T H E I M P A C T O F C H E E S E C O L L O I D A L S T R U C T U R E O N

F L A V O U R D E V E L O P M E N T

David Everett

University of Otago

New Zealand

1

Structure

Flavour Texture

Digestibility Release of nutrients

and bioactive components

Appearance Shelf-life

T H E I M P A C T O F S T R U C T U R E

2

A S I M P L E S T R U C T U R E …

3

solution Number of possible compounds = 3

(disregarding degradation and

polymerisation reactions)

Vastly more

complex

combinations and

permutations

BUT…these types of reactions are difficult to control in a food product

4

A M O R E C O M P L E X S T R U C T U R E …

Mozzarella cheese Mozzarella cheese made

from homogenised milk A: pool of free oil; B: emulsified fat globule

Rowney, M. K., Hickey, M. W., Roupas, P., &

Everett, D. W. (2003). Journal of Dairy Science,

86(3), 712-718.

Scale bars: 10 µm

I N T E R F A C E S I N B O V I N E M I L K

5

Walstra, P., T. J. Geurts, A. Noomen, A. Jelema, and M. A. J. S. van Boekel.

1999. Dairy Technology: Principles of Milk Properties and Processes. Marcel

Dekker, Inc., New York.

Colloidal interfaces

Casein micelle surface—aqueous phase

Milk fat globule membrane—aqueous phase

MFGM—fat globule triacylglycerol core

Minerals—aqueous phase

(β-LG hydrophobic calyx—aqueous phase)

M I L K F A T G L O B U L E ( M F G M ) T O P O L O G I C A L M O D E L

6 Lopez C. et al., Food Chemistry 125: 355-368 (2011).

The phospholipid tri-layer, 4-10 nm thick

37+ enzymes identified in the

MFGM

Xanthine oxidase (XO,

E.C.1.2.3.2) comprises 20% of

MFGM proteins; present at 35

mg/L milk

Complexes with butyrophilin

(20-40% of MFGM proteins)

Location of membrane proteins

far from certain —

asymmetrical

M F G M P H A S E S E P A R A T I O N

7

Liquid ordered regions rich

in sphingomyelin (highly

saturated, longer chain)

and cholesterol

Liquid disordered region

Packs more tightly; less

permeable, laterally

compressed & thicker

Gallier. S., Gragson, D., Jiménez-Flores,

R., Everett, D.W., J. Agric. Food Chem.

58: 4250–4257 (2010)

Packs more loosely, and allows

inclusion of the bulky

fluorescent dye

C O M P A R T M E N T A L I S A T I O N O F E N Z Y M E S

8

Redox enzymes

Xanthine oxidase

Cytochrome C reductase

Hydrolases

Acetylcholine esterase

Alkaline phosphatase

Acid phosphatase

5’-Nucleotidase

Glucose-6-phosphatase

Phosphodiesterase

Adenosine triphosphatase

Lyase

Aldolase

Transferases

γ-Glutamyl transferase

Galactosyl transferase

Gallier, et al., J.

Agric. Food Chem. 58:

4250–4257 (2010)

Casein micelle

Sulfhydryl oxidase

Lactoperoxidase

Superoxide dismutase

Ribonuclease

Serum phase

Lipoprotein lipase

Plasmin

Holt, C., Advances

in Protein

Chemistry, vol. 43:

63-151 (1992)

O X I D A T I O N R E A C T I O N S O F X O

9

Xanthine oxidase XH + H2O + O2 X=O + H2O2

Xanthine dehydrogenase XH + H2O + NAD+ X=O + NADH

Aldehyde oxidase* RCHO + H2O + O2 RCOOH + H2O2 * Found mainly in liver

Uric acid elevated in blood; crystals implicated in gout§

XO is the target of the widely used anti-gout drug, Allopurinol, an isomer of

hypoxanthine and a xanthine oxidase inhibitor § Genetic, diet, and lifestyle

causes; consumption of alcohol,

fructose-sweetened drinks, meat,

seafood; known as “rich man’s

disease, or “the disease of kings”

M A T R I X E F F E C T O N X O A C T I V I T Y X O I N S O L U T I O N A N D I N B U T T E R M I L K

10

XO in buttermilk XO in buttermilk

X A N T H I N E O X I D A S E A C T I V I T Y I N N E W Z E A L A N D C H E E S E S

11 Ali Rashidinejad, PhD thesis, University of Otago (2015)

Located in the cytoplasmic

region of the MFGM

Oxidation of aldehydes to

acids

Increase in n-fatty acids

leading to methyl ketones, γ-

and δ-lactones.

XH + H2O + O2 X=O + H2O2

W A S H I N G O F F A T G L O B U L E S

12

Release of MFGM

Attachment

of non-MFGM

proteins

Washing &

Recombination

Raw cream

Fat

globules

Non MFGM

proteins Dilutio

n

Diluted cream Washed cream

Centrifugat

ion

Washing cream with either simulated milk ultrafiltrate

(SMUF) or water Outer PL

bilayer

XO

Triacylglycerol

core

Outer PL bilayer

Triacylglycerol

core

X A N T H I N E O X I D A S E A C T I V I T Y O N E M U L S I O N S U R F A C E S

13

* Specific activity of XO (mU mg-1 of

protein)

Kathriarachchi, K., PhD thesis, University of Otago (2015)

0"

10"

20"

30"

40"

50"

60"

70"

80"

90"

100"

Raw"cream" SMUF" Water"

Washed"cream"

Emulsion"

Short chain aldehydes are volatile and can be analysed by headspace GC Corresponding acids are not sufficiently volatile at this concentrations to analyse by headspace GC, requiring derivatisation to an ester

Aldehydes acids pairs:

acetaldehyde acetic acid

2-methylpropanal 2-methylpropanoic acid

3-methylbutanal 3-methylbutanoic acid

2-methylbutanal 2-methylbutanoic acid

Aldehyde Acid XO

Formation of Acid

Depletion of Aldehyde

V O L A T I L E S P R O D U C E D B Y X O A C T I O N

14

S U R F A C E O X I D A T I O N O F A L D E H Y D E S

0

3

5

8

10

0 3 6 9 12 15

Depletion of 3

-methylbutanal (mM)

Initial 3-methylbutanal concentration (mM)

0

2

4

6

8

0 3 6 9 12 15

Formation of 3

-methylbutanoic acid (

mM)

Initial 3-methylbutanal concentration (mM)

SMUF cream 0.0597216U/mg

DI cream 0.0638 U/mg

DI emulsion 0.0720U/mg

SMUF emulsion 0.0821851 U/mg

Depletion of aldehyde

Action of xanthine oxidase on reconstructed or washed emulsion surfaces

Formation of acid

Kathriarachchi, K., Leus, M. & Everett, D.W. Int. Dairy Journal, 37: 117-126 (2014)

15

Example reaction: 3-methylbutanal 3-methybutanoic acid

G L U T A M Y L T R A N S F E R A S E A C T I V I T Y I N W A S H E D C R E A M R E N N E T G E L S

16

Changes in absorbance at 410 nm due to production of p-nitroaniline in renneted gels

Containing cream washed 3× with SMUF

Containing recombined emulsions prepared from MFGM extracted from SMUF-washed cream

a

b

a a

b b

Kathriarachchi, K., PhD thesis, University of Otago (2015)

Optimal pH range 8.0 - 9.0

Glutamylization of hydrophobic amino

acids

Potential for reducing bitterness and

increasing sour taste and astringency

Kokumi flavour in Gouda cheese from γ-

glutamyl dipeptides

5-L-glutamyl peptide + amino acid peptide + 5-L-

glutamyl-amino acid

0

30

60

90

120

150

180

210

240

270

300

Gel with washed cream or emulsion

Specific Activity * XO activity in SMUF washed cream gel

XO activity in recombined emulsion gel

* Specific activity of XO (mU mg-1 of

protein)

17 Kathriarachchi, K., PhD thesis, University of Otago (2015)

X A N T H I N E O X I D A S E A N D G L U T A M Y L T R A N S F E R A S E A C T I V I T Y I N

R E N N E T E D G E L S

a

b

d c

R E D O X P O T E N T I A L I N R E N N E T E D G E L S

18

Raw cream washed three times with simulated milk

ultrafiltrate (SMUF) Recombined emulsions prepared from MFGM

extracted from SMUF-washed cream Kathriarachchi, K., PhD thesis, University of Otago (2015)

X A N T H I N E O X I D A S E A N D G L U T A M Y L T R A N S F E R A S E A C T I V I T Y U N D E R D I F F E R E N T R E D O X P O T E N T I A L S

19

Absorbance at 290 nm due to uric acid production in

renneted gels made from raw cream washed 3× with SMUF.

Absorbance at 410 nm due to production of p-

nitroaniline in renneted gels with raw cream

washed 3× with SMUF.

a

c

b

No statistical differences

Kathriarachchi, K., PhD thesis, University of Otago (2015)

E N C A P S U L A T I O N O F P O L Y P H E N O L I C S I N A C H E E S E M A T R I X

Ali Rashidinejad, PhD thesis, University of Otago (2015)

125 ppm 250 ppm 500 ppm

Free 0.69±0.06b 0.63±0.12b 0.57±0.16b

Encapsulated - 0.98±0.02a 0.99±0.05a

Encapsulation of catechin in a cheese matrix

Arrows show microcapsules; scale bar 5 µm

20

E N C A P S U L A T I O N O F P O L Y P H E N O L I C S I N A C H E E S E M A T R I X

The effect of free catechin on xanthine oxidase activity of full-fat cheese

The effect of encapsulated catechin on xanthine oxidase activity of full-fat cheese

21 Ali Rashidinejad, PhD thesis, University of Otago (2015)

R E S E A R C H C O L L A B O R A T O R S

22

Ali Rashidinejad

Pat Silcock

Ofir Benjamin Shahin

Roohinejad Haotian Zheng Pankaj Sharma

University of

Otago Local farm

Keith Gordon Jersey Cow

Kalpana Kathriarachchi

Department of Food Science PhD students

(Otago)

Calif. Poly. State

University

Rafael Jiménez-Flores Jules Kieser Indra Oey

23

Thank you for your

attendance

Otago Harbour, Dunedin, New Zealand

E X T R A S L I D E S

T H E M I L K F A T G L O B U L E M E M B R A N E ( M F G M ) A N E X A M P L E O F A C O M P L E X S U R F A C E S T R U C T U R E

25

Originates from lipid droplet extrusion from the mammary

epithelial cells

Protects milk fat globule from lipolysis and coalescence

Contains components with bioactive functionality

Major component of buttermilk

Keenan, Mather & Dylewski, Physical Equilibria Lipid

Phase, In N.P. Wong, Fundamentals of Dairy

Chemistry, 3rd ed. (1988)

In bovine milk, xanthine oxido-reductase is in the XO form

Capable of oxidising a wide range of aldehydes

X A N T H I N E O X I D A S E ( X O )

26

XO has both bacteriocidal and bacteriostatic properties brought about by—

Production of reactive superoxide and hydrogen peroxide in the gut

Reduction of nitrite to nitric oxide, and to peroxynitrite

Stimulating lactoperoxidase system in milk (reductant + H2O2 oxidant + H2O)

X A N T H I N E O X I D A S E A C T I V I T Y I N P R O C E S S E D M I L K

27

XH + H2O + O2 X=O + H2O2

Located in the cytoplasmic region of

the MFGM

Oxidation of aldehydes to acids

Increase in n-fatty acids leading to

methyl ketones, γ- and δ-lactones.

Kathriarachchi, K., PhD thesis, University of Otago (2015)

No treatm

ent

63°C/3

0 min

72°C/1

5 sec

4°C/1

day

Salt 6%

0

20

40

60

80

100

120

140

160

180

Activity m

U m

L-1

Milk

Recombined

R E C O M B I N E D E M U L S I O N S

28

Images taken by

transmission

electron

microscopy

CM: casein

micelles

MFG: milk fat

globules

Scale bar 5 µm Scale bar 200 nm

MFGM extracted from unwashed cream

MFGM extracted from SMUF-washed cream

MFGM extracted from water-washed cream

Kathriarachchi, K., Leus, M. & Everett, D.W. Int. Dairy Journal, 37: 117-126 (2014)

X A N T H I N E O X I D A S E A C T I V I T Y ( I L L U S T R A T I N G T H E P R O T E C T I V E E F F E C T O F D I F F E R E N T

S U R F A C E S T R U C T U R A L E N V I R O N M E N T S )

0

10

20

30

40

50

60

70

80

90

100

Washed cream or emulsion

Specific Activit

y *

SMUF washed cream MFGM emulsion in SMUF DW washed cream MFGM emulsion in DW

* Specific activity of XO (mU mg-1 of

protein)

29 Kathriarachchi, K., PhD thesis, University of Otago (2015)

P E R C E N T D E P L E T I O N O F A L D E H Y D E S

30 Kathriarachchi, K., Leus, M. & Everett, D.W. Int. Dairy Journal, 37: 117-126 (2014)

G L U T A M Y L T R A N S F E R A S E A C T I V I T Y I N P R O C E S S E D M I L K

31

5-L-glutamyl peptide + amino acid peptide + 5-L-

glutamyl-amino acid

Optimal pH range 8.0 - 9.0

Glutamylization of hydrophobic amino acids

Potential for reducing bitterness and

increasing sour taste and astringency

Kokumi flavour in Gouda cheese from γ-

glutamyl dipeptides

Kathriarachchi, K., PhD thesis, University of Otago (2015)

No treatm

ent

63°C/3

0 min

72°C/1

5 sec

4°C

/1 d

ay

Salt 6%

0

1

2

3

4

5

6

7

8

9

10

Activity m

U m

L-1

Milk

Recombined