LIPID

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Definition: Lipids are organic compounds formed mainly from alcohol and fatty acids combined together by ester linkage.
O R CH2 OH

H2O
R

O R CH2 O C R

Fatty alcohol

HO C

Fatty acid Esterase (lipase)

ester (lipid)

 Lipids are insoluble in water, but soluble in fat or organic solvents (ether, chloroform, benzene, acetone). Lipids include fats, oils, waxes and related compounds. They are widely distributed in nature both in plants and in animals.

Structures of Lipids

Fatty Acids
Long-chain carboxylic acids Insoluble in water Typically 12-18 carbon atoms (even number) Some contain double bonds

Fatty Acid Formulas

The formulas for fatty acids are written as Condensed formulas. Line-bond formulas. For example caprylic acid with 8 carbon atoms. CH3(CH2)6COOH CH3CH2CH2CH2CH2CH2CH2COOH
O OH

Saturated Fatty Acids

Single CC bonds. Molecules that fit closely together in a regular pattern. Strong attractions between fatty acid chains. High melting points Solids at room temperature

Some Saturated Fatty Acids

Unsaturated Fatty Acids

They contain double bond monounsaturated they contain one double bonds . (CnH2n-1 COOH) polyunsaturated they contain more the one double bond (CnH2n-more than 1 COOH).

Properties of Unsaturated Fatty Acids

Contain one or more double C=C bonds Nonlinear chains do not allow molecules to pack closely Few interactions between chains Low melting points Liquids at room temperature

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1-Monounsaturated fatty acids

Palmitoleic acid : It is found in all fats. It has 16 carbons and one double bond located at carbon number 9 and involving carbon 10.
CH3-( CH2 )5CH = CH-(CH2)7 COOH

2-Oleic acid Is the most common fatty acid in natural fats. It is C18:19, i.e., has 18 carbons and one double bond located at carbon number 9 and involving carbon 10.

CH3-(CH2)7- CH=CH (CH2)7-COOH

2-Polyunsaturated fatty acids (Essential fatty acids)

Definition: They are essential fatty acids that can not be synthesized in the human body and must be taken in adequate amounts in the diet. They are required for normal growth and metabolism

 Source: vegetable oils such as corn oil, linseed oil, peanut oil, olive oil, cottonseed oil, soybean oil and many other plant oils, cod liver oil and animal fats. Deficiency: Their deficiency in the diet leads to nutrition deficiency disease. Its symptoms include: poor growth and health with susceptibility to infections, dermatitis, decreased capacity to reproduce, impaired transport of lipids, fatty liver, and lowered resistance to stress.

Function of Essential Fatty Acids:

1. They are useful in the treatment of atherosclerosis by help transporting blood cholesterol and lowering it and transporting triglycerides. 2. The hormones are synthesized from them. 3. They enter in structure of all cellular and subcellular membranes and the transporting plasma phospholipids. 4. They are essential for skin integrity, normal growth and reproduction. 5. They have an important role in blood clotting (intrinsic factor). 6. Important in preventing and treating fatty liver. 7. Important role in health of the retina and vision. 8. They can be oxidized for energy production.

1-Linoleic:
It is the most important since other essential fatty acids can be synthesized from it in the body. CH3-(CH2)4-CH = CH-CH2-CH=CH-(CH2)7-COOH

2-Linolenic acid:
in corn, linseed, peanut, olive, cottonseed and soybean oils.

CH3-CH2-CH=CH-CH2-CH=CH-CH2-CH=CH(CH2)7-COOH

3-Arachidonic acid:
It is an important component of phospholipids in animal and in peanut oil from which prostaglandins are synthesized.
CH3-(CH2)4-CH=CH-CH2-CH=CH-CH2-CH=CH-CH2CH=CH-(CH2)3-COOH

Prostaglandins in the Body

Prostaglandins are Produced by injured tissues. Involved in pain, fever, and inflammation. Not produced when anti-inflammatory drugs such as aspirin inhibit their synthesis.
Copyright 2007 by Pearson Education, Inc. Publishing as Benjamin Cummings

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Waxes Fats and oils (trigycerides) Phospholipids

Lipids with fatty acids

Sphingolipids

WAXES
Fatty acids + Long chain alcohol Important in fruits: 1. Natural protective layer in fruits, vegetables, etc. 2. Added in some cases for appearance and protection.

Beeswax (myricyl palmitate), Spermaceti (cetyl palmitate)

O C30 H61 O C C H31 15
C16 H33 O

O C C H31 15

Triacylglycerols
Glycerol head group HO-CH2CH(OH)-CH2-OH Ester linkage from each hydroxyl to Fatty acid
Fats and oils are Also called triacylglycerols.

Triacylglycerols
In a triacylglycerol, Glycerol forms ester bonds with three fatty acids.

Copyright 2007 by Pearson Education, Inc. Publishing as Benjamin Cummings

25

Formation of a Triacylglycerol
glycerol + three fatty acids triacylglycerol

O CH2 CH CH2 OH OH + OH HO C O HO C O HO C (CH2)14CH3 (CH2)14CH3

O
(CH2)14CH3 CH2 O C

(CH2)14CH3 (CH2)14CH3 + 3H2O

O CH O C O CH2 O C (CH2)14CH3
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Triacylglycerols in Energy Storage & Thermal insulation

Concentrated source of energy
Energy derived from oxidation reactions More completely reduced state yields 2x the energy/g as Carbohydrates

Pure non-aqueous phase

Lipases hydrolize the ester linkages to release Fatty Acids

Triacylglycerols in food
Vegetable Oils unsaturated
- catalytic hydrogenation reduces double bonds - less specific than enzymatic methods makes some trans-fats

Properties of Triglycerides
Hydrogenation
Unsaturated compounds react with H2 Ni or Pt catalyst C=C bonds CC bonds

Hydrolysis
Split by water and acid or enzyme catalyst Produce glycerol and 3 fatty acids

Halogenation
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Hydrogenation
O CH2 O CH CH2 O O C (CH2)5CH CH(CH2)7CH3 O C (CH2)5CH CH(CH2)7CH3 O C (CH2)5CH CH(CH2)7CH3 + 3 H2 Ni

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Product of Hydrogenation
O CH 2 CH CH 2 O O O C O C (CH 2)14CH 3 O C (CH 2)14CH 3 (CH 2)14CH 3

Hydrogenation converts double bonds in oils to single bonds. The solid products are used to make margarine and other hydrogenated items.

Hydrolysis:
They are hydrolyzed into their constituents (fatty acids and glycerol) by the action of super heated steam, acid, alkali or enzyme (e.g., lipase of pancreas). - During their enzymatic and acid hydrolysis glycerol and free fatty acids are produced.
O CH2 O C R1 O CH2 O C R3 H2C OH O R1 C OH O O C OH

O R2

C O C H

Lipase or Acid
3 H 2O

HO C H H2C OH

+ R C OH 2
R3

Triacylglycerol

Glycerol Free fatty acids

Saponification
Alkaline hydrolysis produces glycerol and salts of fatty acids (soaps). Soaps cause emulsification of oily material this help easy washing of the fatty materials
O O CH2 O C R1 O CH2 O C R3 H2C OH HO C H O R1 C ONa O O C ONa

R2 C O C H

+ R C ONa 2
R3

3 NaOH

H2C OH

Triacylglycerol

Glycerol Sodium salts of fatty acids (soap)

Halogenation
Neutral fats containing unsaturated fatty acids have the ability of adding halogens (e.g., hydrogen or hydrogenation and iodine or iodination) at the double bonds. - It is a very important property to determine the degree of unsaturation of the fat or oil that determines its biological value
CH3 (CH2)4 CH CH CH2 CH CH (CH2)7 COOH

Linoleic acid 2 I2
CH3 (CH2)4 CH I CH I CH2 CH I CH I (CH2)7 COOH

Stearate-tetra-iodinate

Glycerophospholipids
Glycerophospholipids are The most abundant lipids in cell membranes. Composed of glycerol, two fatty acids, phosphate and an amino alcohol.
Fatty acid Glycerol Fatty acid PO4 Amino alcohol

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Each glycerophospholipid includes a polar region: glycerol, carbonyl O of fatty acids, Pi, & the polar head group (X)

O R1 C O

H2C CH H2C

C O

R2

P O

glycerophospholipid
polar "kink" due to double bond

non-polar hydrocarbon tails of fatty acids (R1, R2).

non-polar

Sphingosine
Sphingosine is a long-chain unsaturated amino alcohol.
CH3(CH2)12 CH=CHCHOH CHNH2 CH2OH sphingosine
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Sphingolipids
In sphingomyelin, a sphingolipid found in nerve cells There is an amide bond between a fatty acid and sphingosine, an 18-carbon alcohol.

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Steroids
Cholesterol Bile Salts Steroid Hormones

PDB 1N83

cholesterol

Cholesterol, an important constituent of cell membranes, has a rigid ring system and a short branched hydrocarbon tail. Cholesterol is largely hydrophobic. But it has one polar group, a hydroxyl, making it amphipathic.

HO

Cholesterol

PDB 1N83

cholesterol

HO

Cholesterol

Cholesterol in membrane

Cholesterol inserts into bilayer membranes with its hydroxyl group oriented toward the aqueous phase & its hydrophobic ring system adjacent to fatty acid chains of phospholipids.

The OH group of cholesterol forms hydrogen bonds with polar phospholipid head groups.

Interaction with the relatively rigid cholesterol decreases the mobility of hydrocarbon tails of phospholipids. But the presence of cholesterol in a phospholipid membrane interferes with close packing of fatty acid tails in the crystalline state, and thus inhibits transition to the crystal state. Phospholipid membranes with a high concentration of cholesterol have a fluidity intermediate between the liquid crystal and crystal states.

Cholesterol in membrane

Two strategies by which phase changes of membrane lipids are avoided:

Cholesterol is abundant in membranes, such as plasma membranes, that include many lipids with long-chain saturated fatty acids.
In the absence of cholesterol, such membranes would crystallize at physiological temperatures. The inner mitochondrial membrane lacks cholesterol, but includes many phospholipids whose fatty acids have one or more double bonds, which lower the melting point to below physiological temperature.

Cholesterol in Foods
Cholesterol is Synthesized in the liver. Obtained from foods. Considered elevated if plasma cholesterol exceeds 200 mg/dL.
TABLE 17.4

Copyright 2007 by Pearson Education, Inc. Publishing as Benjamin Cummings

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Bile Salts
cholic acid, a bile acid CH3 OH CH3 CH3 O C N H glycine, an amino acid CH2

COO- Na+

Polar region

HO
Nonpolar region

OH

sodium glycocholate, a bile salt

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The function
Emulsification of lipids during digestion. Help in digestion of the other foodstuffs. Activation of pancreatic lipase. Help digestion and absorption of fat-soluble vitamins. Solubilizing cholesterol in bile and prevent gall stone formation. Choleretic action (stimulate their own secretion). Intestinal antiseptic that prevent putrefaction

Steroid Hormones
Steroid hormones Are chemical messengers in cells. Are produced from cholesterol. Include sex hormones such as androgens (testosterone) in males and estrogens (estradiol) in females
low solubility in water transported by proteins, can pass through membranes

FAT SOLUBLE VITAMINS

Vitamin A:

H3 C

CH3
8 9

CH3
5 7 6 4

CH3
3

CH2 OH
2 1

CH3

CH 3 H 3C H 3C CH 3 CH 3

Vitamin D2:
H H CH 2

HO

Vitamin E:
R1 R2 HO R3 CH3 CH3 O (CH2 CH2 CH2 CH2 )2 CH2 CH2CH2 CH(CH 3 )2

ANALYTICAL METHODS TO MEASURE THE CONSTANTS OF FATS AND OILS

1. 2. Acid Value Saponification Value

3.
4. 5. 6.

Iodine Value
Gas Chromatographic Analysis for Fatty Acids Liquid Chromatography Cholesterol Determination

1. Acid Value
Number of mgs of KOH required to neutralize the Free Fatty Acids in 1 g of fat.

AV =

ml of KOH x N x 56 Weight of Sample

= mg of KOH

2. Saponification Value
Saponification - hydrolysis of ester under alkaline condition.

H2 C HC H2 C

O O C O O C O O C

R R R + 3 KOH

H2 C HC H2 C

O H OH O H + O 3R C OK

Saponification Value of Fats and Oils

Fat Milk Fat Coconut Oil Cotton Seed Oil Soybean Oil Lard Saponification # 210-233 250-264 189-198 189-195 190-202

2. Saponification Value Determination

Saponification # --mgs of KOH required to saponify 1 g of fat.

1.
2. 3. 4. 5.

5 g in 250 ml Erlenmeyer.
50 ml KOH in Erlenmeyer. Boil for saponification. Titrate with HCl using phenolphthalein. Conduct blank determination.
SP# = 56.1(B -S) x N of HCl Gram of Sample

B - ml of HCl required by Blank. S - ml of HCl required by Sample.

3. Iodine Number

Number of iodine (g) absorbed by 100 g of oil.

Molecular weight and iodine number can calculate the number of double bonds. 1 g of fat adsorbed 1.5 g of iodine value = 150.

Iodine Value Determination

Iodine Value = (ml of Na2S2O3 volume for blank - ml of Na2S2O3 volume for sample) N of Na2S2O3 0.127g/meq 100

Weight of Sample (g)

CH

CH

+ ICl Iodine chloride

CH Cl

CH I

Excess unreacted ICl

ICl I2 + + KI KCl + I2 + 2 NaI

2 Na2 S2 O3

Na2 S4 O6

Iodine Numbers of Triglycerides

Fatty Acids Palmitoleic Acid # of Double-bonds 1 Iodine # 95

Oleic Acid
Linoleic Acid Linolenic Acid Arachidonic Acid

1
2 3 4

86
173 261 320

4. GC Analysis for Fatty Acids

1.
2. 3.

Extract fat.
Saponify (hydrolysis under basic condition). Prepare methyl ester (CH3ONa).

4.
5.

Chromatography methyl ester.

Determine peak areas of fatty acids. Fatty acids are identified by retention time.

6.

Compare with response curve of standard.

Fatty Acids Methyl Esters:

Res pons e 18:1

14

16

18:2 18

18:3 20 22

21:1

24

Time

GC condition: 10% DEGS Column (from supelco) Column temperature 200C.

5.

TRIGLYCERIDE ANALYSIS BY LIQUID CHROMATOGRAPHY Soybean Oil

Solvent CH3CN/HF
Column 84346 (Waters Associates)

RESPONSE

RETENTION TIME

Oleate-containing triglycerides in olive oil

Fatty Acid Composition Total Acyl Carbons: Unsaturation Equivalent Carbon Number

OL2
O2L OPL O3 OSL

54:5
54:4 52:3 54:3 54:3

44
46 46 48 48

O2P
O2S OPS

52:2
54:2 52:1

48
50 50

OS2

54:1

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6. CHOLESTEROL DETERMINATION
Enzymatic Determination: Cholesterol Oxidase

Choles terol Oxidase etc. + HO O H2 O2

CH3O

OCH3 NH2

CH3O

OCH3 NH

H 2 O2 + H2N

Peroxidase

HN

+ H2 O

0-Dianisidine (Colorless)

Oxidized 0-Dianisidine (Brown color)At 440 nm

Abs orption at 440 nm

g/ml Cholesterol

Cholesterol by GLC 1. 2. Prepare cholesterol butyrate. Analyze by GLC. time in GC - 15 min. sensitivity - 10-7 g.

Spectromertic Absorption Standard Curve of Cholesterol

Abs orption at 4 40 nm

g/ml Cholester ol

Cholesterol by GLC
1. 2. Prepare cholesterol butyrate. Analyze by GLC. time in GC - 15 min. sensitivity - 10-7 g.

LIPID CONTENT ANALYSES

1. Gravimetric Method
(1) Wet extraction - Roese Gottliegb & Mojonnier. (2) Dry extraction - Soxhlet Method.

2.

Volumetric Methods (Babcock, Gerber Methods)

1. Gravimetric Method
(1) Wet Extraction - Roese Gottlieb & Mojonnier.

For Milk: 1) 10 g milk + 1.25 ml NH4OH mix. solubilizes protein and neutralizes. 2) + 10 ml EtOH - shake. Begins extraction, prevents gelation of proteins. 3) + 25 ml Et2O - shake and mix. 4) + 25 ml petroleum ether, mix and shake.

(2) Dry Extraction - Soxhlet Method.

Sample in thimble is continuously extracted with ether using Soxhlet condenser. After extraction, direct measurement of fat - evaporate ether and weigh the flask.

Indirect measurement - dry thimble and weigh thimble and sample.

Soxhlet Method.

2. Volumetric Method (Babcock, Gerber Methods)

Theory:

1. Treat sample with H2SO4 or detergent.

2. Centrifuge to separate fat layer. 3. Measure the fat content using specially calibrated bottles. Methods: 1. Known weight sample.

2. H2SO4 - digest protein, liquefy fat.

3. Add H2O so that fat will be in graduated part of bottle. 4. centrifuge to separate fat from other materials completely.

REACTIONS OF FATS
Hydrolytic Rancidity:

1. Triglyceride -> Fatty acids Specially C4 butyric acid (or other short chain fatty acids) are the real problem.

2. By lipase.

LIPID OXIDATION
Major flavor problems in food during storage are mainly due to the oxidation of lipid.

Lipid Oxidation - free radical reactions.

1.
2.

Initiation.
Propagation.

3.

Termination.

Pentane Formation from Linolenic Acid

14

CH3

(CH )3 2

CH2

13

CH

12

CH

11

CH 2

10

CH

CH

CH 2

COOH

Initiation (metal)

- H.

CH3

(CH )3 2

CH2

.CH
12

12

11

10

CH + O2
11

CH

CH

CH

CH n COOH 2

Propagation CH3 (CH )3 2 CH2 CH O Propagation O

12

10

CH

CH + H.
11

CH

CH

CH n COOH 2

10

CH 3

(CH )3 2

CH2

CH O O H

CH _

CH

CH

CH -

CHn COOH 2

Hydroperoxide Decomposition

.OH
11 10 9

12

CH 3

(CH )3 2

CH2

CH

CH

CH

CH

CH

CHn COOH 2

.
CH 3 (CH )3 2

. CH
2

O + H C

12

11

10

CH + H

CH

CH

CH

CH COOH 2n

Termination CH3

(CH )3 2 Pentane

CH3

ANALYSIS OF FLAVOR QUALITY & STABILITY OF OIL

1.

Peroxide Value
O A. KI + CH 3 C OH HI + CH 3 O C OK

B.

ROOH + 2 HI

I2 +

H2 O +

ROH

C.

I2 + 2 Na2 S 2 O3

2 NaI +

Na2 S4 O6

Peroxide Value =

ml of Na2S2O3 N 1000 Grams of Oil

(milliequivalent peroxide/kg of sample)

2. Active Oxygen Method (AOM) Determined the time required to obtain certain peroxide value under specific experimental conditions.

The larger the AOM value, the better the flavor stability of the oil.

3. TBA Test.
To determine the rancidity degree of meat or fish product.
HS N OH N OH + H O C CH2 C H O

HS N

OH

HO CH CH

N N

SH + 2 H2 O

CH OH

OH Colored P igment