Viji Sarojini ([email protected]) School of Chemical Sciences Towards Food-Grade Anti-freeze Peptides Food and Health Research Symposium March 6, 2018
Jan 27, 2021
Viji Sarojini ([email protected])School of Chemical Sciences
Towards Food-Grade Anti-freeze Peptides
Food and Health Research SymposiumMarch 6, 2018
Preserving the textural and sensory characteristics of frozen food to ensure productquality is a challenge faced by the food industry. The use of antifreeze pre-treatmentis of major interest in this regard. Naturally occurring anti-freeze proteins (AFPs)exhibit properties of ice recrystallization inhibition (RI). The ability of AFPs toinfluence the size, morphology and aggregation of ice crystals can be used in foodtechnology to preserve food texture by reducing cellular damage, to minimise theloss of nutrients by reducing drip, and to lower operational costs. Our recent resultson the use of AFPs in frozen food preservation will be presented in this talk.
Abstract
Kong, et al. J. Agric. Food Chem. 2016, 4327
What is the problem we are trying to address?
Rapid growth of ice
Large crystals
AFPs
• Regulate shape of crystal growth• Inhibit recrystallization• Crystals stay small
Freeze Injury
Storey and Storey, Functional Metabolism:Regulation and Adaptation 2004. John Wiley & Sons
How does it Affect the Food Industry?
Ice RecrystallizationTemperature fluctuations are common in frozen storage
Ice crystals undergo changes in number, size and shape
Water makes up the bulk of the volume in most foods
• Ice-crystal formation not a threat to food quality when crystal size is small.
• Dehydration of cells: Disrupt membranes or cell walls, distort tissuestructure.
• Thawed texture and water-holding properties adversely affected.
• Large ice crystals will aggravate this tissue damage, particularly when theyform intracellularly.
Quality in Frozen Food; Erickson and Huang edited; International Thompson Publishing; Springer Science, 1997
• Help to prevent deleterious changes in foods caused by freezing andthawing processes or frozen storage.
• May be added during processing and product formulation or producednaturally in the living organism from which the food is derived.
• Wide variety
Cryoprotectants
Depends on the nature of the food
Formulated, non-cellular foods• Intimate mixing of cryoprotectants and food material is possible
• to stabilize one important component in the food such as the myofibrillarproteins in surimi
How can Cryoprotectants be Added?
Cellular foods (fruits, vegetables and meats)
Increased shelf-life involves stabilizing cellular structure and controlling water movement to prevent cellular collapse, as well as stabilizing protein
• Sugars, amino acids, polyols, methyl amines, carbohydrate polymers,synthetic polymers (e.g.,polyethylene glycol, PEG)
• Proteins (e.g., bovine serum albumin)
• Inorganic salts (e.g., potassium phosphate and ammonium sulfate).
• But need to be added in high concentration
Cryoprotecting Labile Proteins during freeze thawing
Natural Anti-freeze Peptides
• Type I: found in winter flounder withα-helix structure.
• Type II: found in only one speciesof fish, the sea raven and has atertiary structure with high contentof reverse turns and disulfidebridges.
• Type III: first isolated from the eelpout with an ordered butunclassified structure.
Davies, P. L.; Hew, C. L., Biochemistry of fish antifreeze proteins. The FASEB Journal 1990, 4, (8), 2460
Fish species found to be producing polypeptides which show antifreezeactivity and decrease the freezing point of water by 1-1.5°C.
Mechanism of Inhibition
1. The type I anti-freeze peptide first self stabilizes to form an α-helix structure, then binds to the ice surface.
2. The polar amino acid residues (Thrand Asn) will cluster on one side of thehelix which form the polar face of thehelix and bind to the ice surfaceassisted by hydrogen bonding of thepolar side chains to water molecules.
3. On the other side of the peptide theoutward facing hydrophobic residuesshow a repulsive effect and preventthe addition of new water molecules tothe ice lattice thus arresting ice crystalgrowth.
Davies, P. L.; Hew, C. L., Biochemistry of fish antifreeze proteins. The FASEB Journal 1990, 4, (8), 2460
Ice Morphology Study
Ice morphology:• The basal plane, prism faces • c and three a axis (a1, a2, a3) • Normally, ice growth takes place on each prism
face. Hexagonal crystal grown.
In the presence of AFPs, ice crystalgrowth patterns are changed, a-axisexpansion inhibited, and ice grows byaccumulating specifically at c axis, togive a bipyramidal shape.
Choy L, Daniel S.C., Protein interaction with ice. Eur J. Biochem 1992, 203, 33-42
Measured using Clifton nanoliter osmometer
Thermal Hysteresis
Difference between freezing and melting points
Measured using Clifton nanoliter osmometer
AFPs lower the freezing point ofa solution without anappreciable change to themelting point
Winter Flounder AFP
R7 -D10: 9.064ÅR7- E11: 7.521Å R29- D32: 9.064 ÅR29- E33: 7.521 Å
DTASD AAAAA ALTAA NAKAA AELTA ANAAA AAAAT AR
Thermal hysterisis0.02 ºC
Analogue
Modified Sequence
R11 –D8: 4.063ÅR11- E7: 6.66Å
R33 –D30: 2.684ÅR33- E29: 5.831Å
AFPA0.08 ºC
Kong , Evans, Perera and Sarojini Journal of Peptide Science, 2012
Large ice crystals do not form in presence of the peptides minimising damage to frozen material
Protection from Freeze Damage
Hyper Active Antifreeze Proteins
• Found in 2 insect groups: moths and beetles
• Beetles: larvae of Dendroides canadensisand Tenebrio molitor
• 8-9 kDa.
• They both have right handed β helices structure
• Insect species found to be producing hyperactive antifreeze protein whichshow very high antifreeze activity and decrease of the freezing point ofwater by 5-10°C.
Dendroides canadensis AFP
• At 25°C the DAFPs contain: 46% β -sheet, 39% turn, 2% helix,and 13% random structure.
Complex structure with several disulphide bonds
Solid Phase Peptide Synthesis
2 - chlorotritryl
chloride
linker
= 2 - chlorotritryl
chloride resin
1) DIPEA
1) DCM:MeOH:DIPEA
8:1.5:0.5 MeOH Capping
Loading1) Fmoc-AA1
-COOH
1) 20%
Piperidine
in
DMF
20min
H2N-AAn
....... AA1
CleavageFrom
resin
Target peptide: H2N
-AAn ....... AA1
-COOH
Fmoc / tBu SPPS
- 2 - chlorotrityl
chloride linker
TFA/EDDT/H2O/TIS94/2.5/2.5/1
ClCl
Fmoc-AA1
2 - chlorotritryl
chloride
linker
Deprotection
Fmoc-AA1
2 - chlorotritryl
chloride
linkerH2N
-AA1
Coupling
2 - chlorotritryl
chloride
linkerFmoc
-AA2-AA1
Repeat deprotectionand
coupling
for each
individual
amino acid
PG
PG
PG
PG
PG
PG
2) DMF
wash
2) DMF
wash
2) DMF
wash
1) TBTU,
HOBt,
DIPEA
1Hr
2) DMF
wash
2 - chlorotritryl
chloride
linker
1) 20%
Piperidine
in
DMF
20min2)
DMF
washDeprotection
PG = acid labile
protecting
group
1) Fmoc-AA2
-COOH
PG
PS3 Peptide SynthesizerManual Peptide Synthesis
Fmoc chemistry – Base Labile
Secondary Structure by Circular Dichroism
• In the original AFP, at 25°C the DAFPs contain: 46% β-sheet, 39% turn,2% helix, and 13% random structure. (minimum at ~205 and 222nm)
• But all the peptides (25 µM) show only random coil structure in CD study(minima at ~200nm)
Not active
Ice Crystal Modification
AFPW
Water
DCR26
DCR39
DCR26 lactam
DCR39 lactam
DCR26 Ala
DCR26 reducedDCR39 reduced
Kong, Leung and Sarojini, Crystal Growth and Design. 2016
AFP Food study
AFPs
http://www.google.co.nz/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRw&url=http://foodfacts.mercola.com/carrot.html&ei=IwBIVYDPMOO3mwX27oHgAw&bvm=bv.92291466,d.dGY&psig=AFQjCNGSOm88rFIIjA221pkQNQDntlz9WA&ust=1430868385311430
Quality Attributes Important to Frozen Foods
• Color
• Texture
• Juiciness
• Ultrastructural Evaluation
Quality is Defined by Consumers
Cryo-Scanning Electron Microscopy images of frozen carrot samples.
Cell walls are arrowed.
Frozen Control AFPW 0.1mg/mL DCR26 0.1mg/mL
Protection of Frozen Carrot
Kong et. al J. Agric. Food Chem. 2016, 64, 4327−4335
Treatment ColourL* h*ab C*ab
Fresh 37.5±2.0a 21.7±0.6b 35.1±2.6aNegative control 29.6±2.2b 23.5±1.7ab 32.5±3.8abPositive control 31.5±1.9b 23.1±1.1ab 33.8±2.4abAFPW 29.5±2.0b 22.3±2.2ab 31.0±3.0bDCR26 31.8±1.7b 23.8±2.3a 32.5±3.2abDCR39 29.8±2.6b 23.2±0.9ab 31.0±2.4b
Effect of AFPs treatment on frozen carrot colour
Frozen Carrot Cont
Hardness and Firmness
• Soaking time was found to have aneffect on texture preservation.
• Samples pretreated with AFPs from only4 h to 1 day not showing significantpreservation of hardness and firmness.
• Pretreatment for 2, 4, and 7 daysappeared to preserve both parameterssignificantly.
Increased soaking times result inincreased diffusion of the AFPs intothe carrot samples.
Different parameters – hardness, springiness, fracturability, chewiness etc.
AFP treated samples showedincreased hardness, firmness etc.compared to untreated samples
soaking time
negative control
positive control
AFPW DCR26 DCR39
4 h 46.875 ±4.48ab1
26.875 ±3.624c
23.958 ±4.781cde
25 ± 5.314 cd
21.667 ±2.635cdefg
1 day 45.625 ±1.718ab
22.292 ±1.423cdef
20.208 ±2.753defgh
19.375 ±7.558defgh
20 ± 5.813defgh
2 days 48.333 ±2.453a
23.333 ±3.664cde
21.042 ±3.811cdefg
19.583 ±3.758defgh
18.750 ±3.081efgh
4 days 44.583 ±3.227ab
22.500 ±6.419cdef
16.667 ±2.966fgh
18.750 ±4.835efgh
14.732 ±2.083hi
7 days 41.875 ±6.918b
15.833 ±1.521gh
9.792 ±5.748i
9.583 ±3.155i
Drip Loss
Carrot samples frozen at −20 °C to produce maximum damage
• Glycerol (0.5M) positive control.
• Drip was collected by centrifugal extraction.
• After 4 weeks of frozen storage, mean drip losses of the negative control were significantly greater
Mean Drip Loss (Percent) at different soaking times
The monoterpenes and sesquiterpenes: α-pinene, β-myrcene, γ-terpinene,terpinolene and β-caryophyllene contributing to the carrot aroma and flavourdetected.
• AFP treated carrot samples showed less depreciation in most of these volatilescompared to the untreated ones.
• AFPW treatment showed less degradation in β-caryophyllene, α-pinene, β-myrcene, β-caryophyllene
• Verifies the potential of AFPs in preventing physical damage caused by ice crystalto tissues and cell structures, therefore reducing the loss of water holding capacitywithin food samples and decreasing drip during thawing, preserving some flavoursof fresh carrot after frozen storage.
Volatile analysis
Frozen ControlAFPW
Peptide
DCR26Peptide
Protection of Frozen Pear
0.1mg/ml
Protection of Frozen Strawberry
0.1mg/ml
Frozen ControlAFPW
Peptide
DCR26Peptide
Frozen Control
AFPW
DCR26
DCR39
Frozen Cherries SEM
0.1mg/ml
Kong et. al. LWT - Food Science and Technology 84 (2017) 441-448
How About Toxicity?
No toxicity at 1 mM
• Human Umbilical Vein Endothelial Cells (HUVEC)• Human Derma Fibroblast Cells (HDFA)• Human Embryonic Kidney Cells (HEK 293)
Cell cytotoxicity assays :
1. AFPW, 2. DCR26, 3. DCR39, 4. DCR26 cyclic, 5. DCR39 cyclic
Can they be used in Food?
• Protein Allergenicity?1. In sensitized people2. Potential to induce reactions in susceptible individuals?− Monitor Antibodies specific for AFPs
• PATENTED in Ice Cream
• No analogous products in fruit, berry or meat industries
Codex Alimentarius Commission, 2002
− Food Safety− Consumer acceptance
Quality Research Must be Consumer Driven
AUTOMATED PEPTIDE SYNTHESIS
Resources
Viji Sarojini ([email protected])School of Chemical Sciences
Towards Food-Grade Anti-freeze Peptides
Food and Health Research SymposiumMarch 6, 2018
Slide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Mechanism of InhibitionSlide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31ResourcesSlide Number 33