Manipulating the fatty acid composition including CLA content

of animal products

Professor, Dr. Peter Buttery

Division of Biochemistry & Nutrition

Biosciences School

The University of Nottingham UK

Why Manipulate the fatty acid composition of animal products?

• To reduce the intake of “bad” fatty acids by the consumer

• To increase the intake of “good” fatty acids by the consumer

• To increase sales of such products by improving their “image”

Dietary Reference Values for Fatty AcidsPopulation averages (% total energy)

1991

Saturated Fatty Acids 10%Cis polyunsaturated fatty acids 6%Cis monounsaturated fatty acids 12%Trans fatty acids 2%Total Fatty Acids 30%

Further Recommendations1994

Population average consumption of long chain n-3 polyunsaturated fatty acids should double (from 0.1g/day to 0.2g/day

(not going to discus this here but is important)

Saturated fatty acid (SFA) intake in Great Britain

0

2

4

6

8

10

12

14

16

18

1992 1993 1994 1995 1996 1997 1998 1999 2000

perc

ent

ener

gy f

rom

SF

A

target

Source: National Food Survey 2000

Contribution of Animal Products to Saturated Fatty Acid (SFA) Intake in Great Britain

National Food Survey 2000

Dairy39%

Meat22%

Other39%

012

345

6

SF

A p

er day (g)

milk

& cream

cheese

bu

tter

eggs

beef

lamb

pork

pou

ltry

other m

eat

• Meat contributes about 22% of total and saturated fat intake in the human diet

• Lamb has a high stearate content which gives a waxy texture, producing poor organoleptic properties

• Red meat, as part of a balanced diet, is an important source of protein and iron

• Meat contributes about 22% of total and saturated fat intake in the human diet

• Lamb has a high stearate content which gives a waxy texture, producing poor organoleptic properties

• Red meat, as part of a balanced diet, is an important source of protein and iron

• Change fatty acid profile of diet ~ low fat diet ~ biohydrogenation of unsaturated fat

• Protected fatty acids

• Manipulation of de novo fat synthesis that is reduce the fat content of the carcass

• Change fatty acid profile of diet ~ low fat diet ~ biohydrogenation of unsaturated fat

• Protected fatty acids

• Manipulation of de novo fat synthesis that is reduce the fat content of the carcass

Possible Approaches

Possible Approaches

Reduction of fat

• Breeding• Diet• Growth Hormone *• Beta agonists*• CLA?

• *Not legal in Europe but are in many parts of the world----China?

Fatty acid composition of intramuscular fat in pigs fed different oils

0

510

1520

2530

3540

45

50

Fatty acid (g/100g total)

C14:0 C16:0 C18:0 C18:1 C18:2 C18:3

LFHF (SO)HF (RO)

Entire male Landrace*Large White Pigs (3 per group) were grown from 55kg to 120kgLF diet: no added fatSO diet: 43.5g/kg sunflower oil + 31.5g/kg rape seed oilRO diet: 75g/kg rape seed oilBudd, Salter, Buttery & Wiseman, unpublished data

THE RUMINANT

• Fats over 10% cause problems with rumen function

• Unsaturated fats are hydrogenated in the rumen so difficult to alter the diet.

Adipose Tissue Fatty Acid Deposition in Ruminant Adipose Tissue

DIET

C18:1/C18:2/C18:3

C18:0

DE NOVO SYNTHESIS

C16:0

C18:0

C18:1

Adipose Tissue

Rumen

Fatty acid composition abomasal fluid and adipose tissue of sheep fed on grass nuts

0

10

20

30

40

50

60

70

fatty acid (g/100g F

AM

E)

14:0 16:0 16:1 18:0 c18:1 t18:1 18:2 18:3

dietabomasumsubcutaneous

Sources of Ruminant Milk Saturated Fatty Acids

Acetate/-OH Butyrate

C4:0-C14:0C16:0

C18:0

Adipose TissueDiet

Mammary Gland

C18:1

Comparison of the fatty acid composition of duodenal fluid and milk from cows

0

10

20

30

40

50

60

Fatty acid (g/100g total)

<12

12:0

14:0

14:1

16:0

16:1

18:0

t18:1

c18:1

18:2

18:3

duodenum

milk

Fatty acid synthesis in adipose tissue & mammary gland

Acetyl CoA

C16:0

C18:0

C18:1

Acetyl CoA

C16:0

C18:0

C18:1

Malonyl CA Malonyl CA

Acetyl CoA Carboxylase(ACC)

Fatty Acid Synthase(FAS)

Elongase

Stearoyl CoA Desaturase(SCD)

MAMMARY ADIPOSE

Correlation between SCD mRNA and oleate content of omental adipose tissue of growing sheep

y = 0.3542x - 7.1498

R2 = 0.9505

0

100

200

300

400

500

600

0 200 400 600 800 1000 1200 1400 1600 1800

SCD mRNA (arbitrary units)

Effect of insulin on SCD gene expression & oleate synthesis in ovine adipose tissue explants

0

1000

2000

3000

4000

acet

ate

inco

rp (

nMol

es)

MUFA

SFA

0

0.5

1

1.5

2

2.5

3

SCD

mR

NA

(ar

b un

its)

cont ins dex ins + dex

cont ins dex ins + dex

Ins: 20nM InsulinDex: 10nM Dexamethosone

Effect of feeding forage or concentrate-based diets on acetyl CoA carboxylase(ACC) & stearoyl CoA desaturase(SCD) mRNA

concentrations of subcutaneous adipose tissue in sheep

0

10

20

30

40

50

60

70

80

mR

NA

(ar

b. U

nit

s/g

tiss

ue)

ACC SCD

forageconc (150)conc (350)

P<0.001

Effect of feeding forage or concentrate-based diets on fatty acid composition of subucateous

adipose tissue in sheep

0

5

10

15

20

25

30

35

fatty acid (g/100g)

14:0 16:0 16:1 18:0 c18:1 t18:1 18:2 18:3

grass

conc (150)

conc (350)

PROTECT FAT FROM RUMEN

• Coat the fat so that it escapes the action of the rumen bacteria

• The coat is then broken down in either abomasum or the duodenum

Effect of feeding rumen- protected fish oil on the muscle fatty acid content of lot-fed cattle

Ashes et al (2000) Recent Advances in Animal Nutrition, 129-140

0

5

10

15

20

25

30

35

40

fatt

y ac

id (

g/10

0g)

14:0 16:0 18:0 c18:1 t18:1 18:2(n-6)

18:3(n-3)

20:5(n-3)

22:6(n-3)

control

Max-EPA

Trans-10, cis 12-CLA

Cis- 9, trans 11-CLA

Linoleic acid

Why interested in CLAs?Suggested health benefits to humans

• Altered nutrient partitioning and lipid metabolism

• Antiatherogenic• Anticarcinogenic• Antidiabetic (type II diabetes)• Immunity enhancement• Improved bone mineralization

Production of CLAs in the rumen Linoleic Acid

cis-9, cis-12 18:2

cis-9, trans-11 CLA

trans-11 18:1

Stearic acid

trans-10, cis-12 CLA

trans-10 18:1

Production of CLA

t11 C18:1

Linoleic acid

c9, t11

Stearic acid

Rumen

t11 C18:1

Adipose tissue

c9, t11

Stearic acid

SCD

Linoleic acid

Effect of feeding forage or concentrate-based diets on the CLA content of abomassal fluid, subcutaneous adipose tissue

& L. dorsi muscle of sheep

0

0.2

0.4

0.6

0.8

1

1.2

1.4

FA

ME

(g/

100g

)

forage conc (150) conc (350)

abomassal

subcut

muscle

0

0.2

0.4

0.6

0.8

1

1.2

1.4

FA

ME

(g/

100g

)

forage conc (150) conc (350)

abomassal

subcut

muscle

cis 9, trans 11 trans 10, cis 12

Seasonal variation in cis 9, trans 11-CLA content of milk

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

fam

e (g

/100

g)

feb mar april may june july aug sept oct nov dec

SCD ACTIVITY

• There is some evidence that there is genetic variation

• Some cows seems to produce more CLA in the diet than others.Polymorphisms in the SCD gene?

• We have not been able to find much variation in adipose tissue and liver of sheep

Production of CLA-enriched butterIp et al (1999) J Nutr 129: 2135-2142

0

5

10

15

20

25

30

g/10

0g t

otal

FA

<12:0 12:0 14:0 16:0 18:0 c18:1 t18:1 18:2 CLA

control

enriched

Cows fed 5.3% sunflower oil and selected for CLA production

Effect of CLA on development of Mammary Cancer in rats

TreatmentGroup

Dietary CLAg/100g

Tumourincidence (%)*

Control 0.1 93

CLA-enrichedButter+

0.8 43

*30 rats per group were treated with a chemical carcinogen. Values represent the number of animals with tumoursadapted from Ip et al (1999) J. Nutr 129:2135-2142+ primarily cis-9, trans-10 isomer

Could it be used in humans?

• Rats were fed 20% (w/w) butter• Rats weighed 180g and would probably consume about

10g of food a day (2g) butter• This is equivalent to 11g butter/kg body weight/day• Thus a 70kg man would have to consume 770g

butter/day to get the same amount• More research is needed• Professor Bauman in the USA working in the area

Manipulating supply of CLA to sheep tissues

• Rumen saturates fatty acids therefore need to protect CLA supplement (containing equal levels c-9,t-11 and t-10,c-12) from ruminal degradation to absorption in small intestine

Not protected Protected

CLA C18:0 CLA CLA

Rumen protected CLA

0

10

20

30

40

50

60

70

% r

each

ing

du

oden

um

CLA-80 pCLA-80

c9,t11t10,c12

CLA-80 protected by Trouw Nutrition: Used matrix of saturated fat of vegetable origin and final product produced by prilling, spray drying and spray chilling.

Determined to be ~ 70 % protected in cannulated sheep by dual-phase markers

Proportion of ingested CLA reaching Duodenum

How much do we feed?

• Ostrowska et al., (1999) • Growing pigs fed CLA-55 (mixture of both isomers)

0, 1.25, 2.5, 5, 7.5, 10g CLA/kg diet

• Fat deposition decreased with increasing CLA• Fat:lean decreased with increasing CLA

• Av 80 kg pig @ highest dose = 0.19 g CLA/kg body weight/day

How much do we feed?

• PCLA ~ 66 % effective at bypassing rumen biohydrogenation

• High levels lipid adversely affect rumen function

• Max amount PCLA supplied daily to small intestine of av. 40 kg lamb calculated = 0.28 kg CLA/kg BW

• Predicted that lambs would consume 1 kg DM/day therefore highest PCLA inclusion was 100 g/kg DM

• 25 and 50g/kg DM groups for dose response

Trial Outline

• 36 ewe lambs Inclusion g/kg feed GE/day (MJ)

1. Control (n=6) 18.42

2. Low PCLA (n=5) 25 19.42

3. Med PCLA (n=5) 50 19.86

4. Hi PCLA (n=5) 100 21.30

5. Low Megalac (n=5) 21.7 19.14

6. Med Megalac (n=5) 43.3 19.86

7. Hi Megalac (n=5) 86.6 21.30

• Megalac controls for lipid coating of PCLA

• Fed for 10 wks, control group designed to grow at 180 g/d

Sample analysis

• Fatty acid composition

– Did the CLA get into the animals tissues?

• Carcass characteristics

– Repartitioning effects of CLA?

Subcutaneous CLA content

0

0.5

1

1.5

2

2.5

mol

es/1

00 m

oles

Control 25 50 100

Amount of supplementpCLAMeg

0

0.5

1

1.5

2

2.5

mol

es/1

00 m

oles

Control 25 50 100

Amount of supplementpCLAMeg

cis-9, trans-11 trans-10, cis-12

Omental CLA content

0

0.5

1

1.5

2

2.5

mol

es/1

00 m

oles

Control 25 50 100

Amount of supplementpCLAMeg

0

0.5

1

1.5

2

2.5

mol

es/1

00 m

oles

Control 25 50 100

Amount of supplementpCLAMeg

cis-9, trans-11 trans-10, cis-12

Perirenal CLA content

0

0.5

1

1.5

2

2.5

mol

es/1

00 m

oles

Control 25 50 100

Amount of supplementpCLAMeg

0

0.5

1

1.5

2

2.5

mol

es/1

00 m

oles

Control 25 50 100

Amount of supplementpCLAMeg

cis-9, trans-11 trans-10, cis-12

L. dorsi CLA content

0

0.5

1

1.5

2

2.5

mol

es/1

00 m

oles

Control 25 50 100

Amount of supplementpCLAMeg

0

0.5

1

1.5

2

2.5

mol

es/1

00 m

oles

Control 25 50 100

Amount of supplementpCLAMeg

cis-9, trans-11 trans-10, cis-12

Liver CLA content

0

0.5

1

1.5

2

2.5

mol

es/1

00 m

oles

Control 25 50 100

Amount of supplementpCLAMeg

0

0.5

1

1.5

2

2.5

mol

es/1

00 m

oles

Control 25 50 100

Amount of supplementpCLAMeg

cis-9, trans-11 trans-10, cis-12

Effect of dietary CLA on carcass fat

100

150

200

250

300

Fat

(g/

kg)

Control 25 50 100

Amount supplement

pCLAMeg

Carcass characteristics

• No change in carcass composition Carcass cold weight Back fat thickness Omental and perirenal depot whole weights Muscle weight (L. Dorsi, V. Lateralis, S.

Tendenosus) Eye muscle depth or width Liver weight

BUT definite incorporation of CLA into tissues

Accumulation of CLA(t10,c12) in subcutaneous adipose tissue

0

0.2

0.4

0.6

0.8

CL

A(t

10,c

12)g

/100

g F

A

0 0.2 0.4 0.8 1.2 1.4 1.6 2.8 5.6

Dietary CLA(t10,c12) (g/kg diet)

pigsheep

Pig data: adapted from Ostrowska et al (2003)

Effect of dietary CLA on carcass fat

100

150

200

250

300

350

Fat

(g/

kg)

0 0.2 0.4 0.8 1.2 1.6

dietary CLA(t10,c12) (g/kg)

100

150

200

250

300

Fat

(g/

kg)

0 1.4 2.8 5.6

dietary CLA(t10,c12) (g/kg)*

pCLAMeg

PIGS SHEEP

Adapted from Ostrowska et al (2003)

*Corrected for protection

Conclusion

• Tissue CLA content increased but no effect on carcass

• Maybe ruminant adipose tissue responds differently to monogastrics?

Conclusions

• Animal products continue to supply a major proportion of dietary saturated fatty acids

• SCD plays a major role in determining the nature of fatty acids synthesized in tissues

• It is possible to increase the concentration of “healthy” fatty acids (e.g. n-3 PUFA & CLA) in meat & dairy produce but whether the changes can be great enough to have a significant impact on human health remains to be established

Acknowledgement

• Professor Andrew Salter

• Dr Sean Richards

• Dr Zoe Daniel

• Dr Richard Wynn