function of lipids in food
- long chain fatty acids (mostly saturated, contribute to texture; solid, wax, paraffin)
- texture
- creaminess
- Storage of fat soluble flavour agents
- agents of rancidity - short chain fatty acids
- flavour agents
- contributes to acidity
- agents of rancidity
What are the components of glycerides (2)
Glycerol: Water soluble alcohol
Fatty acids: chains of carbon with a carboxylic acid at one end and methyl group at another
- hydroxyl group in glycerol molecule attaches to carboxyl group of FA through ester bond
- majority of the foods we consume are triglycerides
Types of lipids (4)
- Fatty acids (Unsaturated and saturated)
- Glycerides (can be mono, di, triglygeride depending on how many hydroxyl groups are linked to the FA; glyceride has 3 OH)
- Waxes: fatty acids + long chain alcohol
- Phospholipids
Cis –> trans fatty acids
through hydrogenation; bend lipids, increases rigidity of membranes and increase melting point
- by nature, majority is cis (more nutritious but trans is cheaper and more stable)
- cis: R group on the same side
Methods for quantifying lipids
- Acid value
- Saponification value
- Iodine value
- GC analysis for fatty acids
- HPLC
Discuss acid value
number of mgs of KOH required to neutralize the free fatty acids in 1g of fat
- measure of the free fatty acids (FFA) present in the fat or oil
- increase in acid value indicates rancidity (decomposition leads to high content of FFA)
Discuss saponification value + method
Saponification value is the mg of KOH required to saponify 1g of fat
- measure of the average molecular weight (or chain length) of all the fatty acids present
Method
- add 5g fat in 250ml conical flask
- add 50ml KOH in flask
- boil for saponification
- titrate with HCL using phenolphthalein
- conduct blank determination (back titration)
Discuss iodine value
Amount of iodine (g) absorbed by 100g of oil.
- The unsaturated fatty acids of the glyceride absorbs a definite amount of iodine
- the iodine value is the measure of the degree of unsaturation of fatty acids. it is constant for a particular oil or fat but depends on the method of analysis applied
discuss general procedure for lipid preparation for GC
- main principle of GC is to change the fat into methylated FA by adding methyl group (methyl esterification) bc FAME is very heat resistant and inlet of GC usually around 260C; or else sample will be burnt at inlet
1. extract fat
- saponification (breakdown of ester bond between FA and glycerol under basic conditions; NaOH, KOH)
* ensure process is complete at high temperatures to ensure all ester bonds are broken - Methyl esterification (derivation to FAME)
- Na has replaced carboxyl group of FA so now replace Na to prepare fatty acid methyl ester to ensure that the extracted FA are heat resistant (FAME very heat resistant)
* use plenty of methanoic SO2/methanol at high temperature to ensure Na fully replaced - separate FAME using organic solvent (hexane)
- centrifuge; will end up with two phases. Collect the upper phase (organic) which is our FAME extract
- to ensure that this methylated FA is free from any contaminant/moisture, treat upper phase with NaOH and centrifuge again (to avoid accumulation of solidified material in GC vile)
- once again, take upper phase and transfer to GC vile and seal with cap
* **ensure isolated supernatent is washed again with NaOH to ensure that any contaminants/moisture is removed from purified sample
* ensure centrifugation and separation of two phases is done properly and - chromatography of methyl esters
- Determine peak areas of fatty acids (FA are identified by retention time)
- compare retention time with RT of standard FA solution to find out which FA was isolated
* * SCFA will emerge first followed by the longer FA
Discuss HPLC of fatty acids
Normally use silver ion HPLC
-stationary phase contains negatively charged resin and +vely charged ion attached to the resin
(Cation exchange + silver ions)
> column is filled with adsorbent material which has a very small particle size (large SA for sample molecules)
- mobile phase: hexane/ acetonitrile
> placed in solvent reservoir which is attached to the pump, which pumps the mobile phase into the column with a high pressure - just before the hplc column there is an injector which allows introduction of the sample in the column
Lipid oxidation + analysis
lipid oxidation can lead to rancidity, loss of nutrients (destruction of vitamins A, D and E) and essential fatty acids and possible formation of toxic compounds and coloured compounds
Mechanism of lipid oxidation
- initiation: the formation of free radicals (breakdown of double bond)
RH + O2»_space; R* + OH*
- Reaction is thermodynamically difficult with an activation energy of 35kcal/mol; need catalyst, light and heat exposure
- propagation: the free radical chain reactions
R* + O2»_space; * + ROO*
ROO* + RH»_space; R* + ROOH
ROOH»_space; RO* + HO*
-ROOH is a hydroperoxide (not a radical), one of the major initial oxidation products that decompose to form compounds responsible for off flavours and odours
- termination: The formation of non-radical products
R* + R*»_space; RR
R* + ROO*»_space; ROOR
ROO* + ROO*»_space; ROOR + O2
- both free radicals react with each other and become more stable
Methods in measuring lipid oxidation
- Primary oxidation products
- Peroxide value (PV) - secondary oxidation products
- anisidine value
- thiobarbituric acid (TBA)
- Kreis test
- iodine value
Discuss peroxide value + method
PEROXIDES: the main initial product of autoxidation (oxidation in the presence of oxygen)
- PV is an indicator of the primary oxidation products in a given fat/oil and hence provides initial evidence of rancidity
- usually expressed in terms of milli-equivalents of oxygen per kg of fat
- measured based on their ability to oxidise ferrous to ferric ions
- Ferrous chloride and ammonium thiocyanate are added to a solution of the sample in chloroform/methanol mixture, the amount of ferric thiocyanate complex produced after a fixed reaction time with the peroxide present in the sample is determined colorimetrically at 500nm
- ferric chloride used to prepare standard curve; convert OD using the standard curve
Discuss the uncertainty of peroxide value
- accuracy is questionable as the results vary with details of the procedure used and the test is extremely sensitive to temperature changes
- there is a temporal variation as during the course of oxidation, peroxide value reaches the peak and then declines
Discuss anisidine value
- Peroxides in oxidized oils are unstable intermediates which decompose into various carbonyl and other secondary oxidation products, principally
2-alkenals and 2,4-dienals
> > > the anisidine value gives a measure of these secondary oxidation products
- analytical procedure involves treating the oil or fat with iso-octane solution, with p-anisidine (para) reagent and the level of reaction product is determined spectrophotometrically at 350nm
discuss advantages and disadvantages of thiobarbituric acid value (TBA)
specific for malonaldehyde
ADV:
- simple and its results are highly correlated with sensory evaluation score
- TBA test is applicable for comparing samples of a single material at different states of oxidation
DISADV:
- non-specific and insensitive for the detection of low levels of malonaldehyde
- other TBA reacting substances such as sugars and other aldehydes can interfere with the malonaldehyde-TBA reaction (malonaldehyde doesnt only come from fat (breakdown of sugars and proteins also produce) so when have large value of TBA, it could not be the actual representation of malonaldehyde from fat)
** main disadvantage is that it doesnt distinguish between malonaldehyde from sugar oxidation and lipid oxidation
Principle of TBA testing
- Involves the reaction between the final secondary product of FA oxidation as a result of frying at high temperature with TBA
- Reaction of one molecule of malonaldehyde and two molecules of TBA to form a red malonaldehyde- tba complex which can be quantified spectrophotometrically (530nm)
Discuss how impurities can be measured
the unsaponifiable matter content is a measure of the proportion of lipid material other than fatty acids; non fat compounds present in oil and fat
the organic materials may consist of impurities such as mineral oil, sterols, tocopherols, carotenoids and higher aliphatic hydrocarbons and pigments
Method for unsaponifiable matter content
- UMC determined by the refluxing a weighed quantity of fat with ethanolic potassium hydroxide (to make sure all fat is extracted)
- the soap solution formed is diluted and extracted with an organic solvent (diethylether which is not water soluble but can dissolve impurities) in a separation funnel (2 phases, impurities (organic) and soap). Draw out the aqueous phase and stop when reach soap.
- the solvent is then evaporated and the extract is dried to a constant weight
- the unsaponifiable matter content is expressed as a percentage of the original sample
- the unsaponifiable matter methodology is also used to separate the oil from its constituent components
Discuss iodine value quantification
IV determination uses the Wij’s methods
Iodine is added to the oil in the form of iodine monochloride
it reacts with the double bonds in the fatty acids, forming additional compounds.
the excess halogen is then determined by a back titration and the amount of iodine in grams, absorbed by 100g of the oil is calculated
The IV may therefore be expressed as a percentage
characteristics of lipids
- non polar end (hydrophobic)
- fat soluble tail - polar end (hydrophilic)
properties of saturated FA
- inverse relationship between chain length and solubility
2. longer the chain, the higher the MP
polyunsaturated acid
- linoleic acid (2db)
- linolenic acid (3db)
- arachidonic acid (4dp)
- high nutritional value as all 4 double bonds are cis
