LIPID

Ahmad rifaiSiwi shintaraYulihartono

Definition:• Lipids are organic compounds formed mainly

from alcohol and fatty acids combined together by ester linkage.

CH 2R

Fatty alcoholOH C R

Fatty acidHO

O

+

H2O

CH 2R O C R

O

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

4

5

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)6—COOH

CH3—CH2—CH2—CH2—CH2—CH2—CH2—COOH

OH

O

Saturated Fatty Acids• Single C–C 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

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).

Unsaturated Fatty Acids

11

Properties of UnsaturatedFatty 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

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 C18:1∆9, 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-CH2-CH=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.

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

Lipids with fatty acids

Waxes

Fats and oils (trigycerides)

Phospholipids

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

C30H61 O C C 15H31

O

C16H33 O C C 15H31

Triacylglycerols

• Glycerol head group HO-CH2-CH(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.

25

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

Formation of a Triacylglycerolglycerol + three fatty acids triacylglycerol

OHCH2

OH

OHCH2

CHO

(CH2)14CH3CHO

O

(CH2)14CH3CHO

O

(CH2)14CH3CHO

O

O

C (CH2)14CH3

CH O

O

C (CH2)14CH3

CH2 O

O

C (CH2)14CH3

CH2

26

+ 3H2O

+

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

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Properties of Triglycerides

Hydrogenation• Unsaturated compounds react with H2 • Ni or Pt catalyst• C=C bonds C–C bonds

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

Halogenation

Hydrogenation

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CH

CH2

CH2 O

O

O

C

O

(CH2)5CH CH(CH2)7CH3

C

O

(CH2)5CH CH(CH2)7CH3

C

O

+

(CH2)5CH CH(CH2)7CH3

H23Ni

Product of Hydrogenation

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

CH

CH2

CH2 O

O

O

C (CH2)14CH3

O

C (CH2)14CH3

O

C (CH2)14CH3

O

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.

CH 2 O

C HO

CH 2

C

C

O C

R1

R3

R2

O

O

O

3 H2O

H2C OH

C HHO

H2C OH

OHCR1

O

OHCR3

O

+ OHCR2

OLipase or Acid

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

CH 2 O

C HO

CH 2

C

C

O C

R1

R3

R2

O

O

OH2C OH

C HHO

H2C OH

ON aCR1

O

ON aCR3

O

+ ON aCR2

O

Triacylglycerol Glycerol Sodium salts of fatty acids (soap)

3 NaOH

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

CH (CH 2)7 COO HCHCH 2CH

Linoleic acidCH(CH 2)4CH 3

2 I2

CH (CH 2)7 COO HCHCH 2CH

Stearate-tetra-iodinate

CH(CH 2)4CH 3

II I I

Glycerophospholipids

Glycerophospholipids are• The most abundant lipids in cell

membranes. • Composed of glycerol, two fatty acids,

phosphate and an amino alcohol.

35

Glycerol

PO4Amino alcohol

Fatty acid

Fatty acid

O P O

O

O

H2C

CH

H2C

OCR1

O O C

O

R2

X

glycerophospholipid

Each glycerophospholipidincludes a polar region:

glycerol, carbonyl O of fatty acids, Pi, & the polar head group (X)

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

Sphingosine

Sphingosine is a long-chain unsaturated amino alcohol.

CH3−(CH2)12 −CH=CH−CH−OH

│ CH−NH2

│

CH2−OH

sphingosine38

Sphingolipids

In sphingomyelin, a sphingolipid found in nerve cells

• There is an amide bond between a fatty acid and sphingosine, an 18-carbon alcohol.

39

cholesterol PDB 1N83

Steroids

• Cholesterol• Bile Salts• Steroid Hormones

C holesterolH O

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.

cholesterol PDB 1N83

Cholesterolin 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.

C holestero lH O

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.

Cholesterolin 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.

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TABLE 17.4

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

Bile Salts

OH

COO- Na+CH2

H

N

O

COHCH3

CH3

HO

CH3

glycine, an amino acid cholic acid, a bile acid

sodium glycocholate, a bile salt

47

Nonpolar region

Polar region

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: CH2OH

CH3 CH3

CH3

CH3H3C

12

34

5

67

89

Vitamin D2:

Vitamin E:

HO

CH2

HH

H3C

H3C CH3

CH3

CH3 O

R1

R2

HO

R3

CH 3(CH 2CH 2CH 2CH 2)2CH 2CH 2CH 2CH(CH 3)2

CH 3

ANALYTICAL METHODS TO MEASURE THE CONSTANTS OF FATS AND OILS

1. Acid Value

2. Saponification Value

3. Iodine Value

4. Gas Chromatographic Analysis for Fatty Acids

5. Liquid Chromatography

6. 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 56Weight of Sample

= mg of KOH

2. Saponification Value

Saponification - hydrolysis of ester under alkaline condition.

O

C R

O

O

C R

C R

O

H2C O

HC O

H2C O

KOH

H

H

H

H2C O

HC O

H2C O

R C OK+ 3 + 3

Milk Fat 210-233

Coconut Oil 250-264

Cotton Seed Oil 189-198

Soybean Oil 189-195

Fat

Saponification #

Lard 190-202

Saponification Value of Fats and Oils

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

1. 5 g in 250 ml Erlenmeyer.

2. 50 ml KOH in Erlenmeyer.

3. Boil for saponification.

4. Titrate with HCl using phenolphthalein.

5. Conduct blank determination.

B - ml of HCl required by Blank.

S - ml of HCl required by Sample.

SP# = 56.1(B -S) x N of HCl

Gram of Sample

2. Saponification Value Determination

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 = (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 CH CH

Cl I

ICl

Io d in e ch lo rid e

+ ICl K I K Cl

I2

I2

N a2S2O3 N a2S4O6 N aI

+

+ 2 2+

+

Excess unreacted ICl

Iodine Value Determination

Iodine Numbers of Triglycerides

Palmitoleic Acid 1 95

Oleic Acid 1 86

Linoleic Acid 2 173

Linolenic Acid 3 261

Fatty Acids # of Double-bonds Iodine #

Arachidonic Acid 4 320

4. GC Analysis for Fatty Acids

1. Extract fat.

2. Saponify (hydrolysis under basic condition).

3. Prepare methyl ester (CH3ONa).

4. Chromatography methyl ester.

5. Determine peak areas of fatty acids.

Fatty acids are identified by retention time.

6. Compare with response curve of standard.

Fatty Acids Methyl Esters:

14

18:1

18:2 2018:3

22

21:1 2416 18

T im e

Res pons e

GC condition: 10% DEGS Column (from supelco)

Column temperature 200C.

5. TRIGLYCERIDE ANALYSIS BY LIQUID CHROMATOGRAPHY

Soybean Oil

Solvent CH3CN/HFColumn 84346 (Waters Associates)

RESPONSE

RETENTION TIME

Oleate-containing triglycerides in olive oil

OL2 54:5 44

O2L 54:4 46

OPL 52:3 46

O3 54:3 48

OSL 54:3 48

O2P 52:2 48

O2S 54:2 50

OPS 52:1 50

Fatty Acid Composition

Total Acyl Carbons: Unsaturation

Equivalent Carbon Number

OS2 54:1 52

6. CHOLESTEROL DETERMINATION

Enzymatic Determination: Cholesterol Oxidase HO O

H2O2

Ch o lestero l Ox id aseetc. +

H2O2

CH 3O OCH 3

H2N NH 2 HN NH

OCH 3CH 3O

H2OP ero x id ase+ +

0-Dianisidine Oxidized 0-Dianisidine(Colorless) (Brown color)At 440 nm

Cholesterol by GLC1. Prepare cholesterol butyrate.2. Analyze by GLC.

time in GC - 15 min.sensitivity - 10-7 g.

g /m l Ch olest erol

A b sorp t ion at 4 4 0 n m

Spectromertic Absorption Standard Curve of Cholesterol

Cholesterol by GLC1. Prepare cholesterol butyrate.2. Analyze by GLC.

time in GC - 15 min.sensitivity - 10-7 g.

g /m l Ch olest erol

A b sorp t ion at 4 4 0 n m

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. Initiation.

2. Propagation.

3. Termination.

Pentane Formation from Linolenic Acid +

+

_

.+

.-

+

CH 3 ( CH2)3 CH2 CH CH CH CH CH CH2 COOH

CH 3 ( CH2)3 CH2 CH CH CH2 CH CH CH2 COOH

.

H

.

CH 3 ( CH2)3 CH2 CH CH CH CH CH CH2 COOH

O

O

H

O

O

CH 3 ( CH2)3 CH2 CH CH CH CH CH CH2 COOH

CH 3 ( CH2)3 CH2 CH CH CH CH CH CH2 COOH

O

I ni t i at i on ( m et al )

Pr opagat i on

Pr opagat i on.

O2

H

OH.Hydr oper oxi deDecom posi t i on

CH 3 ( CH2)3 CH2 H C CH CH CH CH CH2 COOH

CH 3 ( CH2)3 CH3

O.

H.Ter m i nat i on

Pent ane

14 13 12 11 10 9

12 11 10 9

12 11 10 9

12 11 10 9

12 11 10 9

12 11 10 9

n

n

n

- n

n

n

ANALYSIS OF FLAVOR QUALITY & STABILITY OF OIL

1. Peroxide Value

KI CH3 C OH HI CH3 C OK

O O

ROOH HI I2 H2O ROH

I2 Na2S2O3 NaI Na2S4O6

A.

B.

C.

+

+

+

+

+

+

+

2

2

2

Peroxide Value = ml of Na2S2O3 N 1000

(milliequivalent peroxide/kg of sample) Grams of Oil

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. N

N

HS

OH

OH

C CH2 C

O

H

O

H

OH

OH

HS

N

N

OH

SH

N

N

CH CH CH

HO

H2O

Colored Pigment

+

+ 2