Lipids

1. Lipids

2. Class Presentations Will be Discussed at the End of Class Exams back next Monday No class this Wednesday !!!!!

3. Lipids Main functions of lipids in foods Energy and maintain human health Influence on food flavor Fatty acids impart flavor Lipids carry flavors/nutrients Influence on food texture Solids or liquids at room temperature Change with changing temperature Participation in emulsions

4. Lipids Lipids are soluble in many organic solvents Ethers (n-alkanes) Alcohols Benzene DMSO (dimethyl sulfoxide) They are generally NOT soluble in water C, H, O and sometimes P, N, S

5. Lipids Neutral Lipids Triacylglycerols Waxes Long-chain alcohols (20+ carbons in length) Cholesterol esters Vitamin A esters Vitamin D esters Conjugated Lipids Phospholipids, glycolipids, sulfolipids �Derived� Lipids Fatty acids, fatty alcohols/aldehydes, hydrocarbons Fat-soluble vitamins

6. Lipids Structure Triglycerides or triacylglycerols Glycerol + 3 fatty acids >20 different fatty acids

7. Lipids 101 Fatty acids- the building block of fats A fat with no double bonds in it�s structure is said to be �saturated� (with hydrogen) Fats with double bonds are referred to as mono-, di-, or tri- Unsaturated, referring to the number of double bonds. Some fish oils may have 4 or 5 double bonds (polyunsat). Fats are named based on carbon number and number of double bonds (16:0, 16:1, 18:2 etc)

8. Lipids Oil- liquid triacylglycerides �Oleins� Fat- solid or semi-solid mixtures of crystalline and liquid TAG�s �Stearins� Lipid content, physical properties, and preservation are all highly important areas for food research, analysis, and product development. Many preservation and packaging schemes are aimed at prevention of lipid oxidation.

9. Nomenclature The first letter C represents Carbon The number after C and before the colon indicates the Number of Carbons The letter after the colon shows the Number of Double Bonds �The letter n (or w) and the last number indicate the Position of the Double Bonds

10. Saturated Fatty Acids

12. Mono-Unsaturated Fatty Acids

13. Poly-Unsaturated Fatty Acids

16. Lipids Properties depend on structure Length of fatty acids (# of carbons) Position of fatty acids (1st, 2nd, 3rd) Degree of unsaturation: Double bonds tend to make them a liquid oil Significantly lowers the melting point Hydrogenation: tends to make a solid fat Significantly increases the melting point Unsaturated fats oxidize faster Preventing lipid oxidation is a constant battle in the food industry

20. Lipids 101 Fatty acid profile- quantitative determination of the amount and type of fatty acids present following hydrolysis. To help orient ourselves, we start counting the number of carbons starting with �1� at the carboxylic acid end.

21. Lipids 101 For the �18-series� (18:0, 18:1, 18:2, 18:3) the double bonds are usually located between carbons 9=10 12=13 15=16.

22. Lipids 101 The biomedical field started using the OMEGA (w) system (or �n� fatty acids). With this system, you count just the opposite. Begin counting with the methyl end Now the 15=16 double bond is a 3=4 double bond or as the medical folks call it�.an w-3 fatty acid

23. Tuning Fork Analogy-TAG�s Envision a Triacylglyceride as a loosely-jointed E Now, pick up the compound by the middle chain, allowing the bottom chain to hang downward in a straight line. The top chain will then curve forward and form an h Thus the �tuning fork� shape Fats will tilt and twist to the lowest free energy level

24. Lipids Lipids are categorized into two broad classes. The first, simple lipids, upon hydrolysis, yield up to two types of primary products, i.e., a glycerol molecule and fatty acid(s). The other, complex lipids, yields three or more primary hydrolysis products. Most complex lipids are either glycerophospholipids, or simply phospholipids contain a polar phosphorus moiety and a glycerol backbone or glycolipids, which contain a polar carbohydrate moiety instead of phosphorus.

25. Lipids

26. Other types of lipids Phospholipids Structure similar to triacylglycerol High in vegetable oil Egg yolks Act as emulsifiers

27. Where Do We Get Fats and Oils? �Crude� fats and oils are derived from plant and animal sources Several commercial processes exist to extract food grade oils Most can not be used without first �refining� before they reach consumers During oil refining, water, carbohydrates, proteins, pigments, phospholipids, and free fatty acids are removed.� Crude fats and oils can therefore be converted into high quality edible oils In general, fat and oil undergo four processing steps: Extraction Neutralization Bleaching Deodorization Oilseeds, nuts, olives, beef tallow, fish skins, etc. Rendering, mechanical pressing, and solvent extraction.

28. Fats and Oils: Processing Extraction Rendering Pressing oilseeds Solvent extraction

29. Fats and OilsFurther Processing Degumming Remove phospholipids with water Refining Remove free fatty acids (alkali + water) Bleaching Remove pigments (charcoal filters) Deodorization Remove off-odors (steam, vacuum)

30. Where Do We Get Fats and Oils? Rendering Primarily for extracting oils from animal tissues.� Oil-bearing tissues are chopped into small pieces and boiled in water.� The oil floats to the surface of the water and skimmed.� Water, carbohydrates, proteins, and phospholipids remain in the aqueous phase and are removed from the oil.� Degumming may be performed to remove excess phospholipids. Remaining proteins are often used as animal feeds or fertilizers.

31. Where Do We Get Fats and Oils? Mechanical Pressing Mechanical pressing is often used to extract oil from seeds and nuts with oil >50%.� Prior to pressing, seed kernels or meats are ground into small sized to rupture cellular structures.� The coarse meal is then heated (optional) and pressed in hydraulic or screw presses to extract the oil. Olive oils is commonly cold pressed to get extra virgin or virgin olive oil. It contains the least amount of impurities and is often edible without further processing. Some oilseeds are first pressed or placed into a screw-press to remove a large proportion of the oil before solvent extraction.

32. Where Do We Get Fats and Oils? Solvent Extraction Organic solvents such as petroleum ether, hexane, and 2-propanol can be added to ground or flaked oilseeds to recover oil.� The solvent is separated from the meal, and evaporated from the oil. Neutralization Free fatty acids, phospholipids, pigments, and waxes exist in the crude oil These promote lipid oxidation and off-flavors (in due time) Removed by heating fats and adding caustic soda (sodium hydroxide) or soda ash (sodium carbonate).� Impurities settle to the bottom and are drawn off.� The refined oils are lighter in color, less viscous, and more susceptible to oxidation (without protection). Bleaching The removal of colored materials in the oil. Heated oil can be treated with diatomaceous earth, activated carbon, or activated clays. Colored impurities include chlorophyll and carotenoids Bleaching can promote lipid oxidation since some natural antioxidants are removed.

33. Where Do We Get Fats and Oils? Deodorization The final step in the refining of oils. Steam distillation under reduced pressure (vacuum). Conducted at high temperatures of 235 - 250�C. Volatile compounds with undesirable odors and tastes can be removed. The resultant oil is referred to as "refined" and is ready to be consumed. About 0.01% citric acid may be added to inactivate pro-oxidant metals.

34. Fats and OilsFurther Processing Hydrogenation Add hydrogen to an oil to �saturate� the fatty acid double bonds Conducted with heated oil Often under pressure In the presence of a catalyst (usually nickel) Converts liquid oils to solid fats Raises melting point

35. Hydrogenating Vegetable oils can produce trans-fats

36. The cis- and trans- forms of a fatty acid

38. Fats and Oils in Foods SOLID FATS are made up of microscopic fat crystals. Many fats are considered semi-solid, or �plastic�. PLASTICITY is a term to describe a fat�s softness or the temperature range over which it remains a solid. Even a fat that appears liquid at room temperature contains a small number of microscopic solid fat crystals suspended in the oil�..and vice versa PLASTIC FATS are a 2 phase system: Solid phase (the fat crystals) Liquid phase (the oil surrounding the crystals). Plasticity is a result of the ratio of solid to liquid components. Plasticity ratio = volume of crystals / volume of oil Measured by a �solid fat index� or amount of solid fat or liquid oil in a lipid As the temperature of a plastic fat increases the fat crystals melt and the fat will soften and eventually turn to a liquid.

39. Fat and Oil: Further Processing Winterizing (oil) Cooling a lipid to precipitate solid fat crystals DIFFERENT from hydrogenation Plasticizing (fat) Modifying fats by melting (heating) and solidifying (cooling) Tempering (fat) Holding the fat at a low temperature for several hours to several days to alter fat crystal properties (Fat will hold more air, emulsify better, and have a more consistent melting point)

40. Lipid Oxidation

41. Effects of Lipid Oxidation Flavor and Quality Loss Rancid flavor Alteration of color and texture Decreased consumer acceptance Financial loss Nutritional Quality Loss Oxidation of essential fatty acids Loss of fat-soluble vitamins Health Risks Development of potentially toxic compounds Development of coronary heart disease

42. LIPID OXIDATION and Antioxidants Fats are susceptible to hydrolyis (heat, acid, or lipase enzymes) as well as oxidation. In each case, the end result can be RANCIDITY. For oxidative rancidity to occur, molecular oxygen from the environment must interact with UNSATURATED fatty acids in a food. The product is called a peroxide radical, which can combine with H to produce a hydroperoxide radical. The chemical process of oxidative rancidity involves a series of steps, typically referred to as: Initiation Propagation Termination

43. Simplified scheme of lipoxidation

44. Initiation of Lipid Oxidation There must be a catalytic event that causes the initiation of the oxidative process Enzyme catalyzed �Auto-oxidation� Excited oxygen states (i.e singlet oxygen): 1O2 Triplet oxygen (ground state) has 2 unpaired electrons in the same spin in different orbitals. Singlet oxygen (excited state) has 2 unpaired electrons of opposite spin in the same orbital. Metal ion induced (iron, copper, etc) Light Heat Free radicals Pro-oxidants Chlorophyll Water activity

45. Considerations for Lipid Oxidation Which hydrogen will be lost from an unsaturated fatty acid? The longer the chain and the more double bonds�.the lower the energy needed.

47. Propagation Reactions

48. Propagation of Lipid Oxidation

49. Termination of Lipid Oxidation Although radicals can �meet� and terminate propagation by sharing electrons�. The presence or addition of antioxidants is the best way in a food system. Antioxidants can donate an electron without becoming a free radical itself.

50. Antioxidants and Lipid Oxidation BHT � butylated hydroxytoluene BHA � butylated hydroxyanisole TBHQ � tertiary butylhydroquinone Propyl gallate Tocopherol � vitamin E NDGA � nordihydroguaiaretic acid Carotenoids

51. Chemical Tests for Lipid Characterizations

52. Measure of the degree of unsaturation in an oil or the number of double bonds in relation to the amount of lipid present Defined as the grams of iodine absorbed per 100-g of sample. The higher the amount of unsaturation, the more iodine is absorbed. Therefore the higher the iodine value, the greater the degree of unsaturation.

53. A known solution of KI is used to reduce excess ICl (or IBr) to free iodine R-C-C = C-C-R + ICl ? R-C-CI - CCl-C-R + ICl [Excess] (remaining) Reaction scheme: ICl + 2KI ? KCl + KI + I2 The liberated iodine is then titrated with a standardized solution of sodium thiosulfate using a starch indicator I2 + Starch + thiosulfate = colorless endpoint (Blue colored)

54. Iodine Value Used to characterize oils: Following hydrogenation During oil refining (edible oils) Degree of oxidation (unsaturation decreases during oxidation) Comparison of oils Quality control

57. Chemical Tests Saponification Value

58. Saponification is the process of breaking down or degrading a neutral fat into glycerol and fatty acids by treating the sample with alkali. Heat Triacylglyceride ---> Fatty acids + Glycerol KOH Definition: mg KOH required to titrate 1g fat (amount of alkali needed to saponify a given amount of fat) Typical values: Peanut = 190, Butterfat = 220

59.

60. Chemical Tests for Oxidation Lipid Oxidation Hydrolysis Peroxide Value Oxidation Tests

61. LIPID OXIDATION

62. Degree of hydrolysis (hydrolytic rancidity) High level of FFA means a poorly refined fat or fat breakdown after storage or use.

63. Oxidation is a very complex reaction - no one test will measure all of the reactants or products. Some assays measure intermediates while others measure end products.

64. Measures peroxides and hydroperoxides in an oil which are the primary oxidation products (usually the first things formed). The peroxide value measures the �present status of the oil�. Since peroxides are destroyed by heat and other oxidative reactions, a seriously degraded oil could have a low PV.

65. KI + peroxyl radical yields free Iodine (I2) The iodine released from the reaction is measured in the same way as an iodine value. I2 in the presence of amylose is blue. I2 is reduced to KI and the endpoint determined by loss of blue color. 4I + O2 + 4H 2I2 + 2H2O

66. Thiobarbituric acid (reactive substances) TBA OR TBARS Tests for end products of oxidation � aldehydes, Malonaldehyde (primary compound), alkenals, and 2,4-dienals A pink pigment is formed and measured at ~530 nm. TBARS is firmly entrenched in meat oxidation research and is a method of choice. TBARS measure compounds that are volatile and may react further with proteins or related compounds. High TBA = High Oxidative Rancidity

67. Good indictor of the end products of oxidation (if there are any). Standard method in many industries. Aldehyde formation from lipid oxidation. Nonenal is also a common end-product

68. Conjugated Fatty Acids During oxidation, double bond migration occurs and conjugated fatty acids are formed. They absorb light efficiently and can be monitored in a spectrophotometer.

69. TECHNIQUES OF MEASURING OXIDATIVE STABILITY Induction Period: is defined as the length of time before detectable rancidity or time before rapid acceleration of lipid oxidation

70. MEASURING OXIDATIVE STABILITY Active Oxygen Method - Air is bubbled through oil or fat at 97.8�C. Time required to reach peroxide value of 100 meq/kg fat determined. (method replaced by OSI) Oil Stability Index � automated Rancimat (instrumental method). Air bubbled through sample (110�C). Oil degrades to many acidic volatiles (e.g. formic acid) which are carried by the air into a water trap. Conductivity of the water can then be assessed.

71. Free Radicals

72. What are free radicals? Where are free radicals from? How damaging are free radicals? How do we control free radicals?

73. What are free radicals? Any molecular species capable of independent existence, which contains one or more unpaired valence electrons not contributing to intramolecular bonding�.is a free radical.

74. Where do they come from? Free radicals are produced by oxidation/reduction reactions in which there is a transfer of only one electron at a time, or when a covalent bond is broken and one electron from each pair remains with each atom.

75. How damaging are free radicals? ROS may be very damaging, since they can attack: Lipids in cell membranes Proteins in tissues or enzymes Carbohydrates DNA These cause cell membrane damage, protein modification, and DNA damage. Thought to play a role in aging and several degenerative diseases (heart disease, cataracts, cognitive dysfunction, and cancer). Oxidative damage can accumulate with age.

77. Our Body vs. Our Food Biological radicals Food-based radicals Where do these 2 areas cross?

78. Functional Foods Concept Certain food ingredients have health benefits beyond basic nutrition Recent development only: since ~1975 The concept that �non-nutrients� were beneficial has taken off since then First idea in scientific community: antioxidant compounds may protect against chronic diseases

79. Free Radicals Early 1950�s: cell damage is due to reactive oxygen species called �free radicals� Unstable, �damaged� molecule that is missing an electron Highly reactive; reacting to some measurable extent with any molecule they come in contact with In living systems, cell injury or disease In foods, quality-degrading impact

80. Reactive Oxygen Species (ROS) Primary target list: protein, lipid, DNA, and carbohydrates End results: cancer, CHD, stroke, arterial disease, rheumatoid arthritis, Parkinson�s/Alzheimer disease, cataracts, macular degeneration�.many more Aging by slow oxidation?

81. The Defense Minimize contact between free radicals and important systems (like cellular components) Cell membranes are one of our best barriers Metal chelation system in-place Protease enzymes are in place to remove damaged proteins for replacement by new �Repair enzymes� help to restore DNA �Antioxidant enzymes�-superoxide dismutase, catalase, glutathione peroxidase

82. Best Defense�A Good Offense �Nutrients� that can�t be synthesized in vivo: vitamin C, vitamin E, (pro)vitamin A �Non-nutrients�: polyphenolics/carotenoids Diet is only source�.are they �essential�? What about conditions of �oxidative stress�? This is a condition when pro-oxidants outnumber antioxidants (I.e. decreased immune response, environmental factors, hypertension, poor diet).

83. Foods and the Antioxidant Link Soy- isoflavones, polyphenolics Tea- polyphenolics, flavans Coffee- polyphenolics Wine- polyphenolics Rosemary- carnosic acid, rosmaric acid Citrus- flavonoids Onions- sulfur cpds, flavonoids Berries- flavonoids, polyphenolics Vegetables- carotenoids, polyphenolics

84. Antioxidants in Food Systems

85. Oxidative Stress-the food remedy Diet: Inflammation- tocopherol Smoke- ascorbic acid Physical stress- carotenoids Pollution- carotenoids Environment: Radiation- glutathione Carcinogens- antioxidant enzymes, diet modification

86. Oxidative Stress and Foods Tocopherol- vegetable oil, whole grains, vegetables, fish/poultry Ascorbic acid- citrus, berries, tomato, leafy veggies, brassicas (broccoli, cauliflower) Carotenoids- yellow/orange fruits and veggies, tomatoes, green leafy veggies. Polyphenolics- coffee/tea, grains, all fruits and vegetables

87. Magic Bullets�for our body? Most likely not� Will increasing the intake of antioxidants modulate disease prevention? Will we live longer with no health problems? Lung cancer and �-carotene: Whoa� Antioxidant compounds have demonstrated benefits (both acute and long-term) of preventing or postponing the onset of many degenerative diseases, but clinical trials are full of holes, and conclusive evidence of �the bullet� is still not with us.

88. Magic Bullets�for our foods? Will increasing the use of antioxidants in foods modulate all oxidative damage? Will food products �live� longer with no quality problems? Pro-oxidant nature of ascorbic acid: Whoa� Ascorbic acid does not always act linearly in food systems In the presence of metal ions (ie. Fe/Cu) it can generate reactive oxygen species (peroxides) or free radicals (hydroxyl radicals)

89. Causes and Effects �-carotene and lung cancer: small, but significant increase with smokers Tocopherols and CHD: protect lipoproteins or inhibit blood clotting (which initiates heart attacks) Tocopherols and Alzheimer�s: reducing oxidative stress by supplementation Cataracts and vitamins (A,C,E): inverse association Macular degeneration and carotenoids: inverse Vitamin C and the Common Cold: shorter, milder colds

90. Structure-Based AOX Polyphenolics

91. Structure of flavonoids

92. B-Ring Substitutions

93. Quercetin 4 �OH groups

94. Catechin 4 �OH groups

95. Cyanidin4 �OH groups

96. Structurally Similar Compounds

97. Importance of the 3-OH group

98. Importance of the 4-Oxo Function Works with the 2-3 double bond in the C-ring and is responsible for electron delocalization from the B-ring. 3-OH and 5-OH substitutions with the 4-oxo function are best for maximum AOX properties

99. Importance of the 2-3 db

100. More on the Phenolic Acids

102. Antioxidants in Food Systems

103. What Makes a Good Antioxidant? Polyphenolics- Radical scavengers Number of hydroxy groups (-OH) Location of hydroxy groups (on benzene ring) Presence of a 2-3 double bond (flavylium ring) 4-oxo function (flavylium ring) Synergistic/antagonistic reactions with other antioxidant compounds

104. What Makes a Good Antioxidant? Carotenoids The number of conjugated double bonds (9+ is best) Substitutions on �-ionone group (on the end) Radical scavengers R* + CAR => R- + CAR*+ Chain breakers ROO*+ CAR => ROO-CAR* ROO-CAR* + ROO* => ROO-CAR-ROO Singlet oxygen quenchers 1O2* + 1CAR => 3O2 + 3CAR*

105. Tocopherol Alpha-tocopherol = Vitamin E beta and gamma forms also Synergist with carotenoids and selenium and is regenerated by vitamin C Efficiency determined by the bond dissociation energy of the phenolic -OH bond The heterocyclic chromanol ring is optimized for resonance stabilization of an unpaired electron.

106. Antagonism-Synergism-Metals Many antioxidant work for and against each other An antioxidant in a biological system my be regenerated In mixed ROS�inefficiency of one antioxidant to quench all the different radicals. No way of knowing if the �better� antioxidant for a particular radical is doing all the work or not. Will a better antioxidant for a given food system �beat out� a lesser antioxidant (antagonistic response) in order to quench the radicals.

107. Example: Factors Affecting AOX of Bell Peppers Chemical interactions In vitro models Find synergistic/antagonistic effects Free metal ions Diluted isolates Add metal chelator

108. AOX with Quercetin Interactions(�-Carotene Bleaching)

109. AOX with Luteolin Interactions(�-Carotene Bleaching)

110. Theoretical Quercetin �Regeneration� Scheme

111. Theoretical Luteolin �Regeneration� Scheme

112. Antioxidant Activity after Dilution

113. Antioxidant Activity with Chelator

114. Antioxidant Methods

115. HAT and SET Reactions Hydrogen Atom Transfer (HAT) vs. Single Electron Transfer (SET) Antioxidants can work in one of two ways (HAT or SET). End result is the same for both, differing in kinetics and side rxns. HAT and SET rxns may occur in parallel Determined by antioxidant structure and properties Solubility and partition coefficient System solvent, system pH

116. HAT HAT-based methods measure the classical ability of an antioxidant to quench free radicals by hydrogen donation (AH = any H donor)

117. SET SET-based methods detect the ability of a potential antioxidant to transfer one electron to reduce any compound, including metals, carbonyls, and radicals. Also based on deprotonation, so pH dependent

118. HAT vs SET HAT Selectivity in HAT rxs are determined by the bond dissociation energy of the H-donating group in the antioxidant Antioxidant reactivity or capacity measurements are therefore based on competition kinetics. Reactions are solvent and pH independent and are very fast Common reducing agents (Vitamin C) are an interference SET Usually slow and can require long times to reach completion Antioxidant reactivity is based on a percent decrease, rather than kinetics Very sensitive to ascorbic acid and other reducing agents. Trace amounts of metal ions will interfere, and cause over-estimation and inconsistent results.

119. Antioxidants and Radicals Four sources of antioxidants: Enzymes Superoxide dismutase, glutathione peroxidase, and catalase Large molecules albumin, ferritin, other proteins Small molecules ascorbic acid, glutathione, uric acid, tocopherol, carotenoids, phenols Hormones estrogen, angiotensin, melatonin Multiple free radical and oxidant sources O2, O2�-, HO?, NO?, ONOO-, HOCl, RO(O)?, LO(O) Oxidants and antioxidants have different chemical and physical characteristics.

120. Complex Systems: Singlet Oxygen Carotenoids are not good peroxyl radical quenchers compared to polyphenolics Carotenoids are exceptional singlet oxygen quenchers compared to polyphenolics However, singlet oxygen is not a radical and does not react via radical mechanisms Singlet oxygen reacts by its addition to fatty acid double bonds, forming endoperoxides, that can be reduced to alkoxyl radicals, that initiate radical chain reactions. Now we have multiple reaction characteristics and multiple mechanisms No single assay will accurately reflect all of the radical sources or test all the antioxidants in such a complex system.

121. Method Selections for Antioxidants Controversy exists over standard methods for antioxidant determination Historical use and peer-review acceptance is critical Use my multiple labs to highlight strength, weakness, and effectivness New methods take time to adopt and accept An �ideal� method: Measures chemistry actually occurring in potential application Utilizes a biologically relevant radical source Simple to run Uses a defined endpoint and chemical mechanism Instrumentation is readily available Good within-run and between-day reproducibility Adaptable for both hydrophilic and lipophilic antioxidants Adaptable for multiple radical sources Adaptable for high-through-put analysis Understanding of the range of use and recognition of interfering agents

122. HAT assays ORAC Oxygen Radical Absorbance Capacity Measures inhibition of peroxyl radical induced oxidations in chain breaking activity by H atom transfer TRAP Total Radical-Trapping Antioxidant Parameter Measures the ability to interfere with peroxyl radicals or stable free radicals

123. SET assays FRAP Ferric Reducing Antioxidant Power The reaction measures the reduction capacity of a ferric compound to a color end-product CUPRAC Copper Reduction Assay Variant of FRAP assay using Cu instead of Fe Folin-Ciocalteu assay Reduction of oxidized iron and molybdenum