Lipid oxidation in emulsified foods: An overview of recent progress

Transcription

1 Lipid oxidation in emulsified foods: An overview of recent progress Charlotte Jacobsen Research group for Bioactives analysis and application Division of Food Technology

2 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 2 DTU Food, Technical University of Denmark

3 Lipid oxidation Unpleasant flavors, Nutrient loss, Texture, Color, Functionality Food emulsions: Mayonnaise, salads & dressing, yoghurt, milk Bulk oil Fish Lipid oxidation is one of the most important chemical degradation processes Oxidized lipids 3 DTU Food, Technical University of Denmark Rancidity

4 Oxidation and analysis of oxidation Hydroperoxides: peroxide value (PV) or conjugated dienes Volatiles: Anisidine value, TBARS, GC/MS (HS, DHS, SPME) Volatile oxidation products Sensory evaluation Rancid off-flavour 4 DTU Food, Technical University of Denmark

5 Factors that can affect lipid oxidation in emulsified foods: 1. Ingredients (Amount, type and quality) 3. The surface charge Water Oil 4. Viscosity Emulsifier at the interface (Structure/thickness) Emulsifier in the aqueous phase AO (Antioxidative properties) AO Fe 2+ Fe ph AO 5. Oil droplet size / surface area 6. Processing conditions 7. Antioxidants 5 DTU Food, Technical University of Denmark

6 The role of emulsifiers Berton-Carabin, C., Ropers, M.-H., Genot, C. (2014). Comprehensive Reviews in Food Science and Food Safety, 13, DTU Food, Technical University of Denmark

7 Effect of oil quality 7 DTU Food, Technical University of Denmark

8 Fish oil enriched milk 3 Fishy taste [0-9] 2 Oil quality affects the food quality seems that PV needs to be 0.1 meq/kg fish oil 1 0 Milk FRO 0.1 FRO 0.5 FRO 1.0 FRO 2.0 Day 8 Day 4 Day 1 Milk with a fish oil and rapeseed oil mixture Let et al., Int. Dairy J. 15: DTU Food, Technical University of Denmark

9 Effect of food composition 9 DTU Food, Technical University of Denmark

10 Effect of fish oil concentration and emulsifier type in fish oil enriched mayonnaise (20 o C) 1-penten-3-one [ng / g] Storage time [days] Oxidation increased with increasing fish oil conc. MP_14%FO M_14%FO M_10%FO M_4%FO MP_STD M_STD Previous data: Egg yolk and low ph main factors responsible for lipid oxidation Milk protein resulted in increased oxidation Rancid (Flavour) M_STD M_4%FO M_10%FO M_14%FO MP_STD MP_14%FO Storage time [Months] 10 DTU Food, Technical University of Denmark

11 Effect of the Ingredients: Mayonnaise-based Shrimp Salad 2-pentenal [ng / g sample] Storage time [days] S_FO_S Shrimp & FO Shrimp & asparagus Shrimp, asparagus & FO S_STD S_STD_FO S_FO_A Asparagus & FO Salads without asparagus were more oxidised Salads without shrimps were the least oxidised Sørensen, Nielsen & Jacobsen, 2010, Eur. J. Lipid Sci. Technol. 112, Intensity (T-Rancid) Shrimp & asparagus S_STD Shrimp, asparagus & FO Shrimp & FO S_STD_FO S_FO_S Storage time [days] The panel could not discriminate between standard salad with or without fish oil 11 DTU Food, Technical University of Denmark

12 Comparing Milk and Drinking yoghurt: Effect of Ingredients in Yoghurt Intensity of fishy off-flavour (0-9): Week 0 Week 1 Week 3 Milk 5.4 ± ± ± 1.0 Yoghurt (CA+P+FS) Yoghurt (CA+P) Yoghurt (CA) 0.0 ± ± ± ± ± ± ± ± ± 0.9 Yoghurt 0.5 ± ± ± 1.2 CA: Citric acid; P: Pectin; FS: Fruit preparation and sugar Fish oil enriched milk oxidised much faster than fish oil enriched yoghurt Ingredients added to yoghurt did not affect oxidation Nielsen, Klein & Jacobsen, 2009, Eur. J. Lipid Sci. Technol. 111, DTU Food, Technical University of Denmark

13 Comparing Milk and Yoghurt: Antioxidant Assays on Peptide Fractions in Yoghurt Radical scavenging (DPPH) Metal chelation Reducing power Crude protein Peptide fractions (KDa) > < Farvin, Nielsen, Baron & Jacobsen, 2010 Food Chem. 123, DTU Food, Technical University of Denmark

14 Comparing Milk and Yoghurt: Oxidative Stability of Milk with Peptide Fractions from Yoghurt 1-pentene-3-ol (ng/g emulsion) Storage time (Days) M+FO M+FO+<3KDa M+FO+3-10KDa M+FO+CPP M+RO Low molecular weight peptides reduce oxidation Farvin, Nielsen, Baron & Jacobsen, 2010 Food Chem. 123, DTU Food, Technical University of Denmark

15 LC-MS/MS characterisation of peptides Absorbance at 210 nm >30 kda kda 3-10 kda Fractions > 3 kda were dominated by peptides from caseins. Phosphorylated peptides were not found. Fraction < 3 kda <3 kda Farvin, Nielsen, Baron & Jacobsen, 2010, Food Chem. 123, dominated by free amino acids 15 DTU Food, Technical University of Denmark

16 Optimising processing conditions - Temperature and pressure - Type of equipment 16 DTU Food, Technical University of Denmark

17 Effect of Emulsification Conditions: Milk Affected by Temperature and Pressure Temp. 50C/Pressure 5MPa 2-hexenal 10 [ng/g milk] 5 Temp. 72C/Pressure 5 MPa Temp. 50C/Pressure 22.5 MPa Temp. 72C/Pressure 22.5 MPa Days of storage at 2 C Homogenization with a low temperature and a low pressure leads to more oxidation than a high temperature and a high pressure! Let, Jacobsen, Sørensen & Meyer (2007), J. Agric. Food Chem. 55: DTU Food, Technical University of Denmark

18 Homogenisation Conditions Affect Protein Composition at the Interface Pressure [MPa] Temperature [ C] β -lactoglobulin α s1 -casein α s2 -casein Reference, no treatment Sørensen, Baron, Brüggemann, Pedersen & Jacobsen (2007) J. Agric. Food Chem. 55: DTU Food, Technical University of Denmark

19 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 19 DTU Food, Technical University of Denmark

20 Effect of emulsification equipments Microfluidizer vs. two-stage Valve Homogenizer Delivery emulsions 20 DTU Food, Technical University of Denmark

21 3b Bord NaCas a - Average, 24. juni :22:46 3a Bord NaCas a - Average, 24. juni :16:38 08:58: juni 24. Average, - a NaCas Micro 1b 08:52: juni 24. Average, - a NaCas Micro 1a 4b Bord Valle a - Average, 24. juni :35:26 4a Bord Valle a - Average, 24. juni :29:05 09:10: juni 24. Average, - a Valle Micro 2b 09:04: juni 24. Average, - a Valle Micro 2a Effect of emulsification Equipment Fish oil-in-water emulsion Ingredients: 10% fish oil 1% emulsifier 89% buffer Type of emulsifier Oil droplet size Microfluidizer CAS_M 69 MPa, 3 passes WPI_M Fe 2+ Fe 3+ Transition metal ions Two-stage valve homogenizer CAS_H WPI_H 80 MPa 50 MPa 4 passes 3 passes Processing conditions Storage with iron for 14 days Volume (%) Particle Size Distribution CAS_M CAS_H Particle Size (µm) Volume (%) Particle Size Distribution WPI_M WPI_H Particle Size (µm) 21 DTU Food, Technical University of Denmark

22 Emulsification Equipment Results: PCA Biplot PCA Scores [Model 1] PCA Loadings [Model 1] Scores PC#2 (12.715%) CAS_Ha CAS_Mb CAS_Hb CAS_Ma WPI_Mb WPI_Ma WPI_Hb WPI_Ha WPI_Ha Loadings PC#2 (12.715%) PV PV_D4 PV_D0 PV_D7 PV_D10 PV_D14 Propanal_D7 1-penten-3-one_D0 t.c-2.4-heptadienal_d0 2-Butenal_D0 t.t-2.4-hexadienal_d0 t.t-2.4-heptadienal_d0 t.t-2.6-nonadienal_d0 4-Heptenal_D7 1-penten-3-one_D7 Hexanal_D0 Propanal_D0 4-Heptenal_D0 t.t-2.6-nonadienal_d14 Heptanal_D0 4-Heptenal_D14 2-Butenal_D7 Pentanal_D7 2-Hexenal_D0 t.t-2.6-nonadienal_d7 t.c-2.4-heptadienal_d7 t.t-2.4-hexadienal_d7 Pentanal_D0 Propanal_D4 2-Hexenal_D7 t.t-2.4-heptadienal_d7 4-Heptenal_D4 2-Butenal_D14 t.c-2.4-heptadienal_d Hexanal_D7 4-Heptenal_D10 2-Butenal_D4 Heptanal_D7 Hexanal_D4 1-penten-3-one_D4 2-Hexenal_D4 Heptanal_D14 1-penten-3-one_D10 t.t-2.6-nonadienal_d4 t.t-2.4-hexadienal_d4 t.t-2.4-heptadienal_d Pentanal_D4 2-Hexenal_D14 Hexanal_D14 1-penten-3-one_D14 2-Butenal_D10 t.c-2.4-heptadienal_d10 t.t-2.6-nonadienal_d10 t.c-2.4-heptadienal_d4 Heptanal_D4 t.t-2.4-hexadienal_d1 2-Hexenal_D10 Heptanal_D10 Pentanal_D10 t.t-2.4-hexadienal_d10 Hexanal_D10 Pentanal_D14 Propanal_D10 Propanal_D Scores PC#1 (58.670%) Loadings PC#1 (58.670%) CAS vs WPI: Higher PV in CAS but less increase in volatiles than Protein WPI in Metal chelation aqueous phase: WPI_M vs WPI_H: Protein at the interface WPI_M: 2.86 mg/ml Structure and thickness of interfacial layer WPI_H: 4.96 mg/ml Horn et al. (2012) Food Chemistry. 134, DTU Food, Technical University of Denmark

23 Effect of antioxidants in omega-3 enriched foods 23 DTU Food, Technical University of Denmark

24 Antioxidants Antioxidants = Compounds that prevent or delay lipid oxidation 1) Primary antioxidants / Radical scavenging Inactivate reactive radicals 2) Secondary antioxidants Bind oxygen Chelate metal ions Quench singlet oxygen Regenerate other antioxidants DTU Food, Technical University of Denmark

25 Lipid oxidation O 2 Radical scavengers I X XH LH LH L LOO Metal chelator LOOH Me n+ 25 Under certain conditions, antioxidants are also found to be prooxidants 25 DTU Food, Technical University of Denmark Secondary oxidation products and off flavors

26 Polar paradox hypothesis Interfacial phenomena in antioxidant activity Bulk oil Emulsion (o/w) Air Air Oil Oil Hydrophilic Amphiphilic Lipophilic Antioxidants Oil Water 26 DTU Food, Technical University of Denmark Porter., Toxicol Ind Health, 9: Latin American and Oil Frankel Congress et al., November J Agric 2015 World Congress on Oils Food and Fats Chem, 42:

27 Bulk oil Physical structures Schematic illustration of some association colloids formed by minor constituents: Physical structures: Lipid oxidation reactions Chen et al., Crit Rev Food Sci Nutr, 51: Alter effectiveness of AO 27 DTU Food, Technical University of Denmark

28 AO in fish oil enriched mayonnaise Pentenal [ng/g mayonnaise] No antioxidant Lactoferrin ( ppm) & Phytic acid ( ppm) EDTA (6 & 24 ppm) Storage time [weeks] Nielsen et al., J. Agric. Food Chem. 52: DTU Food, Technical University of Denmark

29 Results AO in Antioxidants fish oil enriched Similar dressing experiment in dressing 16 PV [meq O 2 /kg oil] Weeks at room temperature t,t-2,4-heptadienal [ng/g milk] Weeks at room temperature AscP- high NoAox AscP- low γ-toco EDTA, EDTA+γ- Toco 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 Very good stability if all 3 antioxidants are added together 29 DTU Food, Technical University of Denmark Let et al., J. Agric. Food Chem. 55:

30 AO in fish oil enriched milk PV [meq O 2 /kg oil] Toco t,t-2,4-heptadienal [ng/g milk] Toco NoAox, EDTA EDTA+Toco 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 AscP AscP+EDTA AscP+Toco AscP+Toco+EDTA RapeseedOil Additional exp: γ-tocopherol seems to work - α-tocopherol does not 30 DTU Food, Technical University of Denmark Let et al., J. Agric. Food Chem. 53:

31 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 Dressing 25% fat Anti 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 31 DTU Food, Technical University of Denmark

32 Cut-off effect (Model emulsions, o/w) Conjugated Autoxidizable Triene (CAT) Assay Emulsion (o/w): Phosphate buffer (ph 7.2), Brij (17 µm), Tung oil Phenolic compound + (stripped, fatty 115 alcohol µm) = phenolipid AAPH (1 mm) as initiator (Laguerre et al., Anal. Biochem., 380: ) Chlorogenic acid Rosmarinic acid Optimal alkyl chain - Rosmarinates & Chlorogenates: C 8 & C 12 Laguerre et al., J. Agric. Food Chem., 57: DTU Food, Technical University of Denmark Laguerre et al., J. Agric. Food Chem., 58:

33 Cut-off effect (Model emulsions, o/w) Antioxidant capacity Decreased Oxidation Antioxidant (CCL) location in an emulsion Increased lipophilicity Aqueous phase 0 CCL n Alkyl chain length CCL = Critical chain length Modified from Laguerre et al., In: Lipid Oxidation: Challenges in Food systems, DTU Food, Technical University of Denmark

34 Caffeates as antioxidants Efficacy of Caffeates Caffeates with medium chain length Conjugated Autoxidizable Triene (CAT) Assay Emulsion (o/w): Phosphate buffer, Brij, Tung Oil. AAPH as initiator Sørensen et al J. Agric. Food Chem. 62, DTU Food, Technical University of Denmark

35 Effect of lipophilisation of caffeic acid on oxidation in fish oil enriched milk Milk enriched with 0,5% of fish oil (total fat 1.5%) 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) Alemán et al Food Chemistry 167, DTU Food, Technical University of Denmark

36 Volatiles - Milk Con 3 days CA C 0 6 days CA C 12 CA C 16 9 days CA C 8 Not defined Day 12 Con CA C 20 CA C 1 CA C 0 CA C 4 CA C 16 CA C 12 Induction time CA C 8 CA C 20 General trend of volatile development Lowest concentration: CA C 4 and CA C 1 CA C 4 CA C 1 36 DTU Food, Technical University of Denmark

37 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 C 0, CA C 1, CA C 4, CA C 8 (+200), CA C 12, CA C 18 Storage at 20 o C 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 Food Chemistry 167, DTU Food, Technical University of Denmark

38 Volatiles - Mayonnaise 9 days CA C 0 CA C 1 CA C days Con CA C 4 CA C 8 CA C 12 CA C 18 Day 28 Con CA C 0 CA C 18 CA C 1 Induction time CA C 8 CA C 4 CA C 12 General trend of volatile development Lowest concentration: CA C 8 (200), CA C 12, CA C 4 and CA C 8 CA C 12 CA C DTU Food, Technical University of Denmark

39 Discussion Mayonnaise Milk No effect No effect CA C 0, CA C 1 The, CA cut-off C 18 effect is influenced by the food system It is not possible to predict optimal chain length in Less efficient food antioxidants products based on CAT Less assay efficient antioxidants CA C 0, CA C 8 - C 20 Most efficient antioxidants Most efficient antioxidants CA C 4, CA C 8, CA C 12 CA C 1, CA C 4 Increased conc. increased effect 39 DTU Food, Technical University of Denmark

40 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: /j.jff DTU Food, Technical University of Denmark

41 Natural antioxidants in mayonnaise salads (tuna) 2-pentenal [ng / g] Reference Thyme 1% 100 Rosemary 1% Oregano 1% Storage time [days] Formation of volatiles: Reference > Thyme > Rosemary > Oregano Sørensen et al., Eur. J. Lipid Sci. Technol. 112: DTU Food, Technical University of Denmark

42 Antioxidant effects of extracts from seaweed (Fucus vesiculosus) in mayonnaise 12 Peroxide value Water Penten-3-ol PV (meq O2/kg oil) ng/ g mayonnaise EtC1 EtC2 AcC1 AcC2 WOC1 WOC2 WYC Days Day WYC2 REF Honold et al. Submitted to Food Chemistry Acetone + Ethanol 42 DTU Food, Technical University of Denmark

43 Evaluation of compounds contributing to activity in mayonnaise Oxidation products later stage of storage Phenolic compounds Astaxanthin Fucoxanthin 43 DTU Food, Technical University of Denmark

44 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 44 DTU Food, Technical University of Denmark

45 Acknowledgements Caroline Baron Nina Skall Nielsen Mette Bruni Let Anna F. Horn Ann-Dorit Moltke Sørensen Anne S. Meyer Anna Klein Lis Berner Inge Holmberg Trang Vu Jane Jørgensen Mercedes Aleman Ditte B Hermund Philipp J Honold Sabeena Farvin 45 DTU Food, Technical University of Denmark