The Science behind “grass fed”

Diarmuid (JJ) Sheehan

Teagasc Food Research Centre Moorepark, Ireland

Food Research Programme

• 283 Research

Staff &

Students at 2

locations

Dairy Meat

ODI, April 4th 2019

Overview of Presentation

• Context in Ireland

• Irelands dairy system – grass based production

• Milk composition

• Influence on compositional parameters

• Products

• Butter

• Cheese

• Cheddar

• Maasdam

• Conclusions along the way

• Acknowledgements

ODI, April 4th 2019

Typical dairy scene in Ireland

Strategy – to

maximise milk

production from grass

“Ireland’s agricultural

system is based on the

use of pasture

as a low-cost primary

feed source, where

cows are

calved in the spring

and maintained

outdoors for the

majority of their

lactation”

ODI, April 4th 2019

3.2

3.3

3.4

3.5

3.6

3.7

3.8

3.9

0

200

400

600

800

1000

1200

Feb

Mar

Apr

May

Jun

Jul

Aug

Sep

t

Oct

Nov

Dec

Jan

Feb

Pro

tein

(%

)

Milk

inta

ke

(mill

ion liters

)

Months

Seasonal variation in milk production

MilkProtein

Compact

Calving

practice

Grass-based

feeding system

(CSO, 2017)

Project Introduction

ODI, April 4th 2019

Seasonal Irish milk production

Grass based production:

Problems:

• Most milk enters late

lactation at same time

• Processor needs large

capacity

• Processors have redundant

capacity

• Product mix- bulk and

storable

Advantages ?

• Can Ireland gain advantage

in the market?

• What is the science behind

grass fed ?

• Can we back up the

marketing ?

0%

2%

4%

6%

8%

10%

12%

14%

16%

Jan

Fe

b

Mar

Apr

May

Ju

n Ju

l

Aug

Se

p

Oct

Nov

Dec

Ireland EU

Milk Intake by Month

Ireland -v- EU

ODI, April 4th 2019

ODI, April 4th 2019

Impact of Different Grass/Clover/Ration Diets on the

Volatile profile, Sensory Characteristics and

Functionality of Milk and Milk Products

ODI, April 4th 2019

ODI, April 4th 2019

Experimental Design

ODI, April 4th 2019

30 Unit Automatic Milking Parlor

3 segregated 5,000L Refrigerated Tanks

Experimental Design

ODI, April 4th 2019

Feeding System

TMR GRS CLV SE P-value

Milk Yield L/d 27.71 20.98 24.59 0.14 < 0.001

Milk Solids kg/d 2.24 1.78 1.99 0.01 < 0.001

Protein kg/d 0.94 0.76 0.87 0.01 < 0.001

Fat kg/d 1.31 1.02 1.12 0.03 < 0.001

TMR GRS CLV

Effect of diet on milk composition

ODI, April 4th 2019

13

0

5

10

15

20

25

30

35

TMR GRS CLV

g/1

00g o

f fa

t

Palmitic AcidP < 0.05

P < 0.05

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

TMR GRS CLV

g/1

00g o

f fa

t

Linoleic Acid

P < 0.05

P < 0.05

Effect of diet on milk fatty acid profile

ODI, April 4th 2019

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

TMR GRS CLV

g/1

00g o

f fa

t

α-Linolenic AcidP < 0.05

P < 0.05 P < 0.05

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

TMR GRS CLV

g/1

00g o

f fa

t

CLA (c9t11)P < 0.05

P < 0.05

Effect of diet on milk fatty acid profile

ODI, April 4th 2019

Pasture based feeding has a beneficial effect on milk fatty

acid profile

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

TMR GRS CLV

g/1

00g o

f fa

t

Omega 3

P < 0.05

P < 0.05 P < 0.05

00.20.40.60.8

11.21.41.61.8

2

TMR GRS CLV

g/1

00g o

f fa

t

Omega 6P < 0.05

P < 0.05 P < 0.05

➢ Essential fatty acids.

➢ Precursors to eicosanoids roles in inflammation

➢ n3 derived eicosanoids possess anti-

inflammatory

➢ n6 derived eicosanoids possess pro-

inflammatory properties (Patterson et al., 2012)

➢ Western diet has resulted in ⇡ n6 fatty acid (Molendi-

Coste et al., 2010)

➢ Concomitant increases in chronic inflammatory

diseases (Patterson et al., 2012)

➢ non-alcoholic fatty liver disease,

cardiovascular disease,

➢ obesity,

➢ inflammatory bowel disease, rheumatoid

arthritis and Alzheimer’s disease

➢ Foods rich in n3 FA could be beneficial in reducing

risk of such diseases (Benbrook et al., 2013)

ODI, April 4th 2019

a

bb

a

b

b

a

bb

a

aa

Effect of diet on milk mineral composition

ODI, April 4th 2019

Conclusions

I. Variations in milk composition could be linked to both stage of lactation and

feeding system utilized.

II. Pasture-based feeding ⇡ fat and ⇡ protein content.

III. GRS feeding systems produced milks with better quality ⇡ true protein

concentrations.

IV. Pasture feeding had a beneficial effect on nutritional status of milks

➢ ⇡ CLA, ⇡ Omega 3 fatty acids ⇣ Omega 6 fatty acids than TMR

➢ TMR milk ⇡ thrombogenic index than that of pasture milk.

V. Fatty acid profiling could be used as verification of milks derived from fresh

pasture over that of TMR systems.

VI. Diet has a significant effect on major and trace minerals in milk.

ODI, April 4th 2019

Pasture derived butter is more yellow in colour

Butter colour:

TM

R

GR

AS

S

CL

OV

ER

0

2 0

4 0

6 0

8 0

1 0 0 C o p y o f L - V a lu e

L* S

co

re

L-Value

TM

R

GR

AS

S

CL

OV

ER

-5

-4

-3

-2

-1

0

A * - V a lu e

A* S

co

re

a-Value

TM

R

GR

AS

S

CL

OV

ER

0

1 0

2 0

3 0

4 0

5 0

B * - V a lu e

B*

Sc

ore

* * *

** * *

TMR GRS CLV

0

1 0

2 0

3 0

4 0

5 0

0

2

4

6

B - V a lu e a n d -C a ro te n e

B* S

co

re

-C

aro

ten

e c

on

ten

t

mg

/Kg

B * V a lu e

-C a ro te n e

T M R G R A S S C L O V E R

ODI, April 4th 2019

TMR diet produces harder butter

0

5

1 0

1 5

2 0

B u tte r H a r d n e s s a t 2 0 ° CH

ar

dn

es

s (

N)

T M R G R A S S C L O V E R

P , 0 .0 0 2

P , 0 .0 0 1 P , 0 .0 0 1

P<0.05

P<0.05

ODI, April 4th 2019

Feeding system has a significant effect on butter sensory properties

ODI, April 4th 2019

Grass/Clover feeding

Grass feeding

TRM feeding

Raw milk Cream separation

Skim milkUltrafiltration Cream

Permeate Retentate

Standardization to PFR 1

Pasteurization

Milk standardisation for cheese manufacture

ODI, April 4th 2019

Standardisation of Milks for Studies

ODI, April 4th 2019

• Temp 32oC, pH 6.5

• Rennet level 0.16ml/L

• Cutting, G' 35 Pa

• Stirring 22 rpm

5 15 25 35 45 55 65 75

Curd sampling time after cutting, min

Curd sample

11 litre Cheese vat Conditions employed

27 curd-making

trials (TMR, CLO,

GRA milk) were

conducted over 3

week period

RCMt =CMt

CMo× 100

Data transformation to relative curd moisture

(RCM) and relative whey expelled (RWE)

RWE =CMo − CMt

CMo100

Syneresis experiments

ODI, April 4th 2019

Model no Type Equation

4 MM-model RCMt = RCMo × (1 −RWEmax

100 H+t× t)

5 Exponential decay RCMt = RCM∞ + a × exp(−kt)

6 Inverse time RCMt = RCM∞ + a/(1 + akt)

7 Exponential decay RCMt = RCMoexp(−kt)

8 Logarithmic function RCMt = RCMo(1 − k𝑙𝑛 t )

9 Exponential decay RCMt = RCMo − RCM5(1 − exp −kt)

10 Power-law RCMt = RCMo − RCM5(1 − t−k)

AICCp =𝑛 ∗ lnσ𝑆𝑆𝐸

σ𝑛+ 2𝑝 + 2𝑝(𝑝 + 1)/(𝑛 −𝑝− 1)

Akaike’s information criterion for model fit

where, n= number of data points, SSE = sum of squared

residuals; and p = number of model parameters

Models for Moisture Loss

ODI, April 4th 2019

82

84

86

88

90

92

94

96

0 10 20 30 40 50 60 70 80

RC

M %

Stirrting time, min

No significant differences in moisture loss kinetics of curd

during cheesemaking from GRA, CLO and TMR milk

RCMt = RCMo(1 − k𝑙𝑛 t )Feed Rate constant (k)min-1

TMR 0.0369 (±0.0024)a

CLO 0.0360 (±0.0022)a

GRA 0.0372 (±0.0025)a

80

82

84

86

88

90

92

94

96

80 85 90 95 100

Pre

dic

ted,

RC

M%

Measured, RCM %

TMR (□), CLO (∆) and GRA fed (○)

R2=0.93

Moisture Loss Kinetics

ODI, April 4th 2019

Minor components in milk and whey

ODI, April 4th 2019

ConclusionsButter:

I. Influence of diet on hardness, colour and sensory properties

Cheese:

I. If standardising on a fat basis: i.e. adjusting milk fat levels usingprotein as a baseline – varying milk protein levels may influencecoagulation/syneresis processes

II. If standardising on a fat and protein basis- feed had no impact oncoagulation & syneresis

III. Milk NPN levels may vary- so if using total protein – it may not account for influence of NPN

IV. No significant impact on yield (next slides)I. But Clover will impact on NPN levels-

II. Some trials (High clover in sward) resulted in high NPN levels- did influence yield-

III. On a small no of trials not statistically significant- but on large scale/ over a season of cheese making – would be of consequence

ODI, April 4th 2019

Cheese Studies

ODI, April 4th 2019

Impact on quality characteristics, nutritional composition,

sensory and volatile properties of full-fat Cheddar &

Massdammer cheeses

Objectives:

➢ Examine the effects of cows feeding system on the composition and quality of

Cheddar and Maasdam –type Cheese.

➢ Methodology:

➢ Bulk milk samples collected from am and pm milking of each herd over 3 days

(approx. 1000L/herd) for manufacture of Cheese using pilot plant facilities in

Moorepark Technology Ltd

➢ Cheese analysis over 270/150 d storage period included:

TextureColour

FAME & FFA

Cheese

Proteolysis

Sensory &

Volatile

properties

ODI, April 4th 2019

Milk coagulation Cutting Stirring Draining Pressing Brining

Ripening time, Days

Tem

pera

ture

°C

0 10 20 150

48

23

31 97

Sampling points for cheese

Ripening

380 L

Rennet cultures and

CaCl2 Washing

Warm room

Maasdam Cheese-making

ODI, April 4th 2019

Milk compositions- Maasdam

Raw milk Standard milk

Component GRA CLO TMR GRA CLO TMR

Fat ( %,w/w) 4.25a 4.13

a 4.29

a 3.06

A 3.10

A 3.04

A

Protein ( %,w/w) 3.60a 3.56

a 3.45

b 3.63

A 3.62

A 3.60

A

Lactose ( %,w/w) 4.63b 4.72

a 4.74

a 4.67

A 4.69

A 4.73

A

NCN ( %,w/w) 0.12a 0.12

a 0.11

a 0.12

A 0.12

A 0.12

A

NPN ( %,w/w) 0.03a 0.03

a 0.03

a 0.03

A 0.03

A 0.03

A

Casein no. 79.30a 78.50

a 79.17

a 78.90

A 78.10

A 79.40

A

PFR 0.85a 0.86

a 0.80

b 1.19

A 1.17

A 1.18

A

Means (n=3, N=9) in a row with different superscripts (a-b)/ (A-B) differs (P < 0.05). TMR refers to

milk produced from cows fed total mixed ration in indoor. CLO and GRA refer to milk produced from

cows grazed in grassland containing grass with clover and grass, respectively.

NPN refers to Non-protein nitrogen and NCN refers to Non-casein Nitrogen.

ODI, April 4th 2019

Composition of cheeses- Maasdam

Before Brining Post- brining

Composition GRA CLO TMR

GRA CLO TMR

Moisture ( %,w/w) 47.57a 47.37

a 47.24

a

45.63 A

45.35 A

45.45 A

Protein ( %,w/w) 24.76 a 24.32

a 24.12

a

25.12 A

24.68 A

24.32 A

Fat ( %,w/w) 25.06 a 24.51

a 24.70

a

24.95 A

24.78 A

25.17 A

FDM ( %,w/w) 47.79 a 46.56

a 46.81

a

45.89 A

45.34 A

46.13 A

MNFS ( %,w/w) 63.47 a 62.74

a 62.73

a

60.80 A

60.29 A

60.73 A

Salt ( %,w/w) - - -

1.21 A

1.49 A

1.30 A

S/M ( %,w/w) - - -

2.64 A

3.29 A

2.86 A

PFR

1.01 A

1.00 A

0.97 A

Ash (g/100 g) 2.68a 2.55

a 2.70

a 3.42

B 3.46

B 3.74

A

Ca (mg/100g) 875.86a 856.16

a 898.36

a 942.40

B 912.57

B 969.72

A

Ca (mg/100 g protein) 3557.3a 3376.24

a 3600.21

a

3755.17

A 3677

A 3988.60

A

Means (n=3, N=9) in a row with different superscripts (a-b)/ (A-B) differs (P < 0.05). TMR, CLO and

GRA refer to cheese produced from milk of cows fed total mixed ration in indoor, grazed in grassland

containing grass with clover , and grazed in pasture containing grass only, respectively.

FDM refers to Fat in dry mater and MNSF refers to moisture in non-fat substances.

ODI, April 4th 2019

Whey composition and loss- Maasdam

Composition GRA CLO TMR

Fat (%, w/w) 0.40a 0.40

a 0.37

a

Protein (%, w/w) 0.96a 1.01

a 1.02

a

Fines (mg/kg) 394.67a 275.23

a 408.93

a

TS (%, w/w) 6.63a 6.55

a 6.63

a

Loss

Fat (% of milk fat) 11.42a 11.10

a 10.68

a

Protein (% of milk protein) 23.35 a 24.16

a 24.60

a

Means (n=3) in a row with different superscripts (a-b) (P < 0.05). TMR, CLO and GRA refer to cheese

produced from milk of cows fed total mixed ration in indoor, grazed in grassland containing grass with

clover , and grazed in pasture containing grass only, respectively.

ODI, April 4th 2019

Yield - MaasdamFeed GRA CLO TMR

Mass balance (input/output*100)

Total weight 97.81 97.37 97.57

Fat 97.51 97.75 95.25

Protein 100.22 101.20 100.68

Moisture 99.02 98.28 98.69

Recovery

Fat (% of milk fat) 89.41a 89.60

a 91.89

a

Protein (% of milk protein) 76.49a 76.78

a 74.61

a

Yield, %

Actual yield, Ya 10.88a 11.15

a 10.94

a

Moisture adjusted yield, Yma 10.76a 11.08

a 10.85

a

Ymafpam 10.12a 10.34

a 10.28

a

Yafpam 10.24a 10.41

a 10.37

a

Ymafcam 10.89a 11.11

a 11.02

a

ODI, April 4th 2019

Feeding system alters the colour of Cheddar

cheese

3535 ODI, April 4th 2019

Chapter Results:

Tom F. O'Callaghan Viva Voce 09/02/20183636

TM

R

GR

S

CL

V

0 .0

0 .5

1 .0

1 .5

2 .0

Ch

ee

se

-Ca

ro

ten

e (

mg

/Kg

)

P = 0 .0 2 0

P < 0 .0 0 1P < 0 .0 0 1

➢ b* values highly correlated with β-carotene

content (P < 0.001, Pearson r = 0.948),

➢ L* values, negatively correlated with the β-

carotene content (P = 0.004; r −0.841)

Feeding system alters the colour of Cheddar

cheese

ODI, April 4th 2019

Colour

components

GRA CLO TMR

L (Whiteness) 79.36±0.97b 80.37±0.37ab 82.17±0.37a

A (Greenness) -2.83±0.47a -3.29±0.50a -2.72±0.37a

B (Yellowness) 28.89±0.14a 29.10±0.91a 24.19±0.55b

GRA and CLO cheese had more yellowness than TMR

0

1

2

3

4

5

6

7

Ivory Colour Smoothshinyappearance

Eye quality Eyedistribution

Ireegular eyes Defects Rind

Grass

Clover

TMR

aa

b

a a

b

Appearance attributes

Feeding system alters the colour of Maasdam cheese

ODI, April 4th 2019

Pasture feeding has a beneficial effect on Cheddar

cheese nutritional composition

3838

FA Triglycerides TMR GRS CLV Treatment

Butyric Acid (C4:0) 3.59 ± 0.15 3.58 ± 0.14 3.68 ± 0.14 0.768

Caproic Acid (C6:0) 1.96 ± 0.06 1.88 ± 0.04 1.93 ± 0.07 0.415

Caprylic Acid (C8:0) 1.09 ± 0.03 1.03 ± 0.02 1.07 ± 0.04 0.297

Capric Acid (C10:0) 2.41 ± 0.06 2.26 ± 0.05 2.35 ± 0.08 0.166

Undecanoic Acid (C11:0) 0.04 ± 0.00 0.04 ± 0.00 0.03 ± 0.00 0.013

Lauric Acid (C12:0) 2.79 ± 0.06 2.63 ± 0.07 2.71 ± 0.09 0.152

Tridecanoic Acid (C13:0) 0.07 ± 0.00 0.06 ± 0.01 0.05 ± 0.00 0.014

Myristic Acid (C14:0) 8.72 ± 0.35 8.80 ± 0.22 7.55 ± 2.27 0.599

Myristoleic Acid (C14:1) 0.93 ± 0.01 1.01 ± 0.02 0.94 ± 0.03 0.021

Pentadecanoic Acid (C15:0) 0.83 ± 0.02 1.00 ± 0.03 0.94 ± 0.04 0.004

Palmitic Acid (C16:0) 26.62 ± 0.83 23.27 ± 0.66 22.80 ± 1.19 0.012

Palmitoleic Acid (C16:1) 1.43 ± 0.04 1.39 ± 0.04 1.29 ± 0.06 0.091

Heptadecanoic Acid (C17:0) 0.52 ± 0.02 0.61 ± 0.04 0.61 ± 0.02 0.042

Stearic Acid (C18:0) 6.15 ± 0.48 6.97 ± 0.92 7.37 ± 0.69 0.300

Vaccenic Acid (C18:1t11) 1.43 ± 0.11 3.23 ± 0.26 2.74 ± 0.18 < 0.001

Oleic Acid (C18:1n9c) 13.78 ± 0.53 14.23 ± 1.70 14.49 ± 1.17 0.843

Linolelaidic Acid (C18:2n6t) 0.15 ± 0.01 0.36 ± 0.03 0.42 ± 0.02 < 0.001

Linoleic Acid (C18:2n6c) (LA) 1.45 ± 0.09 0.49 ± 0.04 0.69 ± 0.03 < 0.001

α-Linolenic Acid (C18:3n3) (ALA) 0.26 ± 0.03 0.62 ± 0.09 0.98 ± 0.08 < 0.001

CLA (c9t11) 0.49 ± 0.02 1.45 ± 0.12 1.13 ± 0.06 < 0.001

CLA (c12t10) 0.11 ± 0.00 0.10 ± 0.01 0.10 ± 0.00 0.579

Eicosenoic Acid (C20:1) 0.02 ± 0.01 0.07 ± 0.01 0.08 ± 0.01 0.003

Eicosatrienoic Acid (C20:3n6) 0.08 ± 0.01 0.01 ± 0.01 0.03 ± 0.00 0.001

Behenic Acid (C22:0) 0.02 ± 0.00 0.03 ± 0.01 0.03 ± 0.00 0.309

Erucic Acid (C22:1n9) 0.05 ± 0.00 0.02 ± 0.00 0.03 ± 0.00 < 0.001

Tricosanoic Acid (C23:0) 0.01 ± 0.01 0.10 ± 0.01 0.11 ± 0.01 < 0.001

Arachidonic Acid (C20:4n6) 0.00 ± 0.00 0.05 ± 0.01 0.06 ± 0.00 < 0.001

0

0.5

1

1.5

2

2.5

3

3.5

4

TMR GRS CLV

g/1

00g o

f fa

t

Vaccenic Acid (C18:1t11)P < 0.05

P < 0.05

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

TMR GRS CLV

g/1

00g o

f fa

t

CLA (c9t11)P < 0.05

P < 0.05

ODI, April 4th 2019

Pasture feeding has a beneficial effect on Cheddar cheese nutritional

composition

3939

FA Triglycerides TMR GRS CLV Treatment

Butyric Acid (C4:0) 3.59 ± 0.15 3.58 ± 0.14 3.68 ± 0.14 0.768

Caproic Acid (C6:0) 1.96 ± 0.06 1.88 ± 0.04 1.93 ± 0.07 0.415

Caprylic Acid (C8:0) 1.09 ± 0.03 1.03 ± 0.02 1.07 ± 0.04 0.297

Capric Acid (C10:0) 2.41 ± 0.06 2.26 ± 0.05 2.35 ± 0.08 0.166

Undecanoic Acid (C11:0) 0.04 ± 0.00 0.04 ± 0.00 0.03 ± 0.00 0.013

Lauric Acid (C12:0) 2.79 ± 0.06 2.63 ± 0.07 2.71 ± 0.09 0.152

Tridecanoic Acid (C13:0) 0.07 ± 0.00 0.06 ± 0.01 0.05 ± 0.00 0.014

Myristic Acid (C14:0) 8.72 ± 0.35 8.80 ± 0.22 7.55 ± 2.27 0.599

Myristoleic Acid (C14:1) 0.93 ± 0.01 1.01 ± 0.02 0.94 ± 0.03 0.021

Pentadecanoic Acid (C15:0) 0.83 ± 0.02 1.00 ± 0.03 0.94 ± 0.04 0.004

Palmitic Acid (C16:0) 26.62 ± 0.83 23.27 ± 0.66 22.80 ± 1.19 0.012

Palmitoleic Acid (C16:1) 1.43 ± 0.04 1.39 ± 0.04 1.29 ± 0.06 0.091

Heptadecanoic Acid (C17:0) 0.52 ± 0.02 0.61 ± 0.04 0.61 ± 0.02 0.042

Stearic Acid (C18:0) 6.15 ± 0.48 6.97 ± 0.92 7.37 ± 0.69 0.300

Vaccenic Acid (C18:1t11) 1.43 ± 0.11 3.23 ± 0.26 2.74 ± 0.18 < 0.001

Oleic Acid (C18:1n9c) 13.78 ± 0.53 14.23 ± 1.70 14.49 ± 1.17 0.843

Linolelaidic Acid (C18:2n6t) 0.15 ± 0.01 0.36 ± 0.03 0.42 ± 0.02 < 0.001

Linoleic Acid (C18:2n6c) (LA) 1.45 ± 0.09 0.49 ± 0.04 0.69 ± 0.03 < 0.001

α-Linolenic Acid (C18:3n3) (ALA) 0.26 ± 0.03 0.62 ± 0.09 0.98 ± 0.08 < 0.001

CLA (c9t11) 0.49 ± 0.02 1.45 ± 0.12 1.13 ± 0.06 < 0.001

CLA (c12t10) 0.11 ± 0.00 0.10 ± 0.01 0.10 ± 0.00 0.579

Eicosenoic Acid (C20:1) 0.02 ± 0.01 0.07 ± 0.01 0.08 ± 0.01 0.003

Eicosatrienoic Acid (C20:3n6) 0.08 ± 0.01 0.01 ± 0.01 0.03 ± 0.00 0.001

Behenic Acid (C22:0) 0.02 ± 0.00 0.03 ± 0.01 0.03 ± 0.00 0.309

Erucic Acid (C22:1n9) 0.05 ± 0.00 0.02 ± 0.00 0.03 ± 0.00 < 0.001

Tricosanoic Acid (C23:0) 0.01 ± 0.01 0.10 ± 0.01 0.11 ± 0.01 < 0.001

Arachidonic Acid (C20:4n6) 0.00 ± 0.00 0.05 ± 0.01 0.06 ± 0.00 < 0.0010

100

200

300

400

500

600

TMR GRS CLV

g o

f C

heddar

Cheese

Cheese consumption to reach .8g CLA/day

T M R G R A S S C L O V E R

0 .0

0 .2

0 .4

0 .6

0 .8

C

LA

g/1

00

g o

f C

he

dd

ar

Ch

ee

se

P , < 0 .0 0 1

P , 0 .0 0 3P , < 0 .0 0 1

min 0.8 g of CLA/d to attain

benefits associated with CLA

based on animal models of

therapeutic doses (Siurana and Calsamiglia,

2016)

531g 182g 226g

T M R G R S C L V

0

5 0

1 0 0

1 5 0

2 0 0

Ha

rd

ne

ss

(N

)

P , 0 .0 1 3

P , 0 .0 2 6

➢ Oleic-to-palmitic acid ratio was negatively

correlated with cheese hardness (P = 0.031; r

= −0.714) and chewiness (P = 0.024; r =

−0.735).

➢ Palmitic acid was significantly and positively

correlated with hardness and chewiness

attributes (P = 0.005; r = 0.836 and P = 0,007; r

= 0.816 respectively).

➢ The increased CLA content of the pasture-

derived cheese was also negatively correlated

with hardness (P = 0.002; r = 0.877), and

chewiness (P = 0.004; r = −0.849).

ODI, April 4th 2019

Proteolysis of Cheddar Cheese

4040

9 0 1 8 0 2 7 0

1 0

1 5

2 0

% p

H 4

.6/T

N

T M R

G R S

C L V

T im e (d ) R ip e n in g

Cheese FAA content over ripening Cheese proteolysis over ripening

While ripening time had a

significant effect on the

level of FAA and

proteolysis. There was no

significant difference

between feeding systems

ODI, April 4th 2019

Ripening time (d)

0 20 40 60 80 100 120 140 160

% p

H 4

.6-S

N /

TN

0

5

10

15

20

25

30

Ripening time (d)

0 20 40 60 80 100 120 140 160

To

tal F

AA

(m

g/k

g o

f che

ese

)

0

2000

4000

6000

8000

10000

12000

0 20 40 60 80 100 120 140 160

Pro

pio

na

te,

mg/k

g c

he

ese

0

2000

4000

6000

8000

Ripening time (d)

GRACLO

TMR

P = 0.09P = 0.99

P = 0.96P = 0.21

Ripening time (d)

0 20 40 60 80 100 120 140 160

pH

5.1

5.2

5.3

5.4

5.5

5.6

5.7

5.8

5.9

P = 0.09P = 0.99

Biochemical Changes in Maasdam cheeses

ODI, April 4th 2019

0 20 40 60 80 100 120 140 160

Ace

tate

, m

g/k

g c

he

ese

0

500

1000

1500

2000

2500

3000

3500

4000

Ripening time (d)Ripening time (d)

0 20 40 60 80 100 120 140 160

Buty

rate

, m

g/k

g c

he

ese

-20

0

20

40

60

80

100

120

140

160

180

GRACLO

TMRAcetate in CLO cheese < Acetate in GRA or TMR cheese

Butyrate in TMR cheese > Butyrate in CLO or GRA cheese

P <0.01 P <0.01

Acetate and Butyrate levels in Maasdam cheeses

ODI, April 4th 2019

0

200

400

600

800

1000

1200

1400

FF

A,

mg

/kg

GRA

CLO

TMR

Lipolysis in Maasdam at cheese 97 d

0

500

1000

1500

2000

2500

3000

3500

GRA CLO TMR

tota

l FFA

, mg/

kg c

he

ese

cheese samples

a

a a

aC4=butyric acid

C6= caproic acid

C8= caprylic acid

C10= capric acid

C12= Lauric acid

C14= myristic acid

C16= Palmitic acid)

C18= stearic acid

C18:1= Oleic acid

C18:2= Lenoleic acid

C18:3=γ-Linolenic acid

0

5

10

15

20

25

30

35

40

45

%F

FA

/To

tal

FF

A

GRA

CLO

TMR

aa

ODI, April 4th 2019

Instrumental -Texture of Cheddar Cheese

ODI, April 4th 2019

“Measuring” Flavour

4545

• Sensory Analysis

• Trained panellists familiar with the products evaluate the most important characteristics that define the product

• Hedonics/Acceptance

• Ranked Descriptive Analysis

• Flavour chemistry (Kilcawley Lab Teagasc)

• Identify the volatile compounds by Gas Chromatography Mass Spectrometry

• Untargeted approach

• HS-SPME & SE

Dr Kieran Kilcawley

ODI, April 4th 2019

Feeding system has a significant effect on Cheddar

sensory properties

4646 ODI, April 4th 2019

0

1

2

3

4

5

6

7

Ivory Colourshiny appearance

Eye quality

Eye distribution

Appearance

Liking of Aroma

Liking of Flavour

Liking of Texture

Liking of Taste

Overall Acceptability

Sweaty/sour Aroma

Dairy Sweet Aroma

Nutty Aroma

Pungent Aroma

Off-aroma

Diacetyl AromaFruity Aroma

Creamy AromaSweet Taste

Salt Taste

Sour Taste

Bitter Taste

Firmness in mouth

Rubbery Texture

Smooth Texture

Oily Texture

Sticky Texture

Nutty Flavour

Cowy/Barny flavour

Dairy Flavour

Diacetyl flavour

Buttery Flavour

Fruity/Estery FlavourAstringent Aftertaste

* *

*

**

* P < 0.05

Feeding system has a significant effect on

Maasdam sensory properties

ODI, April 4th 2019

Conclusions

I. No effect of feed system observed on Cheddar and Maasdam cheese

composition and yield (once milks standardised to fat and protein levels)

II. Higher levels of NPN in Clo derived milks- could influence yield on a larger study

III. Pasture derived feeding systems lead to production of Cheddar cheeses with a

healthier fatty acid profile, e.g. ⇡ n3 fatty acid

IV. Alterations in the fatty acid profile of the cheese resulted in pasture derived

cheese having reduced hardness scores at room temperature.

V. No effect of diet on proteolysis levels.

VI. Differences observed in short chain volatile acids

VII. Feeding system and ripening time had a significant effect on the sensory profile of

the Cheddar and Maasdam cheeses (e.g. color, texture, etc.,)

4848 ODI, April 4th 2019

Grated cheese sample

Freeze dried powder

HR MAS NMR

CPMG pulse

sequence spectrum

NOESY pulse

sequence spectrum

Extraction H NMR spectrum

Metabolomic study of Maasdam Cheese

H NMR spectrum CPMG spectrum NEOSY spectrumODI, April 4th 2019

GRA

CLO

TMR

GRA

CLO

TMR

Le

u

pro

p2

-3 b

uta

nedio

l

La

c

Le

u Ala

Lys

Ace

pro

Me

tP

rop

pro

4 a

mynobuty

rate

Glu

, P

roS

ucc

Glu

Met

Lys

Cho

l

Gly

cin

e

Me

tho

nol

2-3

bu

tan

edio

l

Glu

, L

eu

, glu

tam

ine

, Lys

L-p

hy

La

c

Formate L-phy

Tyr

Tyr

Pro

π-methylH

Carn

itin

e,B

eta

ine

Oro

tic

Asp

ara

gin

e

Ph

en

yl

Met

Pro

Cre

atin

ine

31 compounds

ODI, April 4th 2019

Representative Spectra

Glycerol

Fatty acids

UFA

NOESY

spectrum

CPMG

spectrum

Ala

Ace

tate

Suc

Car

nit

ine

Gly

cine

Met

Lac

Phe

Form

ate

ODI, April 4th 2019

Cheese Metabolome

CPMG

spectrum

NEOSY

spectrum

ODI, April 4th 2019

Table 3. Effect of feeding systems1 on volatile compounds in Maasdam cheese at 97 day of ripening.

Volatile compounds Experimental cheese groups (peak area)

Std RI GRA SE CLO SE TMR SE P

Acid

Acetic acid 629 680 4.3*10^7a 2.1*10^6 3.9*10^7

a 1.3*10^6 4.0*10^7

a 3.9*10^6 0.45

Propanoic acid 725 720 4.1*10^7a 1.0*10^7 3.2*10^7

a 1.1*10^7 3.5*10^7

a 8.4*10^6 0.79

Butanoic acid 787 809 1.4*10^7a 9.9*10^5 8.8*10^6

a 2.7*10^6 1.6*10^7

a 2.2*10^6 0.11

3-Methylbutanoic acid 838 847 1.7*10^6 a 2.8*10^5 1.6*10^6

a 5.8*10^5 2.9*10^6

a 7.9*10^5 0.28

2-Methylbutanoic acid 831 853 2.2*10^6 a 2.3*10^5 2.1*10^6

a 1.0*10^6 3.9*10^6

a 9.3*10^5 0.28

Hexanoic acid 1005 980 8.0*10^6 a 4.0*10^6 3.2*10^6

a 3.0*10^6 1.0*10^7

a 1.0*10^7 0.74

Octanoic acid 1163 1164 1.4*10^6 a 1.9*10^5 1.2*10^6

a 2.2*10^5 2.6*10^6

a 8.5*10^5 0.2

Alcohols

1-Propanol 549 537 4.5*10^5 a 1.2*10^5 1.1*10^5

a 1.1*10^5 3.4*10^4

a 3.4*10^4 0.05

3-Methyl-1-butanol 731 740 6.1*10^5 a 2.4*10^5 4.5*10^5

a 1.3*10^5 4.9*10^5

a 1.8*10^5 0.83

2-Methyl-1-butanol 743 747 6.3*10^5 a 1.5*10^4 1.9*10^6

a 8.3*10^5 8.6*10^5

a 4.8*10^5 0.28

2,3-Butanediol 802 814 4.1*10^6 a 6.4*10^5 3.5*10^6

a 1.0*10^6 4.2*10^6

a 3.1*10^5 0.75

Aldehydes

Benzaldehyde 997 971 2.9*10^6 a 1.4*10^6 1.7*10^6

a 7.6*10^5 2.1*10^6

a 1.8*10^6 0.83

Benzene acetaldehyde 1048 1053 2.7*10^4 a 8.5*10^3 2.3*10^4

a 3.2*10^3 2.8*10^4

a 6.6*10^3 0.85

Esters

Ethyl propanoate 714 712 1.2*10^6 a 1.6*10^5 8.9*10^5

a 5.7*10^4 1.1*10^6

a 1.9*10^5 0.4

Ethyl isobutyrate 767 770 7.6*10^4 a 7.6*10^4 3.7*10^4

a 3.7*10^4 5.5*10^4

a 5.5*10^4 0.89

Ethyl butanoate 799 802 1.5*10^6 a 5.7*10^5 1.7*10^6

a 6.5*10^5 7.9*10^5

a 8.9*10^4 0.44

Ethyl hexanoate 1002 998 1.6*10^5 a 2.9*10^4 1.5*10^5

a 3.4*10^4 2.4*10^5

a 4.2*10^4 0.22

Ethyl octanoate 1198 1194 7.5*10^4 a 2.1*10^4 6.7*10^4

a 2.2*10^4 1.3*10^5

a 3.3*10^4 0.23

Hydrocarbons

Toluene 723 759 9.4*10^5 a 2.7*10^5 1.7*10^6

a 2.7*10^5 1.5*10^5

a 1.6*10^4 <0.01

o-Xylene 872 875 5.8*10^4 a 1.3*10^4 5.2*10^4

a 1.5*10^4 4.7*10^4

a 1.8*10^4 0.89

ODI, April 4th 2019

Continued Table 3. Effect of feeding systems1 on volatile compounds in Maasdam cheese at 97 day of ripening.

Experimental cheese groups (peak area)

Std RI GRA SE CLO SE TMR SE P

1-Octene 788 799 3.4*10^5 a 1.9*10^5 6.6*10^5

a 3.6*10^5 1.7*10^5

a 8.8*10^4 0.4

Ketones

2-Butanone 599 576 6.1*10^7 a 1.3*10^7 4.3*10^7

a 5.5*10^6 3.5*10^7

a 4.5*10^6 0.16

Acetoin 738 744 1.1*10^7 a 4.3*10^6 1.0*10^7

a 5.2*10^6 7.9*10^6

a 2.5*10^6 0.87

2-Heptanone 891 892 1.9*10^6 a 1.1*10^5 1.7*10^6

a 1.3*10^5 2.1*10^6

a 3.1*10^5 0.42

2-Nonanone 1094 1092 2.8*10^5 a 3.4*10^4 2.5*10^5

a 2.4*10^4 2.9*10^5

a 4.6*10^4 0.79

Lactones

δ-Decalactone 1504 1504 1.5*10^4 a 8.0*10^3 2.5*10^4

a 4.6*10^3 3.5*10^4

a 5.8*10^3 0.16

Sulfur compounds

Dimethyl disulfide 754 756 1.3*10^5 a 3.5*10^4 6.7*10^4

a 2.1*10^4 9.2*10^4

a 1.2*10^4 0.28

Carbon disulfide 536 530 1.5*10^5 1.2*10^4 1.3*10^5 2.1*10^3 1.1*10^5 8.5*10^3 0.06

Dimethyl sulfide 497 515 0. 0. 5.4*10^4 1.2*10^4 0. 0. 0.002

Dimethyl trisulfide 983 980 3.6*10^4 a 1.3*10^4 1.4*10^4

a 5.8*10^3 1.8*10^4

a 4.1*10^3 0.23

Terpenes

D-Limonene 1033 1037 3.0*10^4 a 2.6*10^3 3.2*10^4

a 7.3*10^2 3.0*10^4

a 2.6*10^3 0.6

α-Pinene 940 941 8.4*10^3 a 4.5*10^3 9.9*10^3

a 5.0*10^3 6.4*10^3

a 3.3*10^3 0.85

Volatile compounds with P < 0.05 signficiantly differ between each treatment. Data presented are the means from 3 replicate trials.

1GRA =

Grass only, CLO = Grass with clover, TMR = total mixed ration, SE = standard error.

ODI, April 4th 2019

VOC GRA CLO TMR P-values

Toluene 944,012a 1,678,522a 152,455b 0.04

AcidAcetic acid

Propionic acid

Butanoic acid

3-Methylbutanoic

acid

2-Methylbutanoic

acid

Hexanoic acid

Octanoic acid

Alcohol1-Propanol

3-Methyl-1-

butanol

2-Methyl-1-

butanol

2,3-Butanediol

AldehydeBenzaldehyde

Benzeneacetaldehyde

EsterEthyl propionate

Ethyl isobutyrate

Ethyl butanoate

Ethyl hexanoate

Ethyl octanoate

HydrocarbonsToluene

o-Xylene

1-Octene

Ketones2-Butanone

Acetoin

2-Heptanone

2-Nonanone

Lactoneδ-Decalactone

SulfurDimethyl Sulfide

Carbon disulfide

Dimethyl disulfide

Dimethyl trisulfide

Volatiles Compounds (VOC)

ODI, April 4th 2019

Summary findings of NMR/GCMS study

I. NMR showed differences were mainly in lipid contents

II. Levels of toluene were higher in Maasdam cheeses prepared from

GRA- or CLO-fed milk compared to those prepared from TMR-fed

milk.

III. Overall, feed-induced variation in metabolite content of Maasdam

cheese was low, suggesting that metabolites produced by starter

cultures and secondary PAB cultures have a much greater role and

can mask the majority of metabolites derived from milk/feed during

fermentation.

ODI, April 4th 2019

Food for Health Ireland 3

Leading Pasture Dairy Pillar

• Look to create a deeper understanding of the advantages and disadvantages of pasture feeding relative to milk and dairy products

• Further profiling of Irish commercial dairy products

• Identification and confirmation of bio-markers for grass fed milks

• Human intervention trials with Grass-fed and non`-Grass-Fed dairy products

Where to next!

Science to support the impact of primary production

practices on the composition and quality of milk and

dairy products across the supply chain

ODI, April 4th 2019

Acknowledgements▪ Ram Raj Panthi - PhD

▪ Dr. Tom O’Callaghan

▪ Dr. Kieran Kilcawley

▪ Dr. Ulrich Sundekilde ( U. of Aarhus, Dk)

▪ Dr. Deirdre Henessy

▪ Prof Lisbeth Goddik

▪ Organisers of ODI conference.

ODI, April 4th 2019

The Science behind “grass

fed”Diarmuid (JJ) Sheehan

Teagasc Food Research Centre Moorepark, Ireland