lipid oxidation in emulsified foods: an overview of recent progress · 2017-06-11 · lipid...

Lipid oxidation in emulsified foods: An overview of recent progress Charlotte Jacobsen Research group for Bioactives – analysis and application Division of Food Technology [email protected]

November 2015 Latin American Oil Congress World Congress on Oils and Fats

2 DTU Food, Technical University of Denmark

Outline • Introduction • Factors affecting oxidation in complex emulsified foods • Effect of oil quality • Effect of ingredients • Optimization of production process • Introduction to antioxidants in emulsified foods • Effect of antioxidant addition including lipophilisation • Conclusions • Acknowledgements



Lipid oxidation

Unpleasant flavors, Nutrient loss, Texture, Color, Functionality

Lipid oxidation is one of the most important chemical degradation processes

Oxidized lipids Rancidity

Food emulsions: Mayonnaise, salads & dressing, yoghurt, milk

Fish

Bulk oil



Oxidation and analysis of oxidation

Volatile oxidation products Rancid off-flavour

Hydroperoxides: peroxide value (PV) or conjugated dienes

Volatiles: Anisidine value, TBARS, GC/MS (HS, DHS, SPME)

Sensory evaluation



Factors that can affect lipid oxidation in emulsified foods:

Oil Water

1. Ingredients (Amount, type and quality)

3. The surface charge

2. pH

5. Oil droplet size / surface area

4. Viscosity

6. Processing conditions

7. Antioxidants

AO

AO

AO

+ +

+ +

Fe 3+

Fe 2+

Emulsifier at the interface (Structure/thickness)

Emulsifier in the aqueous phase (Antioxidative properties)



The role of emulsifiers

Berton-Carabin, C., Ropers, M.-H., Genot, C. (2014). Comprehensive Reviews in Food Science and Food Safety, 13, 945-977



Effect of oil quality



Milk with a fish oil and rapeseed oil mixture

0

1

2

3

Milk FRO 0.1 FRO 0.5 FRO 1.0 FRO 2.0 Day 1 Day 4

Day 8

Fishy taste

[0-9]

Let et al., 2005. Int. Dairy J. 15:173-182

Fish oil enriched milk

Oil quality affects the food quality

→ seems that PV needs to be

≤ 0.1 meq/kg fish oil



Effect of food composition



Effect of fish oil concentration and emulsifier type in fish oil enriched mayonnaise (20 oC)

0 25 50 75 100 1250

50

100

150

200

250

300

350

M_STD

M_4%FO

M_10%FOM_14%FO

MP_STD

MP_14%FO

Storage time [days]

1-pe

nten

-3-o

ne [n

g / g

]

Rancid (Flavour)

0 40.0

2.5

5.0

7.5

10.0

12.5M_STDM_4%FOM_10%FOM_14%FOMP_STDMP_14%FO

Storage time [Months]

• Oxidation increased with

increasing fish oil conc.

• Previous data: Egg yolk and low pH

main factors responsible for lipid oxidation

• Milk protein resulted in increased oxidation



Effect of the Ingredients: Mayonnaise-based Shrimp Salad

Sørensen, Nielsen & Jacobsen, 2010, Eur. J. Lipid Sci. Technol. 112, 476-487

0 10 20 30 40 50 600

50

100

150

200

250

S_STDS_STD_FO

S_FO_S

S_FO_A

Storage time [days]

2-pe

nten

al [n

g / g

sam

ple]

2 10 24 36 50 570

25

50

75 S_STDS_STD_FOS_FO_S

Storage time [days]

Inte

nsity

(T-R

anci

d)

Shrimp & asparagus Shrimp, asparagus & FO

Asparagus & FO

Shrimp & FO Shrimp & FO

Shrimp & asparagus Shrimp, asparagus & FO

• Salads without asparagus were more oxidised

• Salads without shrimps were the least oxidised

• The panel could not discriminate between standard salad with or

without fish oil



Comparing Milk and Drinking yoghurt: Effect of Ingredients in Yoghurt

• Fish oil enriched milk oxidised much faster than fish oil enriched

yoghurt

• Ingredients added to yoghurt did not affect oxidation

Intensity of fishy off-flavour (0-9):

Nielsen, Klein & Jacobsen, 2009, Eur. J. Lipid Sci. Technol. 111, 337-345

Week 0 Week 1 Week 3

Milk 5.4 ± 1.5 6.0 ± 2.4 7.4 ± 1.0

Yoghurt (CA+P+FS)

0.0 ± 0.1 0.5 ± 1.0 0.4 ± 0.7

Yoghurt (CA+P)

0.4 ± 0.5 0.9 ± 1.4 1.0 ± 1.3

Yoghurt (CA)

0.4 ± 0.5 0.7 ± 1.0 1.3 ± 0.9

Yoghurt 0.5 ± 0.4 0.8 ± 1.0 1.6 ± 1.2

CA: Citric acid; P: Pectin; FS: Fruit preparation and sugar



Comparing Milk and Yoghurt: Antioxidant Assays on Peptide Fractions in Yoghurt

Crude protein

>30 10-30 3-10 <3 Radical scavenging (DPPH)

+++ +++ +++ ++ ++

Metal chelation + + + +++ +++

Reducing power + + + ++ +++

Peptide fractions (KDa)

Farvin, Nielsen, Baron & Jacobsen, 2010 Food Chem. 123, 1081-1089.



Comparing Milk and Yoghurt: Oxidative Stability of Milk with Peptide Fractions from Yoghurt

0 2 4 6 8 10 12 140

1

2

3

4

5

6

7

M+RO

M+FO

M+FO+CPP

M+FO+3-10KDa

M+FO+<3KDa

Storage time (Days)

1-pe

nten

e-3-

ol (n

g/g

emul

sion

)

Low molecular weight peptides reduce oxidation

Farvin, Nielsen, Baron & Jacobsen, 2010 Food Chem. 123, 1081-1089.



LC-MS/MS characterisation of peptides

>30 kDa

10-30 kDa

3-10 kDa

<3 kDa

Absorbance at 210 nm

Fractions > 3 kDa

were dominated

by peptides from

caseins. Phosphory-

lated peptides were

not found.

Fraction < 3 kDa

dominated by free

amino acids

Farvin, Nielsen, Baron & Jacobsen, 2010, Food Chem. 123, 1090-1097.



Optimising processing conditions

- Temperature and pressure

- Type of equipment



Effect of Emulsification Conditions: Milk Affected by Temperature and Pressure

• Homogenization with a low temperature and a low pressure leads to more oxidation than a high temperature and a high pressure!

0 3 8

1 4 7 11 0

5

10

15

Temp. 72C/Pressure 5 MPa

Temp. 50C/Pressure 22.5 MPa

Temp. 72C/Pressure 22.5 MPa

Temp. 50C/Pressure 5MPa

2-hexenal

[ng/g milk]

Days of storage at 2°C

Let, Jacobsen, Sørensen & Meyer (2007), J. Agric. Food Chem. 55:1773-1780



Homogenisation Conditions Affect Protein Composition at the Interface

Sørensen, Baron, Brüggemann, Pedersen & Jacobsen (2007) J. Agric. Food Chem. 55:1781-1789

Pressure [MPa]

Temperature [°C]

β -lactoglobulin α -casein α -casein

Reference, no treatment 4.92 5.18 4.19

5 50 8.01 5.94 5.14

72 10.17 4.28 4.09

15 50 9.36 5.21 5.08

72 14.56 4.55 4.59

22.5 50 9.45 4.56 4.23

72 13.72 3.68 3.74

s1 s2

+ - -



Homogenisation conditions affect surface behaviour of proteins

Casein: green Lactoferrin: red Lipids: blue

Fish oil enriched milk

50 °C, 50 bar Fish oil enriched milk

72 °C, 225 bar



Effect of emulsification equipments Microfluidizer vs.

two-stage Valve Homogenizer Delivery emulsions



Effect of emulsification Equipment Fish oil-in-water emulsion

• Ingredients: – 10% fish oil – 1% emulsifier – 89% buffer

• Storage with iron for 14 days

Type of emulsifier

Transition metal ions

Processing conditions

+ +

+

+

Fe 3+

Fe 2+

Oil droplet size Microfluidizer

Two-stage valve homogenizer

69 MPa, 3 passes

80 MPa 50 MPa

4 passes 3 passes

CAS_M

WPI_M

CAS_H

WPI_H

Particle Size Distribution

0.01 0.1 1 10 100 1000 3000 Particle Size (µm)

0 1 2 3 4 5 6 7 8 9

10

Volu

me

(%)

1a Micro NaCas a - Average, 24. juni 2010 08:52:221b Micro NaCas a - Average, 24. juni 2010 08:58:403a Bord NaCas a - Average, 24. juni 2010 09:16:383b Bord NaCas a - Average, 24. juni 2010 09:22:46

CAS_M

Particle Size Distribution

0.01 0.1 1 10 100 1000 3000 Particle Size (µm)

0 1 2 3 4 5 6 7 8 9

Volu

me

(%)

2a Micro Valle a - Average, 24. juni 2010 09:04:312b Micro Valle a - Average, 24. juni 2010 09:10:184a Bord Valle a - Average, 24. juni 2010 09:29:054b Bord Valle a - Average, 24. juni 2010 09:35:26

CAS_H WPI_M

WPI_H



Emulsification Equipment Results: PCA Biplot

• CAS vs WPI: Higher PV in CAS but less increase in volatiles than WPI – Metal chelation

• WPI_M vs WPI_H: Protein at the interface – Structure and thickness of interfacial layer

-0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

t.t-2.4-Heptadienal_D 2-Hexenal_D14

2-Butenal_D4

t.t-2.4-Heptadienal_D

t.c-2.4-Heptadienal_D

t.t-2.4-Heptadienal_D

2-Butenal_D14

2-Hexenal_D10 t.t-2.4-Hexadienal_D4

4-Heptenal_D4

t.t-2.4-Hexadienal_D1

2-Hexenal_D4 Hexanal_D4 1-penten-3-one_D4 t.t-2.6-nonadienal_D4 Heptanal_D4 Pentanal_D4

2-Hexenal_D0

t.c-2.4-Heptadienal_D4

2-Hexenal_D7 Pentanal_D0

t.t-2.6-nonadienal_D10 2-Butenal_D10 t.c-2.4-Heptadienal_D10

t.t-2.4-Heptadienal_D7

Heptanal_D0


Propanal_D4 t.t-2.4-Hexadienal_D7

4-Heptenal_D0 t.t-2.6-nonadienal_D14

1-penten-3-one_D14

4-Heptenal_D10

Heptanal_D14

Pentanal_D14

Pentanal_D7

Propanal_D0

2-Butenal_D7

1-penten-3-one_D10


Hexanal_D0

t.t-2.4-Heptadienal_D0

Hexanal_D14


4-Heptenal_D14

1-penten-3-one_D7

2-Butenal_D0

t.t-2.6-nonadienal_D7

Pentanal_D10

4-Heptenal_D7 t.t-2.6-nonadienal_D0

Hexanal_D10 Heptanal_D10

Propanal_D14

1-penten-3-one_D0 Propanal_D7

Loadings PC#1 (58.670%)

PCA Loadings [Model 1]

Propanal_D10


Heptanal_D7 Hexanal_D7

PV_D7

PV_D14

PV_D10

PV_D4

PV_D0

Load

ings

PC

#2 (1

2.71

5%)

-8 -6 -4 -2 0 2 4 6 8 10

-4

-3

-2

-1

0

1

2

3

4

5

WPI_Ha

WPI_Hb

WPI_Mb

WPI_Ma

Scores PC#1 (58.670%)

PCA Scores [Model 1]

CAS_Ma

CAS_Ha CAS_Mb

CAS_Hb

Sco

res

PC

#2 (1

2.71

5%)

CAS_Ma

WPI_Mb

WPI_Hb

WPI_Ma

WPI_Ha

CAS_Ha CAS_Mb

CAS_Hb

Protein in aqueous phase:

WPI_M: 2.86 mg/mL

WPI_H: 4.96 mg/mL

Horn et al. (2012) Food Chemistry. 134, 803–810.

PV



Effect of antioxidants

in omega-3 enriched foods



Antioxidants

24

Antioxidants = Compounds that prevent or delay lipid oxidation

1) Primary antioxidants / Radical scavenging

2) Secondary antioxidants

Inactivate reactive radicals

Bind oxygen

Chelate metal ions

Quench singlet oxygen

Regenerate other antioxidants



Lipid oxidation

25

LH

X● XH

L●

O2

LOO●

LOOH

LH

Secondary oxidation products

and off flavors

I

Men+

Metal chelator

Radical

scavengers

Under certain conditions, antioxidants are also found to be prooxidants…



Polar paradox hypothesis Interfacial phenomena in antioxidant activity

Air

Bulk oil Emulsion (o/w)

Air

Water

Oil

Oil Oil

● Hydrophilic

● Amphiphilic

● Lipophilic Antioxidants

● ● ● ●

●

●

●

●

●

●

●

●

●

●

●

●

● ●

● ●

● ●

● ●

● ● ●

●

●

● ●

● ●

Porter., 1993. Toxicol Ind Health, 9:93-122 and Frankel et al., 1994. J Agric Food Chem, 42:1054-1059



Bulk oil – Physical structures

Chen et al., 2011. Crit Rev Food Sci Nutr, 51:901-916

Schematic illustration of some association colloids formed by minor constituents:

Physical structures:

↑ Lipid oxidation reactions

Alter effectiveness of AO



AO in fish oil enriched mayonnaise

0

40

80

120

160

200

0 1 2 3 4

2-Pe

nten

al [n

g/g

may

onna

ise]

Storage time [weeks]

No antioxidant

Nielsen et al., 2004. J. Agric. Food Chem. 52:7690-7699

EDTA

(6 & 24 ppm)

Lactoferrin

(700-2800 ppm)

& Phytic acid

(15-116 ppm)



1. Very good protective effect of EDTA – best single antioxidant 2. Limited but still protective effect of tocopherols 3. Interestingly, no effect of adding both EDTA and tocopherol 4. Prooxidant effect of ascorbyl palmitate in high concentration

Results Antioxidants – Similar experiment in dressing

0

4

8

12

16

0 1 2 3 4 5 6 0

10

20

30

0 1 2 3 4 5 6

Weeks at room temperature Weeks at room temperature

NoAox

AscP- high

EDTA,

EDTA+γ-Toco

AscP- low

γ-Toco

Very good stability if all

3 antioxidants are added together

t,t-2,4-Heptadienal

[ng/g milk]

PV

[meq O2/kg oil]

AO in fish oil enriched dressing

Let et al., 2007. J. Agric. Food Chem. 55:2369-2375



AO in fish oil enriched milk

NoAox, EDTA EDTA+Toco

Toco Toco

0

2

6

10

14

0 2 4 6 8 10 12 0

6

12

18

0 2 4 6 8 10

AscP

AscP+EDTA

AscP+Toco

AscP+Toco+EDTA

RapeseedOil

t,t-2,4-Heptadienal

[ng/g milk]

PV

[meq O2/kg oil]

Days at 2°C Days at 2°C

1. Prooxidant effects of tocopherols when added to fish oil alone 2. No effect of EDTA 3. Ascorbyl palmitate highly protective against oxidation 4. Rapeseed oil highly protective against oxidation

Additional exp:

γ-tocopherol seems to work

-

α-tocopherol does not

Let et al., 2005. J. Agric. Food Chem. 53:5429-5437



Antioxidant effects in fish oil enriched foods • Different antioxidants have very different effects in different

foods • Possibly due to different mechanisms of action and localizations

Tocopherol

Ascorbyl palmitate

Ascorbic acid

EDTA Propyl gallate/

Gallic acid

Lactoferrin Caffeic acid

Milk 1.5% fat

Weak anti Anti - Anti to no

- - -

Milk drink 5% fat

- Pro - Anti - Weak anti to pro

-

Drinking yoghurt 1.5% fat

- - - Anti - - -

Dressing 25% fat

Weak anti Pro - Anti - - -

Mayonnaise 80% fat

Weak anti to pro

Pro Pro Anti Pro Weak anti to pro

-

Energy bars 6.2% fat

Anti to weak pro

Pro - Pro

- - Pro

Pro: Prooxidative; Anti: Antioxidative



Cut-off effect (Model emulsions, o/w)

Laguerre et al., 2009. J. Agric. Food Chem., 57:11335-11342

Phenolic compound + fatty alcohol = phenolipid

Conjugated Autoxidizable Triene (CAT) Assay Emulsion (o/w): Phosphate buffer (pH 7.2), Brij (17 µM), Tung oil (stripped, 115 µM)

AAPH (1 mM) as initiator

Laguerre et al., 2010. J. Agric. Food Chem., 58:2869-2876

Chlorogenic acid Rosmarinic acid

Optimal alkyl chain - Rosmarinates & Chlorogenates: C8 & C12

(Laguerre et al., 2008. Anal. Biochem., 380:282-290)



Aqueous

phase

Cut-off effect (Model emulsions, o/w)

CCL

Ant

ioxi

dant

cap

acity

Alkyl chain length

0 n

Increased lipophilicity

Antioxidant (CCL) location

in an emulsion

CCL = Critical chain length

Decreased Oxidation

Modified from Laguerre et al., 2013. In: Lipid Oxidation: Challenges in Food systems, 261-295



Caffeates as antioxidants

Conjugated Autoxidizable Triene (CAT) Assay

Emulsion (o/w): Phosphate buffer, Brij, Tung Oil. AAPH as initiator Sørensen et al. 2014. J. Agric. Food Chem. 62, 12553–12562

Efficacy of Caffeates

Caffeates with medium chain length



Effect of lipophilisation of caffeic acid on oxidation in fish oil enriched milk

Treatments Concentration [μM] Control - Caffeic acid 100 Methyl caffeate 100 Butyl caffeate 100 Octyl caffeate 100 Dodecyl caffeate 100 Hexadecyl caffeate 100 Eicosyl caffeate 100

Storage: 5ºC,12 days (0, 3, 6, 9 and12)

Milk enriched with 0,5% of fish oil (total fat 1.5%)

Alemán et al. 2015 . Food Chemistry 167, 236-244.



Volatiles - Milk

Induction time

9 days CA C8

Not defined CA C1 CA C4

3 days Con CA C0

6 days CA C12

CA C16

CA C20

Con CA C0

CA C16

Day 12

CA C4 CA C1

CA C12

CA C8

CA C20

General trend of volatile development

Lowest concentration: CA C4 and CA C1



Experimental design: Mayonnaise

Fish Oil Enriched Mayonnaise

• 80 % Fat (64% rapeseed oil, 16% Fish Oil, w/w), Egg yolk as emulsifier

• Antioxidants (solubilized in MeOH), Conc. 100 µM – Con, CA C0, CA C1, CA C4, CA C8 (+200), CA C12, CA C18

• Storage at 20oC for 4 weeks (Sampling days: 0, 3, 9, 12, 15, 21 and 28) Efficacy of the antioxidants

– Lipid hydroperoxides (Spectrophotometric)

– Secondary volatile oxidation products (GC-MS)

– Tocopherols (HPLC)

Alemán et al. 2015 . Food Chemistry 167, 236-244.



Volatiles - Mayonnaise

9 days CA C0

CA C1

CA C8 200

12 days Con CA C4

CA C8

CA C12

CA C18

Induction time

Con CA C0

CA C18

CA C1

Day 28

CA C12 CA C8 200

CA C8

General trend of volatile development

Lowest concentration: CA C8 (200), CA C12, CA C4 and CA C8

CA C4

CA C12



Discussion

Mayonnaise No effect CA C0, CA C1, CA C18 Less efficient antioxidants Most efficient antioxidants CA C4, CA C8, CA C12

Increased conc. increased effect

Milk

No effect Less efficient antioxidants

CA C0, CA C8 - C20 Most efficient antioxidants CA C1, CA C4

The “cut-off” effect is influenced by the food system

It is not possible to predict optimal chain length in food products based on CAT assay



Inhibition effects of ferulates in fish oil enriched milk

□ PV

■ 1-penten-3-one,

○ 1-penten-3-ol,

● 2-pentenal,

♦ 2-hexanal,

▲ 2,4-heptadienal

+ 2,6-nonadienal

Sørensen, A.-D. M., Lyneborg, K.S., Villeneuve, P., Jacobsen, C. (2015) J. Functional Foods doi: 10.1016/j.jff.2014.04.008



Natural antioxidants in mayonnaise salads (tuna)

0 10 20 30 40 50 600

255075

100125150175200225

Storage time [days]

2-pe

nten

al [n

g / g

] Reference

Thyme 1%

Rosemary 1%

Oregano 1%

Formation of volatiles:

Reference > Thyme > Rosemary > Oregano

Sørensen et al., 2010. Eur. J. Lipid Sci. Technol. 112:476-487



Antioxidant effects of extracts from seaweed (Fucus vesiculosus) in mayonnaise

0

2

4

6

8

10

12

0 10 20 30

PV (m

eq O

2/kg

oil)

Days

Peroxide value

0

50

100

150

200

250

300

0 10 20 30

ng/

g m

ayon

nais

e

Day

1-Penten-3-ol

EtC1

EtC2

AcC1

AcC2

WOC1

WOC2

WYC1

WYC2

REF

Water

Acetone + Ethanol Honold et al. Submitted to Food Chemistry



Evaluation of compounds contributing to activity in mayonnaise

Phenolic compounds

Oxidation products – later stage of storage

Astaxanthin

Fucoxanthin



Conclusions • Ingredients can affect lipid oxidation

• Oil quality is more important in some systems than in others

• The emulsifiers used as ingredients and the composition of the

interface in emulsions affect lipid oxidation

• Emulsification processes must be optimised to minimise oxidation

• Type of emulsification equipment can affect oxidation

• The same antioxidant can have very different effects in different food systems

• Polar antioxidants can be modified to have an optimal chain length for each food system

• Natural antioxidants from seaweed have promising effects



Acknowledgements

• Caroline Baron

• Nina Skall Nielsen

• Mette Bruni Let

• Anna F. Horn

• Ann-Dorit Moltke Sørensen

• Anne S. Meyer

• Anna Klein

• Sabeena Farvin

• Lis Berner

• Inge Holmberg

• Trang Vu

• Jane Jørgensen

• Mercedes Aleman

• Ditte B Hermund

• Philipp J Honold

lipid oxidation in emulsified foods: an overview of recent progress · 2017-06-11 · lipid...

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