2. LIPIDS & VITAMINS

3. LIPIDS “Lipids are organic compounds that contain hydrogen, carbon, and oxygen atoms, which forms the framework for the structure and function of living cells.”

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 ( 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)

37. Thank you