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Trends in Interesterification of Fats and Oils

Alejandro G. Marangoni Saeed M. Ghazani

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Disclosure of interest

• Funded by: Natural Sciences and Engineering Council of Canada

• Funded by: Ontario Ministry of Agriculture and Food

• Consultant to several multinational corporations, but not on the topic of interesterification

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Importance of fats and oils to health and well- being:

The highest source of energy for the body compared to carbohydrates and proteins Carriers for oil soluble vitamins (A, E, K) May contain essential and conditionally

essential fatty acids that are not produced at all, or not produced efficiently, by the human body

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Importance of fats and oils to the food industry:

Fats and oils add flavor, lubricity, texture to foods and contribute to the feeling of satiety upon consumption After extraction and refining, they can be

processed into products such as margarine, shortening, salad and frying oils. Processed fats and oils are important

functional ingredients in foods

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Functionality of Oils and Fats

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Spreadability

Laminating ability

Shortening ability

Mechanical Strength

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World Supply of Vegetable Oils

0

10

20

30

40

50

60

2007 2008 2009 2010 2011 2011

Mill

ion

met

ric

tons

Coconut oil

Cottonseed oil

Olive oil

Palm oil

Palm Kernel oil

Peanut oil

Rapeseed oil

Soybean oil

Sunflowerseed oil

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Fatty acid composition of vegetable oils

Gunstone, 2006

Oil Source 8:0 10:0 12:0 14:0 16:0 18:0 18:1 18:2 18:3

Corn - - - - 13 3 31 52 1

Cottonseed - - - - 27 2 18 51 Tr (<1%)

Olive - - - - 10 2 78 7 1

Palm - - - 1 44 4 39 11 Tr

Palm Olein - - - 1 41 4 41 12 Tr

Palm Stearin - - - 1 47-74 4-6 16-37 3-10 -

Canola - - - - 4 2 56 26 10

Soybean - - - - 11 4 22 53 8

Sunflower - - - - 6 5 20 60 Tr

Coconut 8 7 48 16 9 2 7 2 -

Palm Kernel 3 4 45 18 9 3 15 2 -

Typical fatty acid composition (%wt) of major vegetable fats and oils

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Fatty acid composition of animal fats

Gunstone, 2008

Typical fatty acid composition (%wt) of major animal fats

Fat Source 14:0 16:0 16:1 18:0 18:1

(cis +trans) 18:2 Other

Butter 12 26 3 11 28 2 18

Lard 2 26 5 11 44 11 1

Beef tallow 3 27 11 7 48 2 2

Mutton Tallow 6 27 2 32 31 2 0

Chicken Fat 1 22 6 7 40 20 4

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Physical Properties: melting point and solid fat content (SFC)

Ghotra, Dyal and Narine, 2002

Melting point (°C) and SFC profile of natural fats

Fat/Oil Melting Point (oC)

SFC value (@T,°C)

10 21.1 26.7 33.3 37.8

Butter 36 32 12 9 3 0

Cocoa Butter 29 62 48 8 0 0

Coconut oil 26 55 27 0 0 0

Lard 43 25 20 12 4 2

Palm oil 39 34 12 9 6 4

Palm Kernel oil 29 49 33 13 0 0

Tallow 48 39 30 28 23 18

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Most commonly used indicator of functionality: SFC-T profile

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Solid fat content profile of several shortenings

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Physical Properties: Crystal Habit

Ghotra, Dyal and Narine, 2002

Classification of fats and oils according to crystal habit

Beta type Beta-prime type Alpha

Soybean Cottonseed Acetoglycerides

Sunflower Palm

Corn Tallow

Canola Herring

Olive Menhaden

Coconut Milk fat

Palm Kernel sardine

Cocoa butter

Lard

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Triacylglycerol composition….the true indicator of functionality

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8:0 10:0 12:0 14:0 16:0 18:0 18:1 18:2 18:3

Palm - - - 1 44 4 39 11 Tr

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Oils and Fats Modification Methods • Different functionalities (nutritional and physical)

require specific compositions that are usually not found in a single natural fat or oil

• For this reason, fats and oils are often modified in

order to achieve these desired compositions and thus physical and nutritional properties – For example, melting behaviour, solid fat content and

crystal habit are important factors in the formulation of shortening and margarine, while decreasing PUFAs to increase oxidative stability is important in frying oil formulations

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Oils and Fats Modification Methods

Gunstone, 2006

1.Blending 2.Fractionation 3.Hydrogenation 4.Interesterification (chemical or enzymatic) 5.Genetic Improvement (GIO)

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Blending

• Different base-stocks are mixed together to obtain a specific composition, consistency, and/or stability in the final product

These base-stocks may include:

Partially or fully hydrogenated oils Interesterified oils and fats Fractions from winterized or fractionated oils

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Fractionation

• Fractionation (solvent or dry) leads to the separation of fats and oils into two or more fractions with different melting points

• Also it could be used to remove an undesirable minor component such as waxes in oils during dewaxing and winterization processes to produce salad oil

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Fractionation The palm oil industry uses oil fractionation process to alter, extend and improve the functionality of palm oil for use in different food and feed applications.

Kellens, Gibon, Hendrix and Greyt 2007 17

Palm oil, IV 52

Stearin, IV 33 (Vanaspati)

Olein, IV 56 (Frying oil)

Super Olein, IV 65 (Cooking oil) Soft PMF, IV 47

(Margarine) Super stearin, IV <15 (Animal feed)

Mid stearin, IV 45 (Margarine)

Mid olein, IV 54 Hard PMF, IV <36 (Confectionary)

Top olein, IV 70 (Salad oil)

Mid olein, IV 60

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Hydrogenation

Partially hydrogenated fats: • Semi- solid • Stable during deep-frying • Long shelf-life • Excellent functionality

Partially hydrogenated vegetable oils = trans fatty acids

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H2

Ni Heat Agitation

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Genetic Improvement

• New mutants can produce oils with specific compositions such as high saturated, high monounsatuared or low polyunsaturated fatty acid contents

• These GI oils and fats can be used in the formulation of shortening, margarine and frying oils

• Microalgal oils are an exciting new development in this area (Solazyme Inc.)

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Some examples of “Genetically Improved” Vegetable Oils

Fatty acid

High Oleic Sunflower oil (Nusun®)

High Oleic High Stearic Sunflower Oil (Nutrisun®)

High Oleic High Palmitic Sunflower Oil

High Oleic Canola Oil

Low Linolenic Soybean Oil

High Oleic Safflower Oil

C 16:0 4.3 8 27.8 4.7 12.2 3.6 C 16:1 - - 7.1 - - 0.1 C 18:0 3.8 18 1.8 2.6 3.6 5.2 C 18:1 57.9 70 57.7 59.1 24.2 81.5 C 18:2 31.9 4 2.3 23.9 57.2 7.2 C 18:3 - - - 3.5 3.8 0.1

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Interesterification • Interesterification is a chemical reaction that induces the rearrangement

of fatty acids within and between triacylglycerols

• In the food industry, interesterification can be carried out using a chemical catalyst or an enzyme. Sodium methoxide is generally used as a catalyst in chemical esterification, while lipases are used in enzymatic esterification. Chemical interesterification is a random reaction while enzymatic interesterification can be random or regiospecific.

• Used since the late 40’s to modify the crystallization behavior of lard

(induce beta prime tendencies), used successfully to make products such as Becel margarine (Unilever) for several decades now, used since the early 80’s with enzymatic catalysts to make cocoa butter equivalents and other structured lipids.

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Macrae, 1983

Triacylglycerol mixture before and after chemical interesterification

A

B

A

C

B

C

+

A

B

A

A

A

A B

B

B C

C

C

A

A

B B

B

A

C

C

A

B

A

B C

A

C

B

C

A

A

A

C B

B

C C

B

C

A

C

A

B

C

B C

C

B

A

B

C

C

A

B

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A

B

A

C

B

C

+

A

B

A

A

B

C

C

B

C

Triacylglycerol mixtures before and after enzymatic interesterification (1,3-specific lipase)

Macrae, 1983

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Lipase

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Lipase Mediated Reactions

Singh and Mukhopadhyay, 2012 24

Hydrolysis

Esterification

Acidolysis

Alcoholysis

Interesterification

Aminolysis

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Some Commercially Available Lipases and their Industrial Applications

Singh and Mukhopadhyay, 2012

Lipase Source Application

Humicola lanuginose Detergent additive

C. Cylindracea Food processing

C. Rugosa Organic synthesis

R. Miehei and M. miehei Food processing

T. lanugi Detergent additive

A. niger Oleochemistry

Penicilliun roquefortii Food processing

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Comparison of interesterification methods

Chemical interesterification Enzymatic interesterification

Low processing cost (batch reactor) High processing cost (continuous plug-flow reactor, lipase)

High processing loss (oil saponification) Minimum processing loss

Low oxidative stability (tocopherol loss)

No change in oxidative stability

High levels of reaction by-products (MAG, DAG, glycerol)

Low levels of reaction by-products

Flavor reversion problem No flavor reversion

Highly reproducible and easily controlled

More complex operation and control

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Industrial enzymatic interesterification plant (four packed-bed reactors)

Gibon 2011

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Typical batch reaction vessel for chemical interesterification

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Chemical vs. enzymatic esterification

High oleic sunflower oils + fully hydrogenated canola oil

Melted at 85ºC

Stopping reaction by acidic water (4%)

Adding sodium methoxide (0.3%)

Washing blends with dilute basic water (0.1N) (1:8)

Reaction at 85ºC/1h, N2

Mixing with bleaching clay(1.5%) Heated at

85ºC/20min

Adding immobilized Candida antarctica lipase (5%)

Placing flasks in an orbital shaker at 70ºC/24hrs/200rpm

Vacuum filtration and storage at 4ºC

Chemical Enzymatic

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Major fatty

acids

Carbon

No.

Fully hydrogenated

canola oil

High oleic sunflower oil

% of total fat or oil

Palmitic acid 16:0 8.8 5.1 Stearic acid 18:0 88.0 5.9

Oleic acid 18:1 0.08 76.8 Linoleic acid 18:2 0.03 8.0 Linolenic acid 18:3 0.0 0.9

Eicosanoic acid

20:0 1.9 0.0

Interesterified high oleic oils and fully hydrogenated hard-stocks

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Equilibrium composition

25%

12.5%

25%

25%

12.5%

OSO

OOS / SOO

OOO + SSS

SSO / OSS

SOS

OOO + SSS

Sodium methoxide, Lipases

Interesterification of 50:50 mixtures of OOO and SSS

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Triacylglycerol composition (% peak area) of NI ,CI, and EI samples of 40% FHCO/HOSO

DAG PLL PLO OOL OOO OPO PPP OOS POS PPS SOS SSP SSS

NI 0.0 1.0 2.5 17.3 31.4 2.8 1.3 2.7 0.0 0.7 1.7 3.6 31.8

CI <1 1.3 2.6 7.4 17.6 2.1 3.4 32.1 3.6 2.0 19.5 1.6 4.3

EI 5.4 0.0 2.4 5.5 18.8 1.6 3.2 32.9 3.0 1.6 21.0 0.7 1.2

DAG, diacylglycerol; O, oleic acid; L, linoleic acid; M, myristic acid; P, palmitic acid; S, stearic acid

NI= Non-interesterified, EI=Enzymatic interesterified, CI=Chemical interesterified

Ahmadi, Wright, Marangoni 2008

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SFC and DSC of EI,CI and NI blends as a function of temperature

0 10 20 30 40 50 60 70 800

10

20

30

4010% FHCOA

NICIEI

Temperature[°C]

SFC

[%]

0 10 20 30 40 50 60 70 800

10

20

30

4020%FHCOB

Temperature[°C]

0 10 20 30 40 50 60 70 800

10

20

30

40

50

6030%FHCO

C

Temperature[°C]

SFC

[%]

0 10 20 30 40 50 60 70 800

10

20

30

40

50

6040%FHCO

D

Temperature[°C]

32 Ahmadi, Wright, Marangoni 2008

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NI CI EI

0 10 20 30 400

50

100

150

200

250

300

10

90

4.6Å

3.9Å 3.7Å

2θ [°]

Inte

nsity

0 10 20 30

4.6 4.2 3.8

4.11

10

10015.2A

2θ [°]0 10 20 30

100

10

3.8

4.14.24.5

B

2θ [°]

β β + β΄

CI and EI induce structural changes

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Effect of shortening type on the physical specifications of cookies Shortening type

Width (mm) Thickness (mm)

Spread ratio (mm)

Yield force (N)

30% CI-blend 70.55 ± 0.25a 9.69 ± 0.09

a 7.38 ± 0.28

a 16.63 ± 3.97

a

Commercial 67.62 ± 0.11b 10.38 ± 0.05

a 6.54 ± 0.15

b 17.99 ± 5.05

a

Different superscript letters indicate significant differences (P< 0.05) of values within each column.

Triangle test result: No significant differences between cookies made using two types of shortening

Ahmadi, Wright, Marangoni 2008

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Structured Lipids (SLs)

• This term is often used to denote triacylglycerols for which some of the naturally ocuring long-chain fatty acids at the sn-1 and -3 positions have been replaced by medium-chain length fatty acids such as caprylic acid (8:0) or capric acid (10:0), long-chain omega-3 fatty acids (DHA, EPA, DPA), long-chain omega-6 fatty acids (AA), or other “foreign” fatty acids

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Structured Lipids - MCT • Medium chain fatty acids are easily hydrolyzed and

readily absorbed • Medium-chain fatty acids are directly metabolized for

energy rather than accumulated as depot fat • Thus, structured lipids are a concentrated and readily

available source of energy and have a reduced tendency to promote obesity

• MCTs are useful food supplements for people with chronic health conditions such as impaired gastrointestinal function, liver disease or CAD.

• MCTs have also been used as a valuable component in designing infant formula, beverages, snack bars, confectionary products, and supplements for athletes

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Structured Lipids Applications

Human milk fat substitute Oil enrichment with essential or long

chain omega-3 fatty acids (EPA and DHA) Medium-chain fatty acids (MCFAs) for

specific nutritional or medical applications

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Human Milk Fat Composition

Jala et.al., 2012

Fatty acid Total (%w/w) sn-2 %sn-2 sn-1,3

12:0 4.9 5.3 36.0 4.7

14:0 6.6 11.2 57.0 4.3

16:0 21.8 44.8 68.0 10.3

18:0 8.0 1.2 5.0 11.4

18:1 n-9 33.9 9.2 9.0 46.3

18:2 n-6 13.2 7.1 18.0 16.3

18:3 n-3 1.2 Nd. Nd. Nd.

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Human Milk-fat Substitute

Human milk fat contains 20 to 25% palmitic acid (about 70% is esterified to the sn-2 position), while palmitic acid in vegetable oils is esterified to sn-1 and sn-3

Palmitic acid is released from sn-1 and sn-3 positions in free fatty form acid after digestion by gastric and pancreatic lipases. In this form, it is not as readily absorbed and can bind to calcium, forming calcium soap. While, palmitic acid in the sn-2 position is more easily absorbed as a 2-MAG. Calcium soap may contribute to comparatively harder stools and calcium and palmitic acid losses

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Human Milk-fat Substitute

• The aim here is the production of 1,3-oleoyl-2-palmitoyl glycerol as the main component in human milk-fat substitute

• Acidolysis of animal fat (lard) with oleic acid (or oleic acid ethyl ester) using immobilized 1,3-specific lipase

• Human milk contains arachidonic acid (AA), as well as DHA. So, 1,3-arachidonoyl-2-palmitoyl glycerol is synthesized by acidolysis of tripalmitin or lard with AA using immobilized microbial lipase. Same can be done with DHA.

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Lard-based human milk-fat substitutes

Cheong and Xu, 2011

18:1

16:0

18:1

22:6 20:4

Major TAG in lard +

18:1

16:0

18:1

16:0

18:1

16:0

20:4

22:6

16:0

20:4

1,3-specific lipase

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22:6 18:1

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Production of Oil Containing Medium- and Long-Chain Fatty Acids • This oil was first produced by transesterification

of canola oil and medium-chain TAG, and has been on the market in Japan as a ‘‘Food for specified health uses” since 2003

Jala et.al., 2012

Fatty acid Percent (%)

L-L-L 55.1

L-L-M or L-M-L 35.2

L-M-M or M-L-M 9.1

M-M-M 0.6

Triacylglycerol composition of MLCT oil

M medium chain fatty acid, L long chain fatty acid

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• These structured lipids contain both long chain fatty acids (LCFA) and medium chain fatty acids (MCFA) are designed to provide immediate delivery of the MCFA fatty acids and a slower, more controlled release of the LCFA

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Some Commercial MLCT products

Jala et.al., 2012

Brand name Fatty acid profile Application Company

Caprenin 8:0-10:0-22:0 Candy bars and confectionary coating

Procter and Gamble

Captex 8:0-10:0-18:2 Clinical application and cosmetic industry

Abitec Corp.

Neobee 8:0-10:0-LCFA Pharmaceutical in medical beverages or bars

Stepan Company

Impact Randomized 12:0 and 18:2 Pharmaceutical Novartis Nutrition

Laurical 12:0 and 18:1, 18:2, 18:3 Confectionary coating, coffee whitener, whipped toppings and entree fat

Calgene Inc.

Structolipid Mixture of 8:0, 10:0, 16:0, 18:0, 18:1, 18:2 and 18:3

As a rapid source of energy for critically ill patients

Fresenius Kabi, Parenteral nutrition

Resetta oil 8:0, 10:0 and canola oil Cooking oil and salad dressing Nisshin Oillio

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Nutritional Beverages

• Structured lipid beverages provide more rapid energy source for people on modified diets, or who experience involuntary weight loss, and for patients who are recovering from illness or injury – For example, beverages can be formulated from

canola oil and caprylic acid after enzymatic interesterification (acidolysis) with an sn-1,3 specific lipase obtained from Rhizomucor miehei

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Cocoa Butter Equivalents (CBEs)

• Cocoa butter (fat obtained from the mature bean of Theobroma cacao) consists of mainly 23-30% palmitic acid, 32-37% stearic acid, and 30-37% oleic acid (in the form of SOS, POS, POP) which gives the product its characteristic hardness at ambient temperatures, with a sharp transition to the molten state at 30–32°C

• The price of this high-value product often fluctuates, and availability of the best-quality cocoa butter on the market may be uncertain

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Cocoa Butter Equivalents (CBEs)

• Starting materials for the enzymatic reaction can be palm oil mid-fraction (high POP), lllipe butter, Sal fat (high StOSt), olive oil, high-oleic sunflower or canola oils (high OOO) and palmitic or stearic acids or their esters

X

18:1

Y

18:0 / 16:0

16:0

18:1

16:0

16:0

18:1

18:0

+

18:0

18:1

18:0

sn-1,3

lipase + +

X and Y can be any fatty acid

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Cocoa Butter Equivalents (CBEs)

Commercial CBE may be produced by combination of different processing steps, including blending, interesterification, fractionation and refining

Palm oil mid-fraction and stearic acid ethyl ester in hexane

Enzymatic Interesterification (40 °C, 48-72 h)

Separation of lipase and hexane (filtration)

Distillation to remove solvent and ethyl esters

Fractionation to remove high melting fraction

Refining

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Structured Phenolic Lipids

• Phenolic acid properties Natural antioxidant Anti-carcinogenic Anti-inflamatory Anti-Alzheimer’s disease UV-absorbing

• Phenolic acids have a low solubility and stability in hydrophobic media, which consequently reduces its biological efficiency in oils, emulsions, and fats

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Ferulic Acid-based Structured Lipids

• Enzymatic esterification of soybean oil and ethyl ferulate leads to the production of phenol monoacylglycerol and phenol diacylglycerol compounds

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Phenol monoacylglycerol and phenol diacylglycerol Production

Jala et.al., 2012 51

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Conclusion

• Chemical and enzymatic interesterification have different applications in the food industry, from formulation of shortenings and margarines to synthesis of structured lipids for special medicinal and nutritional applications

• Lipase-catalyzed interesterification reactions for the synthesis of functional lipid products will remain an area of interest for many years to come

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References:

1. Gunstone FD (2006) Vegetable oils. In: Bailey’s Industrial Oil and Fat Products, Vol.1, Sixth Edition, John Wiley & Sons, Inc., Publication, 213-268.

2. Gunstone FD (2008) The Major Sources of Oils and Fats. In: Oils and Fats in the Food Industry, John Wiley & Sons, Inc., Publication, 11-25.

3. Ghotra BS, Dyal SD, Narine SS (2002) Lipid shortenings: a review. Food Research International 35,1015–1048.

4. Marc Kellens M, Gibon V, Hendrix M, Greyt WD (2007) Palm oil fractionation. Eur. J. Lipid Sci. Technol. 109, 336–349.

5. Gibon V (2011) Enzymatic interesterification of oils. Lipid Technology 23, 274-277. 6. Macrae AR (1983) Lipase-Catalyzed Interesterification of Oils and Fats. JAOCS 60, 291-294. 7. Singh AK, Mukhopadhyay M (2012) Overview of Fungal Lipase: A Review. Appl Biochem

Biotechnol.166, 486–520. 8. Jala RC, Hu P, Yang T, Jiang Y, Zheng Y, Xu X (2012) Lipases as biocatalysts for the synthesis of

structured lipids. Methods Mol. Biol. 861, 403-33. 9. Cheong LZ, Xu X (2011) Lard-based fats healthier than lard: Enzymatic synthesis, physicochemical properties and applications. Lipid Technology 23, 6-9. 10. Ahmadi L, Wright AW, Marangoni AG (2008) Chemical and enzymatic interesterification of tristearin/ triolein-rich blends: Chemical composition, solid fat content and thermal properties. Eur. J. Lipid Sci. Technol. 110, 1014–1024.

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