Hello everyone, Today we discuss about Lipids (part-1) in Food Analysis subject in M.pharmacy(Pharmaceutical Analysis Department)...

4. • Lipids are one of the major constituents of foods, and are
important in our diet for a number of reasons.
• They are a major source of energy and provide essential lipid
nutrients.
•Nevertheless, over-consumption of certain lipid components
can be detrimental to our health,
e.g. cholesterol and saturated fats.
•In many foods the lipid component plays a major role in
determining the overall physical characteristics, such as flavor,
texture, mouth feel and appearance.
LIPIDS : INTRODUCTION

5. •For this reason, it is difficult to develop low-fat alternatives
of many foods, because once the fat is removed some of the
most important physical characteristics are lost.
•Finally, many fats are prone to lipid oxidation, which leads to
the formation of off-flavors and potentially harmful products.
•Some of the most important properties of concern to the food
analyst are:
Total lipid concentration
Type of lipids present
Physicochemical properties of lipids, e.g., crystallization,
melting point, smoke point, rheology, density and color
Structural organization of lipids within a food

6. Properties of Lipids in Foods:
•Lipids are usually defined as those components that are
soluble in organic solvents (such as ether, hexane or
chloroform), but are insoluble in water.
•This group of substances includes triacylglycercols,
diacylglycercols, monoacylglycercols, free fatty acids,
phospholipids, sterols, caretonoids and vitamins A and D.
•The lipid fraction of a fatty food therefore contains a complex
mixture of different types of molecule.
•Even so, triacylglycercols are the major component of most
foods, typically making up more than 95 to 99% of the total
lipids present.

7. •Triacylglycerols are esters of three fatty acids and a glycerol
molecule.
•The fatty acids normally found in foods vary in chain length,
degree of unsaturation and position on the glycerol molecule.
•Consequently, the triacylglycerol fraction itself consists of a
complex mixture of different types of molecules.
•Each type of fat has a different profile of lipids present which
determines the precise nature of its nutritional and
physiochemical properties.
•The terms fat, oil and lipid are often used interchangeably by
food scientists. Although sometimes the term fat is used to
describe those lipids that are solid at the specified temperature,
whereas the term oil is used to describe those lipids that are
liquid at the specified temperature.

8. They may be classified based on their physical properties at
room temperature (solid or liquid, respectively fats and oils),
on polarity, or on their essentiality for humans, but the
preferable classification is based on their structure.
Based on structure, they can be classified in three major
groups.
1. Simple lipids,
2. Complex lipids,
3. Derived lipids
LIPIDS : CLASSIFICATION

9. 1.Simple lipids
They consist of two types of structural moieties.
They include:
glyceryl esters that is esters of glycerol and fatty acids:
e.g. triacylglycerols, mono- and diacylglycerols;
cholesteryl esters that is esters of cholesterol and fatty acids;
waxes which are esters of long-chain alcohols and fatty acids, so
including esters of vitamins A and D;
ceramides that is amides of fatty acids with long-chain di- or
trihydroxy bases containing 12–22 carbon atoms in the carbon chain:
e.g. sphingosine.

10. 2.Complex lipids
They consist of more than two types of structural moieties.
They include:
phospholipids that is glycerol esters of fatty acids;phosphoric acid, and
other groups containing nitrogen;
phosphatidic acid that is diacylglycerol esterified to phosphoric acid;
phosphatidylcholine that is phosphatidic acid linked to choline, also
called lecithin;
phosphatidyl acylglycerol in which more than one glycerol molecule is
esterified to phosphoric acid: e.g. cardiolipin and diphosphatidyl
acylglycerol;
glycoglycerolipids that is 1,2-diacylglycerol joined by a glycosidic
linkage through position sn-3 with a carbohydrate moiety;
gangliosides that is glycolipids that are structurally similar to ceramide
polyhexoside and also contain 1-3 sialic acid residues; most contain an
amino sugar in addition to the other sugars;
sphingolipids, derivatives of ceramides;
sphingomyelin that is ceramide phosphorylcholine;

11. 3.Derived lipids
They occur as such or are released from the other two major groups
because of hydrolysis that is are the building blocks for simple and
complex lipids.
They include:
fatty acids and alcohols;
fat soluble vitamins A, D, E and K;
hydrocarbons;
sterols.

12. LIPIDS : GENERAL METHODS
OF ANALYSIS OF LIPIDS
•The properties of oils and fats vary along with the degree of
unsaturation, average molecular weight and also acidity from
hydrolysis.
•A number of parameters are used for their analysis which are
included under physical constants and chemical constants.
• Physical constants include viscosity, specific gravity,
refractive index, solidification point etc.
ANALYTICAL PARAMETERS FOR OILS AND FATS

13. Following is a brief idea about some of the analytical parameters
grouped under chemical constants.
1. Iodine value,
2. Saponification value,
3. Acid value,
4. Hydroxyl value,
5. Acetyl value,
6. Unsaponifiable matter,
7. Peroxide value,
8. Kreistest (rancidity index),
9. Ester value,
10. Reichert Messle Value &,
11. Polenski value.

14. 1. Iodine value:
Definition:
Iodine value is the number, which express in grams, a quantity of
halogen, calculated as iodine which is absorbed by 100g of the substance
under the described condition. Iodine value may be determined by iodine
monochloride method, iodine monobromide method, pyridine
monobromide etc.
Significance:
• Iodine value is the measure of unsaturation ( number of double bond )
in fat.
• Iodine number is useful to analyze the degree of adulteration
• On basis of iodine value the oils can be differentiated into non-drying
oil and semidrying oil. Drying oil shows less iodine value, non-drying oil
shows more iodine value and semidrying oil shows moderate iodine
value.

15. Procedure:
•Iodine Monochloride Method: place an accurately weighed quantity of
substance being examined(castor oil) in a dry 250ml capacity iodine
flask.
• Add 1ml of carbon tetrachloride and dissolve in 20ml of iodine
monochloride solution. Insert the stopper and allow to stand in the dark
at a temperature in between 15-25 degrees Celsius for 30 mins.
• Place 15ml of potassium iodide solution and cup top, carefully remove
the stopper, rinse the stopper and sides of the flask with 10ml of water,
shake and titrate with 0.1M sodium thiosulphate using starch as an
indicator.
• The starch solution added towards the end of the titration. Note the ml
required (a).
• Repeat the operation omitting the substance being examined and note
the number of ml required (b). calculate the iodide value with the
following expression. Iodine value = [1.269(b-a)]/w W – Weight in
grams of the substance of the oil.

16. 2. Saponification value:
It is defined as the number of milligrams of KOH required to neutralize
the fatty acids resulting from complete hydrolysis of 1 gm of the sample
of oil or fat.
Significance:
•Saponification value of fat or oil is one of it’s characteristic physical
properties.
• Saponification value occurs in an inverse proportion to the average
molecular weight of fatty acid present in oil.
• Higher saponification number for fats containing short chain fatty
acids.
Saponification value
• This value is normally applied for butter fat, coconut oil in which lower
fatty acids glycerides occur in high content. • It is used for detecting
adulteration • Saponification value is determined by refluxing a known
amount of sample with excess of standard alcoholic KOH

17. Procedure:
•2 gm of the given sample of oil is accurately weighed in a RB flask and
refluxed with 25ml of 0.5M ethanolic potassium hydroxide with a little
pumic powder in a water bath for 30 minutes. •Add 1ml of
phenolphthalein solution and titrate immediately with 0.5M hydrochloric
acid (a ml) •Carry out the blank, omitting the substance under
examination (b ml)
•Calculate the saponification value •Saponification value = 28.05 (b-
a)/w •b – volume of hydrochloric acid consumed in blank titration •a –
volume of hydrochloric acid consumed in sample titration •w – weight of
the sample

18. 3. Acid value:
It is defined as the number of milligrams of potassium hydroxide
required to neutralize the free fatty acids present in 1gm of sample of fat
or oil.
Significance:
Acid value is used as an indication of rancid state. Generally rancidity
causes free fatty acids, which have been liberated by hydrolysis of
glycerides due to the action of moisture, temperature or enzyme lipase.
Acid value
Acid value can be determined by treating sample with solution of KOH
using phenolphthalein as indicator

19. Procedure
•Accurately weigh about 1gm of the oil and to this add 50 ml mixture of
equal volume of ethanol (95%) and ether, previously neutralized with
0.1M of KOH to phenolphthalein solution. •Add 0.1 ml of
phenolphthalein solution and titrate with 0.1M KOH until the solution
remains faintly pink
•n – number of milligrams of potassium hydroxide required •w- weight
of the sample. •The STD for edible fats and oils indicate that the acid
value must not exceed 0.6 . The acid value can be determined by the
formulae; Acid value = 5.61

20. 4. Hydroxyl value:
It is defined as number of milligrams of potassium hydroxide required to
neutralize the acetic acid capable of combining by acetylation with 1 g
sample of fat or oil.
5. Acetyl value:
It is the number of milligrams of potassium hydroxide required to
neutralize acetic acid obtained when 1g of sample acetylated oil is
saponified.
Significance :
Acetyl number is a measure of number of hydroxyl groups present. to
detect adulteration and rancidity.

21. 6. Unsaponifiable Matter:
It is the matter present in fats and oil, which after saponification by
caustic alkali and subsequent extraction with an organic solvent, remains
non-volatile on drying at 8o°C. It includes sterols (phytosterol and
cholesterol), oil soluble vitamins, hydrocarbons and higher alcohols.
Paraffin hydrocarbons can be detected by this method as adulterants.
7. Peroxide Value:
Peroxide Value Is the number which expresses in milli equivalents of
active oxygen that expresses the amount of peroxide containing 1000gms
(kg) of substances (meq/kg). It is a measure of peroxides present in oil.
Aperoxide value is generally less than 10 mEqkg in fresh samples of oil.
Due to temperature or storage, rancidity occurs causing increase in
peroxide values.

22. 8.Kreistest (rancidity index):
Due to rancidity, epihydrin aldehyde or malonaldehyde are increased
which are detected by Kreis test using phloroglucinol which produces
red colour with the oxidized fat.
9.Ester value:
It is defined as number of milligrams of potassium hydroxide required to
combine with fatty acids which are present in glyceride form in 1 g
sample of oil or fat. Difference between saponification value and acid
value is ester value..

23. 10. Reichert messle value:
This value is a measure of volatile water soluble acid contents the fat. It
is defined as number of milli litres N/10 potassium hydroxide solution
required to neutralize the volatile water soluble fatty acids obtained by 5
g fat.
Significance:
Higher content of volatile fatty acids of butter responsible for its higher
reichert-meissl number. It is useful in testing purity/adulteration of butter.
11.Polenski Value:
It is defined as the number of millitres of N/10 potassium hydroxide
solution required to neutralize water-insoluble, steam - distillable acids
liberated by hydrolysis of 5 gm of fat.
Significance:
The Polenski value is an indicator of how much volatile fatty acid can be
extracted from fat through saponification.

24. LIPID CONTENT ANALYSIS
1. Gravimetric Method
(1) Wet extraction – Roese Gottliegb &
Mojonnier.
(2) Dry extraction – Soxhlet Method.
2. Volumetric Methods (Babcock, Gerber
Methods)

25. 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.

26. (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.

27. 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.

28. Objectives of Refining:
1- In refining, physical and chemical processes are combined to remove
undesirable natural as well as environmental-related components from
the crude oil.
2-These components comprise for example phosphatides, free fatty
acids, pigments (such as chlorophyll), odors and flavors (including
aliphatic aldehyde and ketone), waxes as well as heavy metals, pesticides
etc.
3-Removal of undesired products from crude oils
free fatty acids (FFA) phospholipids (gums) oxidised products
metal ions colour pigments other impurities
4- Preservation of valuable vitamines. (vitamina E ortocopherol–natural
anti-oxidants)
5- Minimize oil losses
6- Protection of the oil against degradation
LIPIDS : REFINING OF FATS
AND OILS

29. Methods of Refining :
Chemical Refining:
The Chemical Refining process is used for oils and fats with low FFA
and contains three basic steps:
Neutralizing
Bleaching
Deodorizing
Residual soap and gums removal in neutralizing is accomplished by
either water washing or using a silica adsorbent in bleaching.
Physical Refining:
The Physical Refining process is used for oils and fats with high FFA and
contains three basic steps:
Acid Conditioning or Enhanced Degumming
Bleaching
Stripping and Deodorizing
The degumming process used depends on the oil or fat being refined.

30. Depending on the requirements, the following basic processes are
implemented:
degumming for removal of phosphatides,
neutralization for removal of free fatty acids,
bleaching for removal of color,
deodorization to distill odors and flavors as well as free fatty acids and
winterization for separation of waxes.

31. 1.Chemical Refining:
Neutralizing:
Objective: Removal of free fatty acids
Batch Neutralization : Refining of vegetable oils is essential to ensure
removal of gums, waxes, phosphatides and free fatty acid (F. F.A.) from
the oil; to impart uniform colour by removal of colouring pigments and
to get rid of unpleasant smell from the oil by removal of odiferous
matter.
Refining is carried out either on batch operation or as continuous
operation. With certain oils even physical refining can be carried out
instead of chemical. For processing less than thirty tones of oil per 24
hours, and when oil has F.F .A. content of 1 % or less normally batch
process is recommended. Batch process involves low capital investment,
simplicity of operation and low maintenance, making refining
economically a viable proposition even at capacity as low as 10 tonnes
per 24 hours.

32. Bleaching:
Objective: bleaching for removal of color,
Silica adsorption reduces water consumption, effluent treatment and
bleaching earth consumption
Pre-bleaching oil minimizes bleaching earth consumption
Deodorizing:
With a light color. Deodorization consists of steam sparging the oil under
high vacuum (< 10 mm Hg) at high temperatures (> 200°C). After
deodorization and cooling of the oil, a chelating agent, such as citric
acid, may be added to deactivate trace metals. Antioxidants may also be
added to enhance stability.

33. 2.Physical Refining:
Acid Conditioning or Enhanced Degumming
Degumming
Objective : degumming for removal of phosphatides,
The aim of degumming operation; The emulsifying action of
phospholipids increases oil losses during alkali refining. Gums lead
brown discoloration of oil after heating during deodorization. Salts
may be formed with cooper, magnesium,calcium and iron, accelerating
oxidative degredation of oil.
Certain phospholipids, such as lecithin, find widespread industrial
application. Different degumming processes are carried out to remove
phosphatides. For efficient and economic application of this procedure
appropriate machines and equipments are used.
1.Water degumming
2.Acid degumming
3.Enzymatic degumming
4.Membrane degumming

34.  Acid degumming
There are two type of Acid degumming
1- Dry acid degumming
2- Wet acid degumming
Acid Degumming Process Steps
Heat oil to 60 -70 °C
Acid addition and mixing
Hydration mixing 30 minutes
Centrifugal separation of hydrated gums
Vacuum drying of degummedoil
Gums -recombined in meal

35. Water degumming
Water Degumming Process Steps
 Heat oil to 60 -70 °C
Water addition and mixing
Hydration mixing 30 minutes
Centrifugal separation of hydrated gums
Vacuum drying of degummed oil
Gums -dried for edible lecithin or recombined in meal

36. Enzymatic degumming:
Enzymatic degumming was first introduced by the German Lurgi
Company as the »Enzy Max process« .The EnzyMax process can be
divided into four different steps:
i. the adjustment of the optimal conditions for the enzyme reaction, i.e.
optimal pH with a citrate buffer and the optimal temperature;
ii. the addition of the enzyme solution;
iii. the enzyme reaction;
iv. the separation of lysophosphatide from the oil at about 75 °C.
Enzymes for enzymatic degumming;
Lecitase® 10L (pancreatic phospholipase A2)
Lecitase® Novo (microbial lipase)
Lecitase® Ultra (microbial lipase)