture changes shape to a sphere

Cloud Point

• Measures the temperature at which crystallization begins in a melted fat sample cooled at a controlled rate. It is the temperature at which the liquid just goes cloudy

Smoke Point

• Temperature at which the sample begins to smoke when tested under specified conditions.

Flash Point

• Temperature at which a flash appears at any point on the surface of the sample due to the ignition of volatile gaseous products

Fire Point

• Temperature at which volatiles are released due to the thermal decomposition of the lipids which then proceeds to continuous combustion.