biocatalysis in modification of lipids for food...

Biocatalysis in Modification of Lipids for

Food Industry

Casimir C. Akoh

Department of Food Science and Technology

The University of Georgia

Athens, Georgia USA

Latin American Congress & Exhibition on

Fats and Oils (AOCS), Chile

August 22, 2013

Biotechnology refers to the integrated application of

the disciplines of biochemistry, microbiology, genetics,

and process engineering, particularly recombinant

DNA technology and genetic engineering, to

biologically derived molecules such as cells,

organisms, or enzymes to produce useful products

Biocatalysis or enzyme technology is a major part of

the technological areas of biotechnology. Involves use

of enzymes to catalyze reactions that yield useful

products

Biotechnology and Biocatalysis

Biocatalytic Modifications

Terpenes

Carbohydrates

Fats and Oils

Phytosterols

Enzymes are Work Horses

We employ enzymes to do a job

Conditions for employment must be right

Provide tools to do the job

Right conditions of: temp, cations, water, pH,

enough time – enzyme will accept the job and is

done

Wrong conditions – yo-yo job,

underemployment, or no product

Enzymes

Cyclases Monoterpenes (C10) - GPP → (menthol,

pinene) flavor compounds

Sesquiterpenes (C15) - FPP → (patchoulol,

caryophyllene) fragrance compounds

Proteases – chymotrypsin, subtilisin (serine hydrolase)

Proteins but can synthesize lipids

Lipases (esterases, phospholipases) - lipids, many

substrates

Carbohydrases (e.g., amylase family) – carbohydrate

hydrolysis and synthesis

Mevalonic acid pathway to different terpenes Source: McCaskill and Croteau (1997) Adv Biochem Eng

Biotechnol

mevalonic acid

acetyl-CoA HMG-CoA

IPP DMAPP

GPP

FPP -PP = Pyro-

or

diphosphate

menthol

patchoulol

Cyclization of GPP to monoterpenes by cyclases Source: McCaskill and Croteau (1997) Adv Biochem Eng Biotechnol

GPP acyclic

Cyclization

requires

dication

Mg++ or

Mn++

Fenchol cyclic

Cyclization of FPP to sesquiterpenes by cyclases Source: McCaskill and Croteau (1997) Adv Biochem

Eng Biotechnol

patchoulol

caryophyllene

t-germacradienyl cation

t,t-FPP

Patchoulol Biosynthesis and Mechanism

t,t-FPP

patchoulol

Patchoulol cyclase

Hydride shift mechanism, no

germacrene or bulnesene

intermediates (from 14C and

3H labeled FPP)

Time course of enzymatic synthesis of geranyl and

citronellyl acetates in n-hexane catalyzed by

Candida antarctica lipase, SP435

Source: Claon and Akoh, 1994

Lipase-Catalyzed Flavor Synthesis

Terpene Butyrates Using Various Substrates –

Catalyzed by Lipases

Source: Claon and Akoh, 1994

Our Lipid Research Interests

Structured lipids (SL)

Production of healthier lipids (lower cholesterol, improve immune system, reduce weight and obesity)

Lipids for better functionality in foods (salad dressing, margarine, shortenings, spreads, cocoa butter alternatives, cooking and frying oils)

Trans-free fats

Incorporation of omega-3 fatty acids from fish oil, algae, SDA soybean (infant formula, better n-3/n-6 ratios)

GLA-containing modified lipids

CLA-containing modified lipids

Infant formula (human milk fat substitutes)

Oxidation of SL and SL emulsions

Value addition to palm oil and other lipids

Phytosteryl esters

Terpene esters as flavor materials

General fats & oils chemistry and biochemistry

SL Production

Direct esterification: Glycerol + FA TAG + Water

Acidolysis: TAG1 + FA1 TAG2 + FA2

Alcoholysis: TAG1 + Alcohol1 TAG2 + Alcohol2

Interesterification: TAG1 + TAG2 TAG3 + TAG4

Chemical

Non-specific

Poor control over the

final product

Harsh reaction

conditions

Enzymatic

Both specific and non-specific

Good control

Milder reaction

conditions

Lipase Reactions in Organic Solvent (Synthesis)

Source: Weete, Lai and Akoh (2008) In Food Lipids Book

Reactions in Organic Solvent (Interesterification and Hydrolysis)

Source: Weete, Lai and Akoh (2008) In Food Lipids Book

Cocoa Butter Substitutes

Source: Weber & Mukherjee (2008) In Food Lipids Book

SOS and POS

Moisture Absorption by Lipid Coated Crackers

Source: Sellappan and Akoh (2000), JAOCS 77:1127-1133

UFA

MCFA / LCFA / PUFA

SFA

(Palmitic acid >60 %)

UFA

MCFA / LCFA / PUFA

Human milk fat

TAG

Vegetable oil

TAG

SFA

UFA

SFA

sn-1

sn-2

sn-3

Human Milk fat Analogs (HMF)

P

P

U

P

Human milk fat

U

U

P

Vegetable oil

U

U

U

P

Ca

Pancreatic lipase

sn-1

sn-2

sn-3

Loss of nutrients

P

P Ca P

Pancreatic lipase

Calcium dipalmitate

✗

✗

✗

✗

P= palmitic acid

U= unsaturated fatty acids

sn-2 monopalmitin

P

P

Ca2+

TAG

Background and significance

Triacylglycerol (TAG)

Nagachinta and Akoh, 2013

Human Milk Fat Analogs


OPO

DOD or GOG

Modified Lipids for Infant Formula

SL resembling human milk fat (HMF) containing GLA

SL resembling HMF containing EPA and DHA

Lipozyme RM IM or lipozyme TL IM as biocatalysts

Substrates: Tripalmitin + Hazelnut oil FAs + GLA

OR

Tripalmitin + Fish oil fatty acids

Sahin et al. (2005) J Agric Food Chem 5779-5783

Optimal Condition and Results

Temp = 55 ºC, Time = 24 h, substrate molar

ratio = 14

Results:

HMF enriched with EPA and DHA (45.5%

palmitic, 37.5% oleic, 4.4% linoleic, 6.2% EPA

and DHA)

HMF enriched with GLA (10% GLA, 45% oleic)

Sn-2 position mostly occupied by palmitic acid

(>60%) Sahin et al. (2005) J Agric Food Chem 5779-5783

Phytosteryl Esters

Plant sterols

Lower plasma total cholesterol and LDL-chol by (10-

20%) in humans

Used in margarine, spread, milk, salad dressing

Solubility problem with free sterol

Phytosteryl esters preferred in food formulations (lower

m.p., increased solubility)

Chemical or enzymatic synthesis

In organic solvent or oil/water phase

Kim and Akoh (2007) Food Chem 336-342

Esterification with Free Fatty Acids

Sitosterol

Sitosteryl

oleate

phytosterol - crystalline powder

- restricted fat-solubility

- melting range 140-150°C

phytosteryl ester - similar fat properties as

edible fats

- melting range 44-45°C

Lipase, CRL

Oleic acid

HO

O

O+ H2O


Candida rugosa Lipase-Catalyzed

Synthesis

Phytosterol + oleic acid in hexane (monophasic media)

No water removal

RSM – Factors considered

Temp = 35-55ºC; Time = 4-24 h; substrate molar ratio (oleic

acid:phytosterol = 1-3; lipase = 2-10 weight%

Measure degree of esterification (mol%)

Optimal Condition:

Temp = 51.3ºC

Time = 17 h

Substrate molar ratio = 2.1

Lipase = 7.2%

Degree of esterification = 97 mol%


Enzymes Can be Trained to Perform Better

But first – Normal Reactions

Substrate specific

Regiospecific

Optimal reaction conditions – very good job

Genetic engineering

Okay, will take another substrate

Will tolerate higher temperature

Will handle unusual pH

Cut and paste amino acid regions – chimeric or recombinant

enzymes

Protein engineering

Fusion enzymes – more than one enzyme (e.g., linear and

debranching amylases)

Lipases

Esterify carbohydrates – sugar fatty acid esters as

emulsifiers, antimicrobials, crystal inhibitors

Esterify terpene alcohols

Modify TAGs, DAG, MAG, PLs

Esterify primary and secondary alcohols

Peptide bond formation?

Mono- and Diacylglycerols


partial hydrolysis

esterification

Sugar and Alkyl Glycosides Esters


alkyl glycoside ester

sugar ester

Phospholipid Synthesis


Enzyme Capabilities are Limitless! Not Really

Esterification of 3º (tertiary) alcohols – not easy

If you motivate me, I will do more of the same job

(enzyme reuse, wash and regenerate)

Immobilize me – I will not go away, will stay and work for

you for a long time

pH memory – remembers the last aqueous solution

exposed to for immobilized enzymes

Applied Biocatalysis

Trans-free fats for margarine, shortening, and spreads

Infant formula or milk fat substitutes or analogs

Phytosteryl esters

Sugar esters

Pro-drugs

Chiral intermediates

Resolve racemic mixtures, e.g., (±)-menthol

Flavor esters – terpene esters and other short chain esters

Cocoa butter equivalents, coating lipids

Healthful lipids – omega-3, CLA-, GLA-containing

Esterify branched alcohols

Biodiesel

You can stop enzyme – Economic or Quality

Reasons?

Filter to remove immobilized enzyme

Pour cold water on enzyme - you get a rain

check

Throw enzyme out of office – terminate reaction

Make the conditions for catalysis a living hell

Confuse enzyme – wrong substrate or

conditions

Optimization and Scale-up

One factor at a time – time consuming

Response Surface Methodology, RSM – better,

less # of experiments

Bioreactor and types:

Stirred tank (liquid or semisolid substrates) –

large scale

Packed bed (pump liquid substrates) – large

scale

Test tube/flask – small scale

Packed Bed Bioreactor

Stainless steel column bed

47 mm (internal diameter) x

50 cm long

Pump to feed the substrate

Jacketed bed with

temperature maintained by a

circulating water bath

Temperature and pressure

probes at inlet and outlet,

connected to a computer for

monitoring

Source: Willis and Marangoni (2008)

In Food Lipids Book

Dr Akoh’s Lab

Source: Willis and Marangoni

(2008) In Food Lipids Book

Akoh’s Lab

Recovery/Downstream Processing

Very important – will determine purity, yield, application,

cost

Filter enzyme out

Acidification

TLC and column chromatography – small scale

Short-path or molecular still distillation – large scale

Deodorization – large scale, necessary for food

application

Short-Path Distillation

UIC KDL-4 (UIC Inc., Joliet, IL)

First stage - removal of caprylic

acid (heating oil temp, 75ºC;

cooling water 15ºC; mix holding

temp 40ºC)

Second stage - removal of

long-chain FA (heating oil temp

185ºC; cooling water 20ºC; mix

holding 40ºC)

Pressure in both cases - below

0.02 Torr

Dr Akoh’s Lab

Post Recovery/Processing

Physical, chemical, stability, and sensory

properties

Food and product application

Animal study

Human feeding trial/clinical for some – Goal!

Product in the market

Enzymatic Products - to – Applications

trans-Free Margarines Prepared with

Enzymatically Synthesized

Structured Lipids

Traditional margarine fats

Margarine fats are traditionally produced by

partial hydrogenation process

Contain trans fatty acids (TFAs)

(10-36% of total FA)

TFAs are a risk factor for coronary heart disease

(CHD)

Structured lipids (SLs) are a good

alternative to partially hydrogenated fats

The use of structured lipids (SLs) is one of the

most successful alternatives to the partial

hydrogenation process to reduce or eliminate

TFAs in margarines

SLs are restructured fats/oils where the

composition and positional distribution of FAs are

modified from the native state by chemical or

enzymatic methods

High diversity in FA profile contributes to the formation of β′ crystal

polymorphic form

High diversity in FA composition contributes to

the predominant presence of β′ crystal

polymorphic form over other forms (α and β)

β′ form is the most desirable in margarines

because it gives smooth texture to the products

β form is undesirable in margarines

because it gives grainy texture to the products

Commercial margarine fats separated from…

CTMF: commercial trans

margarine fat

CTFMF: commercial trans-free

margarine fat

FA profiles of lipid substrates (w/w%)

canola oil palm stearin palm kernel oil

fatty acid total sn-2 sn-1,3 total sn-2 sn-1,3 total sn-2 sn-1,3

C6:0 0.4 0.6

C8:0 trace 5.1 1.4 0.9

C10:0 trace 4.3 1.7 5.5

C12:0 0.3 0.1 0.4 52.6 49.8 54.2

C14:0 1.5 1.0 1.8 15.3 15.2 15.4

C16:0 4.7 0.4 6.8 62.1 46.5 69.7 6.8 4.2 8.1

C16:1n-7 0.2 0.1 0.3 0.1 trace 0.2

C18:0 1.8 0.3 2.6 4.6 2.1 5.9 1.6 0.5 2.1

C18:1t trace 0.1

C18:1n-9 56.1 64.1 51.9 24.3 40.1 16.4 12.0 23.4 6.3

C18:1n-7 2.8 1.2 3.7 0.4 0.1 0.6

C18:2t,t

C18:2n-6 21.1 25.4 19.0 6.2 9.9 4.3 1.9 3.8 0.9

C18:3n-3 10.6 7.2 12.4 0.2 0.1 0.2

C20:0 0.5 0.8 0.3 0.1 0.4

C20:1 2.2 1.3 2.5 trace

SFA 7.0 0.7 10.2 68.8 50.2 78.2 86.1 27.2 92.8

USFA 93.0 99.3 89.8 31.2 49.8 21.8 13.9 72.8 7.2

TFA trace 0.1

97%

62%

97%

SLs synthesis

SLs were produced by Lipozyme TL IM-

catalyzed interesterification of the blends of:

Canola oil (CO) - provide oleic acid (cholesterol-lowering

effects)

Palm stearin (PS) - contribute to rigidity

Palm kernel oil (PKO) - contribute to high diversity in

FA and TAG profiles

Blending ratio (CO:PS:PKO, w/w/w)

40:60:0 → SL460

40:50:10 → SL451

40:40:20 → SL442

40:30:30 → SL433

50:30:20 → SL532

60:25:15 → SL621

Margarine formulation

Ingredient w/w %

Lipid phase 80.5

Commercial margarine fats or SLs 80.0

Soy lecithin fluid 0.5

TBHQ 0.01

Aqueous phase 19.5

Distilled water 17.7

Table salt 1.8

Total FA profile (w/w%)

commercial

margarine fat structured lipids

fatty acid CTMF CTFMF SL460 SL451 SL442 SL433 SL532 SL621

C6:0 trace trace 0.1 0.1 0.1 trace

C8:0 trace 0.5 0.9 1.5 0.9 0.7

C10:0 trace 0.4 0.9 1.3 0.8 0.6

C12:0 0.5 0.1 5.6 11.1 16.4 10.5 8.0

C14:0 0.8 0.9 2.5 4.1 5.6 3.8 2.9

C16:0 12.1 26.9 39.9 33.9 28.1 22.5 22.3 19.4

C16:1n-7 trace 0.2 0.2 0.1 0.1 0.1 0.1 0.2

C18:0 8.5 3.9 3.4 3.1 2.9 2.6 2.7 2.5

C18:1t 14.9

C18:1n-9 23.2 35.7 36.7 35.4 34.1 32.9 39.0 42.9

C18:1n-7 2.0 1.2 1.4 1.4 1.3 1.2 1.5 1.7

C18:2t,t 0.1

C18:2n-6 33.8 25.9 11.8 11.5 10.9 10.5 12.2 13.8

C18:3n-3 4.7 3.7 4.0 4.0 4.0 3.9 4.4 5.3

C20:0 0.3 0.3 0.4 0.4 0.3 0.3 0.4 0.4

C20:1 0.4 0.9 1.2 1.2 1.2 1.1 1.3 1.6

SFA 20.9 32.4 44.7 46.4 48.3 50.2 41.4 34.5

USFA 79.1 67.6 55.3 53.6 51.7 49.8 58.6 65.5

TFA 15.0

Melting completion temperature (DSC)

melting completion

temperature (°C)

commercial margarine fat

CTMF 40.1 c

CTFMF 32.0 f

structured lipids

SL460 43.5 a

SL451 40.6 b

SL442 37.0 d

SL433 33.5 e

SL532 33.9 e

SL621 31.7 f

Solid fat content (pulsed NMR)

Polymorphic form of fat crystal at 4 °C (X-ray diffractometer)

polymorphic form level of β′ and β forms

commercial margarine fat

CTMF α + β′ + β β′ >> β

CTFMF α + β′ + β β′ = β

structured lipids

SL460 α + β′ + β β′ = β

SL451 α + β′ + β β′ > β

SL442 α + β′ + β β′ > β

SL433 α + β′ + β β′ > β

SL532 α + β′ + β β′ > β

SL621 α + β′ + β β′ > β

SL532 and SL621 were most suitable for formulating trans-free margarines with desirable nutritional and textural properties

Trans-free

Lower and similar atherogenicity compared to CTMF and

CTFMF, respectively

Good cold-spreadability

Smooth texture

Similar textural properties to CTMF and CTFMF margarines,

respectively

Food and Functional Applications of SL

Beverages, infant formula, milk drinks

Margarine, butter, spreads, shortening, dips – all trans free

Salad dressing, sauces

Frying oil, snack foods

Confectionaries, coating lipid, soft candies

Cocoa butter substitutes, alternatives, equivalents, chocolates

Dairy products, ice cream

Improve melting and crystallization properties of fats

Baking chips, baked goods

Capsules, e.g., DHA, EPA, CLA-containing SL as nutritional supplements or delivery mechanism

Potential Health Benefits of SL

Enhanced absorption of sn-2 fatty acid

Prevention of thrombosis

Improved immune function

Calorie reduction

Improved absorption of other fats

Reduction in serum TAG, LDL cholesterol, and total

cholesterol

Lipid emulsion for enteral and parenteral feeding

Incorporation into mainstream diets

Healthful and digestible fats

Thank You!!

Thank you!

biocatalysis in modification of lipids for food...

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