mastering cheese texture and flavour during...

PLH McSweeneyUniversity College Cork, Ireland

Mastering cheese texture and flavour during ripening

University College Cork (1845)

Food

Food

Food

Food

Food

Food

Food

Food

Food in…BEES/ERIHistory, GeographyFood Business and Development (CUBS)Management and Marketing (CUBS)APCMicrobiologyProcess and Chemical EngineeringMedicine

Food

Food: the strategic priority for UCC

Ireland: the dairy islandIrlanda: la isla lechera

University College Cork

The dairy industry in Ireland

Kerry Group. Market capitalisation US$18.5 bn

Irish owned multinationals

1.4 m dairy cows. Approx

16,500 dairy farmers in Ireland. Average herd size 80cows (and growing). Average cow produces 5,000 litres milk per annum. Average farm

output is ca. 11,000 L/Ha

7,300 m litres of milk

But we consume <10%. Export 80% of production. Growing!

205,000 tonnes of cheese(mainly Cheddar L)

Ingredients…

Make 11% of the world’s

trade in infant

formula

Product mix determined by seasonality of milk supply

Outline…

• Overview of cheese ripening, coagulants

• Calcium equilibrium and cheese texture

• Optical properties of low-fat cheese• Redox potential and cheese


Stages of manufacture


Metabolism of lactate…

Propionate, Acetate, CO2Propionibacterium freudenreichii

Oxidative metabolism

Oxidation toformate, ethanol

and acetateLAB?

Butyrate, CO2, H2Clostridium sp.

H

CCOOHH3C

HO

Ca lactate crystals


O

CO

O

CO

O

CO

OHC

O

O

O

O

H

CH3C

COOHOH

Chymosinand plasmin

Caseins

Intermediate-sized peptides

Proteinases fromLactococcus

Short peptides

Peptidases of Lactococcusand NSLAB

Amino acids

Triglyceride

Fatty acidL-Lactate D-Lactate

Lactose

Fermentationby starter

Racemizationby NSLAB

COOH

CH2

C COOHHO

H2C

COOH

H

CH3C

OHCOOH

Catabo

lism of

lacta

te

VOLATILE FLAVOUR COMPOUNDS

Citrate

Metabolism of citrate

Lipas

e

Fatty acid catabolismAmino acid catabolism

Interactions betweenproducts


Other products

Lipolysis andmetabolism of fatty acids


O

CO

O

CO

O

CO

OHC

O

O

O

O

H

CH3C

COOHOH

Chymosinand plasmin

Caseins



Short peptides


Amino acids

Triglyceride


Lactose



COOH

CH2

C COOHHO

H2C

COOH

H

CH3C

OHCOOH

Catabo

lism of

lacta

te


Citrate


Lipas

e




Other products

Proteolysis


Origins of proteases in cheese

§ Coagulant (chymosin, others)§ Milk (plasmin, somatic cell proteinases)§ Starter bacteria§ Secondary starter§ Non-starter microflora


Coagulant§ Rennet coagulation of milk…§ Proportion of added rennet activity retained in curd§ Little hydrolysis of b-casein in cheese due to hydrophobic

interactions of hydrophobic C-terminal region § Principally responsible for primary hydrolysis of as1-casein



interactions of hydrophobic C-terminal region§ Principally responsible for primary hydrolysis of as1-casein

Chymosin1 199

1 23 24 199

102 199

Chymosin

f1-9, f1-13

Lactocepin

Further hydrolysis

Phe23-Phe24

Leu101-Lys102



interactions of hydrophobic C-terminal region § Principally responsible for primary hydrolysis of as1-casein§ Chymosin assay…

P T E F F R L

NO2

CHYMOSIN

Synthetic heptapeptide substrate

F R L

NO2

Product absorbsat 300 nm


pH4.5 5.0 5.5 6.0 6.5 7.0

Enzy

me

activ

ity u

nit (

mm

ol

pdt.

mg

dry

mat

ter-1

hr-1

)

0

2

4

6

8

10

12

14

% Casein1 2 3 4 5 6 7

Enzy

me

activ

ity u

nit (

mm

ol

pdt.

mg

dry

mat

ter-1

hr-1

)

0.0

0.5

1.0

1.5

2.0

2.5

Tota

l Enz

yme

activ

ity

(mm

ol p

dt. h

r-1)

0

50x103

100x103

150x103

200x103

250x103

Chymosin (IMCU/ml milk)0.05 0.10 0.15 0.20

Enzy

me

activ

ity u

nit (

mm

ol

pdt.

mg

dry

mat

ter-1

hr-1

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Ionic strength of milk (M)0.0 0.2 0.4 0.6 0.8

Casein micelle size (nm)100 150 200 250 300 350 400

Enzy

me

activ

ity u

nit (

mm

ol

pdt.m

g dr

y m

atte

r-1 h

-1)

0.0

0.2

0.4

0.6

0.8

● pH at whey drainage (chymosin)● pH of milk (chymosin)● pH of milk (C. parasitica proteinase)


0

10000

20000

30000

40000

50000

60000

70000

80000

Cast Feta Feta Parmesan Emmental Cheddar Camembert Gouda Port Salut BuffaloMozzarella

PizzaMozzarella

Cheese varieties

Peak

Are

a/m

g dr

y m

atte

r/h


A interesting coagulant…


Characteristics of good rennet substitutes

• High milk clotting : proteolytic ratio• Proper specificity• Good activity in milk• Easily denatured (in whey)• Few suitable enzymes…

Camel chymosinCHY-MAXTM M

??

• No significant differences in composition and pH; suggestion of increased yield

• Primary proteolysis was significantly lower in camel chymosin cheeses large quantitative differences between the peptide profiles of cheeses; however, the levels of amino acids were similar

• Flavour differences• Camel chymosin can be used

successfully to make Cheddar cheese with lower levels of proteolysis but with good flavour.


Inhibition of coagulant

β-CN

αs1-CN

β-CN (f 106-209)

β-CN (f 29-209)

β-CN (f 108-209)

αs1-CN (f 102-199)

αs1-CN (f 24-199)αs1-CN degradationproducts

β-CN (f1-189/192)

NaCN C 0.1 0.25 0.4 2 C 0.1 0.25 0.4 2 C 0.1 0.25 0.4 2

1 d 180 d90 d

β-CN

αs1-CN

β-CN (f 106-209)

β-CN (f 29-209)

β-CN (f 108-209)

αs1-CN (f 102-199)

αs1-CN (f 24-199)αs1-CN degradationproducts

β-CN (f1-189/192)

NaCN C 0.1 0.25 0.4 2 C 0.1 0.25 0.4 2 C 0.1 0.25 0.4 2

1 d 180 d90 d

Urea-polyacrylamide gel electrophoretograms (12.5% T, 4% C, pH 8.9) of bovine sodium caseinate (CN) and the Cheddar cheeses manufactured from milk containing 0 (C), 0.1, 0.25, 0.4 or 2% equine blood serum after 1, 90 and 180 d of ripening.

Bansal et al.

a2-Macroglobulin720 kDa


O

CO

O

CO

O

CO

OHC

O

O

O

O

H

CH3C

COOHOH

Chymosinand plasmin

Caseins



Short peptides


Amino acids

Triglyceride


Lactose



COOH

CH2

C COOHHO

H2C

COOH

H

CH3C

OHCOOH

Catabo

lism of

lacta

te


Citrate


Lipas

e




Other products

Amino acidcatabolism


Catabolism of amino acids

Met Tyr Trp

MGLCBLCGL

TPL TIL

Methanethiol

Phenol

Indole

Aromatic amino acidsBranched-chain amino acids

Methionine

ELIMINATION

a-Keto acid

Aldehyde

Alcohol

Ester Methyl thioester

Carboxylic acid

Acyl-CoA

Hydroxyacid

DimethyldisulphideDimethyltrisulphide

Benzaldehyde(Phe)

Hydroxybenzaldehyde(Tyr)

Indole-3-acetate(Trp)

Cresol, skatole(Tyr, Trp)

TRANSAMINATION

HA-DH

Oxidati

on

a-KADH

a-KADC

aldDHAlcohol DH

?

?

Aminotransferase


Degradation of a-keto acids

H2N CH C

CH2OH

O

OH

a-Keto acid(p-Hydroxy phenylpyruvate)

a-Amino acid(Tyrosine)

Hydroxyacid

AldehydeCarboxylic acid Alcohol

O C C

CH2OH

O

OH

Chemicaldegradation

C

OH

O H

p-Hydroxy-benzaldehyde

Outline…





Ammonia produced at surfaceby proteolysis diffuses into cheese

Migration of soluble Ca, PO43- and lactate towards surface

Lactate metabolised at surfaceCa3(PO4)2 precipitates

Growth of Penicillium camemberti at surface

pH

High pH

Low pH

Dire

ctio

n of

mig

ratio

n

Con

cent

ratio

n gr

adie

nt

Cheese softens

fromsurface towards

core

Textural changes during ripening…• Role of calcium, pH in softening of Camembert-type cheeses is well

established…

• What is the role of calcium in softeningof Cheddar cheese?Biochemical and physicochemicalprocesses… Role of as1-casein?

50

60

70

80

90

100

0 30 60 90 120 150 180Ripening time (d)

Hard

ness

(% d

ecre

ase)

Softening of Cheddar

cheese


Role of chymosin in cheese texture development?§ Chymosin inhibited by pepstatin

(IsoVal-Val-Val-Stat-Ala-Stat)

Useful tool to study cheese texture


2D Graph 1

Ripening time (d)0 30 60 90 120 150 180

Har

dnes

s (N

)

80

120

160

200

240

Inso

lubl

e C

alci

um/T

otal

Cal

cium

(%)

50

55

60

65

70

75

80

Res

idua

l as1

-cas

ein

(%)

0

20

40

60

80

100

Time vs H ctrl Time vs Ca ctrl Time vs as1 ctrl

2D Graph 2

Ripening time (d)0 30 60 90 120 150 180

Har

dnes

s (N

)

80

120

160

200

240

Res

idua

l as1

-cas

ein

(%)

0

20

40

60

80

100

Inso

lubl

e C

alci

um/T

otal

Cal

cium

(%)

50

55

60

65

70

75

80

HardnessResidual as1-caseinInsoluble Calcium

2D Graph 2

Ripening time (d)0 30 60 90 120 150 180

Har

dnes

s (N

)

80

120

160

200

240

Res

idua

l as1

-cas

ein

(%)

0

20

40

60

80

100

Inso

lubl

e C

alci

um/T

otal

Cal

cium

(%)

50

55

60

65

70

75

80

HardnessResidual as1-caseinInsoluble Calcium

Control (100% residual chymosin activity)

Pepstatin (16% residual chymosin activity)

Hardness decreased significantly (p<0.001) irrespective of as1-casein hydrolysis

Calcium solubilization most closely related to initial softening

Hydrolysis of as1-casein plays a role but not as important as solubilization of Ca


G' @ 0.4 Hz

Temperature (oC)

10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0

G' (

Pa)

101

102

103

104

105

106

8.34 g/L6.95 g/L5.56 g/L2.78 g/L1.39 g/L

Loss tangent @ 0.4 Hz

Temperature (oC)

10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0

Loss

tang

ent

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

8.34 g/L6.95 g/L5.56 g/L2.78 g/L1.39 g/L

Storage modulus[CCP] had no significant effect on G´ at 20oCG´ at 70oC increased significantly with increasing [CCP]

Loss tangentLTmax increased significantly with decreasing [CCP]

[CCP] had no significant effect on temperature of LTmaxRheological properties of Cheddar cheese at 70oC as a

function of colloidal calcium phosphate content

Colloidal calcium phosphate content of cheese (g calcium/100 g protein)

1.40 1.60 1.80 2.00 2.20 2.40

Stor

age

and

Loss

mod

ulus

(Pa)

0

50

100

150

200

250

300

350

400

Loss

tang

ent

0.60

0.80

1.00

1.20

1.40

1.60

1.80

G'G"Tan d

Rheological properties of Cheddar cheese at 70oC as a function of colloidal calcium phosphate content

Colloidal calcium phosphate content of cheese (a.u./100 g protein)

0.80 0.90 1.00 1.10 1.20 1.30 1.40

Stor

age

(G')

and

Loss

(G")

mod

uli (

Pa)

0

50

100

150

200

250

300

350

400

Loss

Tan

gent

0.60

0.80

1.00

1.20

1.40

1.60

1.80

G'G"Tan d

G´´ > G´

G´ > G´´

Rheological properties at 70oC

Variation in CCP levels using synthetic Cheddar cheese aqueous phase


Influence of Ca equilibrium on melt

Ø Insoluble Ca decreases during ripening and is responsible for increased melt during early ripening

Ø Cheeses with lower insoluble Ca have increased meltability

Loss

tang

ent

Lowest Insoluble Ca

Highest Insoluble Ca

Cheeses with same pH

(Choi et al. 2008)


Varying the texture of Cheddar cheese by controlling levels of soluble Ca using trisodium citrate or CaCl2 added at salting

Five vats of Cheddar cheese manufactured –

Control (100% NaCl)Experimental 1 (90% NaCl, 10% TSC)Experimental 2 (70% NaCl, 30% TSC)Experimental 3 (90% NaCl, 10% CaCl2)

Experimental 4 (70% NaCl, 30% CaCl2)

Levels of NaCl were varied to maintain a constant ionic strength to avoid different moisture levels

6000

8000

10000

12000

14000

16000

18000

7 14 28 56 112 202Time (days)

Har

dn

ess

(G)

ControlTSC 10%TSC 30%CaCl2 10%CaCl2 30%

University College Cork 35

1. Manufacture of Gouda-type cheesescontaining a Ca-sequestering agent (EDTA) encapsulated in liposomes

2. Determine the effect of EDTA on the textural and rheological properties of Gouda-type cheeses without affecting composition

Liposomes

EDTA addition + mix

UT/Homogenisation/Microfluidisation

Microfiltration

EDTA suspended in buffer + liposome

Outline…





Translucency in low-fat Cheddar Cheese

• Colour is considered the mostimportant attribute to foodappearance.

• Importance of fat content andtemperature in cheese colour:

– Fat increases light scatering,affecting whiteness. (L* values)

– Fat reduction on cheese increasestranslucency (decreases L*values) and hence acceptability

– High temperatures increaseswhiteness. However, it isInfluenced by fat content (meltingeffect)

• Other parameters that affectcolour in cheese:

– Salt content– Calcium content– Homogenisation of cheese-milk– Addition of annatto– Addition of titanium dioxide

Rodrigo A. Ibáñez (Chile)PhD. Student (April 2011 – March 2015)


Translucency of low-fat Cheddar Cheese• L* value used as an indicator of

translucency (the higher the value,the lower the translucency).

• The study of translucent materialshas also been studied by meansof the reflectance of a thin layer ofa sample under a black and awhite background.

• Based on this relation, theKubelka-Munk index (K/S) hasbeen developed (Judd andWyszecki, 1975)

R∞:reflectance of a layer of infinite thickness.R: reflectance of the sample layer above a whitebackground with a known reflectance RgR0: reflectance of the sample with an ideal blackbackground.


Effect of titanium dioxide, annatto and homogenisationon Kubelka-Munk index in half fat Cheddar Cheese

Titanium Dioxide

Homogenisation

Annatto

0, 20 and 40(g TiO2/100 kg cheesemilk)

0, 10 and 20(MPa)

0, 8.25 and 16.5(ml/100 kg cheesemilk)

Outline…





Physicochemical parameters influencing cheese ripening…• pH, aw (≈[NaCl]), T well understood and controlled…• Oxidation-reduction potential largely ignored…

• ORP: measure of ability of chemical species to oxidize or reduce

• ORP of milk ≈ +250 to +350 mV, cheese varies (but very reducing)

• Affects microbial growth, production of aroma compounds by amino acid catabolism


Oxidation-reduction potential and cheese

• ORP affects the type of microorganisms that grow in and on

cheese…

• Evidence that ORP influences which species or strains of

NSLAB proliferate (Boucher et al., 2006).

• ORP contributes to the creation of conditions necessary for

balanced flavour development and influences compounds

formed…


Measurement of ORP in mature hard cheese

Filled with3 M KCl

Platinum Electrode

Reference Electrode

mV

20 mm

-450

-350

-250

-150

-50

50

150

250

350

0 250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 3250

Time (min)

OR

P (m

V)


Cheddar cheesemaking Redox Potential

pHTemperature

20

30

40

50

60

70

80

T (�

C)

-400

-320

-240

-160

-80

0

80

160

0 50 100

150

200

250

300

350

400

OR

P (m

V)

5.0

5.5

6.0

6.5

7.0

7.5

Time (min)

pH

Pasteurization Starter addition

CaCl2 & coagulant addition

Cutting & cooking Whey drainage Milling &salting

-400

-320

-240

-160

-80

0

80

160

410

510

610

710

810

910

1010

1110

1210 1310

1410

1510

ORP

(mV

)

9

Time (min)

-400

-320

-240

-160

-80

0

80

160

410

510

610

710

810

910

1010

1110

1210 1310

1410

1510

ORP

(mV

)

9

Time (min)


Changes during ripening

1 3 5 7 9 11 13 15 17 19 21 23 25 27

OR

P (m

V)

-400

-300

-200

-100

0

100

200

300

0 40 80

Time

Minutes Days

Reference electrode in agar containing saturated KCl gelled in plastic tube with holes

Miniature platinumelectrodes


Influence of microbial growth on ORP in Cheddar cheese extract during simulated cheese pressing

C C10 S2 T °C

-400

-300

-200

-100

0

100

200

300

400

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

OR

P (m

V)

0

5

10

15

20

25

30

35

40

Tem

pera

ture

(°C

)

Time (hour)

pH 5.4 add NaClStrain

L. l. lactis C10 L. l. lactis DL16 L. l. lactis ATCII454 L. l. lactis ML3 L. l. MM510 L. l. lactis ATC29146 L. l. lactis M70 L. l. Crem, Z8 L. l. Crem HP L. l. Crem 45P+ L. l. Crem SW224 L. l. Crem SK11 04 L. l. Crem UC317 L. l. Crem E L. l. Crem R L. l. Crem EB7 L. l. Crem D L. l. Crem W L. l. Crem G L. l. Crem S2 L. l. lactis C2 L. l. lactis 26-2 L. l. SCO213 L. l. Crem A L. l. Crem B L. l. Crem C L. l. Crem F L. l. Crem I L. l. Crem J L. l. Crem Q


ORP control during Cheddar ripening

• Cheddar is a dry salted variety• Redox agents added to the salted curd before

pressing

• Reducing agents:• Cysteine (2%)• Sodium hydrosulfite (0.05%,0.1%)• Oxidizing agent:• Potassium iodate (0.05%,0.1%,1%)


ORP and pH during ripening

5.10

5.30

5.50

5.70

5.90

0 20 40 60 80 100 120 140 160 180 200

pH

Ripening Time (days)

Control KIO3 1% KIO3 0.1% KIO3 0.05% Na2S2O4 0.1% Na2S2O4 0.05% Cys 2%

-0.400

-0.300

-0.200

-0.100

0.000

0.100

0.200

0.300

0 20 40 60 80 100 120 140 160 180 200

OR

P (

V)

Ripening time (days)

Control KIO3 1% KIO3 0.1% KIO3 0.05% Na2S2O4 0.1% Na2S2O4 0.05% Cys 2%


PCA of volatile compounds at 2 month ripening

University College CorkFood Institutional Research

Measure (FIRM)

Thank you!¡Gracias!

mastering cheese texture and flavour during...

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