Viji Sarojini (v.sarojini@auckland.ac.nz)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 (v.sarojini@auckland.ac.nz)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