1 inra-urh, research centre of clermont fd/theix, france,

A L I M E N T A T I O N

A G R I C U L T U R E

E N V I R O N N E M E N T

D. Bauchart, 1st Workshop « Feed to Food »Novi Sad, 29th September-1st October 2009

Effects of dietary n-3 PUFA and antioxidants

on beef fatty acids and lipoperoxidation

in meat-producing cattle1Dominique Bauchart*, 1Mylène Gobert,

1,2Mihaela Habeanu, 3Parafita Emilie, 1Dominique Gruffat, 1Denys Durand

1INRA-URH, Research Centre of Clermont Fd/Theix, France, 2Institute for Animal Biology and Nutrition, Balotesti, Romania,3Association for the development of meat industry (ADIV),

Clermont- Ferrand France.





Introduction

Consumers are now aware of the putative link between meat lipid consumption and human health. An increased intake of n-3 (3) PUFA (from animal products) together with a low ratio of PUFA n-6/n-3 (close to 5) would be beneficial for human health. Since feeding of cattle with linseed-supplemented diets increased n-3 PUFA of lipids in both fresh and cooked meats, beef can be a potential source of n-3 PUFA for humans.

However, in this context, this strategy could modify beef minor FA (CLA, trans 18:1) having various effects on human health.

Moreover, beef lipoperoxidation can be stimulated not only by the high sensibility of n-3 PUFA to reactive oxygen species attacks during oxidative stress, but also by a non adequate status in dietary or endogenous antioxidants. Moreover, beef lipoperoxidation is potentially stimulated i) by a pre-slaughter stress of animals , ii) by technological processes applied to beef.





Objectives

Here are presented some main results of the "Lipivimus" Research Program financed by the French National Research Agency (ANR, 2007-2009) concerning the impact of two dietary lipid sources of n-3 PUFA (linseed, linseed + rapeseed) provided with or without antioxidants (vitamin E, plant extracts rich in polyphenols, PERP) in finishing cull cows on :

i) beef FA characteristics related to their health value for consumers,

ii) beef lipoperoxidation at slaughter or following technological treatments in normal or stressed animals just prior slaughtering.

Beef enriched with n-3 PUFA

Linseed and rapeseed Normand cull

cows





Effects of dietary n-3 PUFA and antioxidants on beef fatty acids





Effects of n-3 PUFA and antioxidants on beef fatty acids

Experimental aspects (I) • Animals and diets

Ø Vit E

Vit E

PERP

Concentrate / straw70 / 30

(Control diet)


+ extruded linseed (40g lipids/kg diet)


+ 1/3 linseed + 2/3 rapeseed

(40g lipids)

Ø

n = 8

Vit E

Vit E PERP

8 8 88 8

Ø stress

8 8

Ø stress

8

72 finishing cull Normand cows





Cows slaughtered at a mean LW of 787kg for the three diets ( body fat score : 3.5 ; BW gain :1.52kg/d) for the 100d-finishing period.


Experimental aspects (II)

150 g of Longissimus thoracis muscle collected 1d post mortem, cut into pieces and freezed in liquid

N2 ground in liquid N2 to produce a

representative and fine powder

Beef powder stored at -20°C until analysis.

• Beef collection and treatments






Analytical aspects Beef fatty acid preparation and analysis

Extraction of total beef lipids: by mixing beef powder with chloroform/methanol (Folch et

al.,1957).

Extraction and methylation of fatty acids (FA) : by transmethylation with Na2 methanolate followed with BF3-

methanol

Total FA composition determination: by gas-liquid chromatography in a CP Sil 88 glass capillary column (100m long x 0.25mm ID, Varian; H2 as the carrier gas). Trans 18:1 analysis

- purification of total trans 18:1 by preparative HPLC- UV detection

identification/ distribution of trans 18:1 isomers by GLC- mass spectrometry





Chemical composition of experimental diets

Diets Control (C)

Linseed (L)

Rapeseed + Linseed (RL)

Dry matter (%) 87 90.6 89

Total proteins (% diet DM) 14 15.2 15.3

Crude cellulose (% diet DM) 10.0 11.1 11.0

Starch + other sugars (% diet DM) 33 23 22

Total minerals (% diet DM) 5.3 7.2 6.8

Total fat (% diet DM) 2 6.7 6.8

C18:3 (g/kg diet DM) 2.1 27.4 12.1





Effects of linseed and rapeseed on beef fatty acids (Habeanu et al, 2009)

Diets Control Linseed

Linseed +

rapeseed

P

Saturated FA (% AG) 47.0 48.2 48.3 0.440

Cis MUFA (% AG) 45.4a 41.6b 40.9b <0.0001

Trans MUFA (% AG) 2.46a 4.08b 5.04c <0.0001

18:3n-3 (% AG) 0.39a 0.61b 0.53ab 0.050

CLA (% AG) 0.32 0.48 0.45 0.080

PUFA n-6/ PUFA n-3 4.54a 3.32b 4.10ab 0.028

Total FA (g/100g LT muscle ) 3.95 3.64 3.63 0.640





Effects of lipid and antioxidant supplements on beef fatty acids

Linseed or linseed + rapeseed supplements did not increase beef lipids.

Higher % of 18:3 n-3 and 9c,11tr 18:2 (CLA) and a lower ratio PUFA n-6/ PUFA n-3 of beef FA with linseed (and linseed +rapeseed at a lower extent) supplement were favorable for the human health,

Addition of antioxidants (vitamin E and PERP) reinforced the beneficial effect of lipid supplements, suggesting a modulation of the microbial FA metabolism by the dietary antioxidants in the rumen.

The large increase in trans MUFA, dominated by trans 18:1, led to the question of their nutritional value. Indeed, 18:1Δ11tr (vaccenic acid) generally abundant in beef would protect against cardiovascular disease (CVD) whereas 18:1Δ9tr (elaidic acid) and 18:1Δ10tr would be rather detrimental by favouring atherosclerosis, CVD inflammation and diabetes.





Effects of lipid supplements on beef trans 18:1 isomers

Purification of total trans 18:1 from beef FAMEBy preparative reversed-phase HPLC: Use of a series of two Kromasil KR100-5C18 inverse phase columns (5µm, 250mm x 10mm) with acetonitrile (eluting solvent, 4mL/min) ; detection at 206 nm.

Separation of trans 18:1 isomersStructural characterization and quantification of each individual trans 18:1 isomer by GLC – Mass Spectrometry

Convertion of methyl trans 18:1 into dimethyl disulfide (DMDS) adducts

Analytical aspects





GLC separation of beef trans 18:1 isomers





Compared effects of linseed vs rapeseed + linseed

on beef trans 18:1 isomers (% total trans 18:1)

(Bauchart et al, 2009)

Trans 18:1 6tr 7tr 8tr 9tr 10tr 11 tr 12tr 13tr 14tr 15tr 16 tr

Diet C 1.3

± 0.9

0.5

± 0.1

1.9

± 0.3

8.5

± 1.5

33.7

± 18.6

36.1

± 14.4

4.3

± 1.2

3.4

± 1.0

4.0

± 1.3

3.4

± 1.8

2.9

± 1.9

Diet L 0.6

± 0.5

0.4

± 0.1

1.6

± 0.4

5.0*

± 0.8

15.6*

± 6.7

33.2

± 11.8

6.1*

± 0.3

8.7*

± 0.8

9.1*

± 0.9

10.9*

± 9.0

8.9*

± 2.7

Diet RL 0.5

± 0.4

0.6

± 0.1

2.3

± 0.6

6.4*

± 0.9

41.1

± 16.4

25.0*

± 12.4

4.9

± 9.2

5.8

± 1.8

5.5

± 1.6

5.0

± 1.8

3.1

± 1.1





Compared effects of linseed vs rapeseed + linseed

on beef trans 18:1 isomers• Direct impact of dietary lipid sources on trans 18:1 isomers

on the health value of beef when dietary FA were dominated by 18:3 n-3 (linseed) or by 18:1 n-9 associated with lower amounts of 18:2n-6 and 18:3n-3 (linseed +rapeseed),

• Higher health value of beef trans 18:1 with the addition of linseed (reduction of Δ9tr and Δ10tr pro-atherogenics),

•

• But, negative effect of linseed and rapeseed on the health value of beef trans 18:1 (reduction of Δ11tr anti atherogenic).





Breeding and technological factors and beef lipoperoxidation





Beef lipoperoxidation : methodological aspects

Technological treatments of beef

Collected just after slaughter, stored under vacuum or in carcass for 12 d at + 4°C and finally cut in steaks as on the classic market,

Packaged at + 4°C in a tray overwrapped with a film i) under air for 4 d. (A), ii) under modified atmosphere (70:30, O2/CO2) for 7 d. (MA), or in a bag iii) under vacuum for 14 d. (V),

Ground into a powder in liquid N2 and stored at -80°C.

Methods for lipoperoxidation characterization

Malondialdehyde (MDA) production measured by HPLC (fluorescence detection) or hydroxyalkenals (HHE and HNE) by GC- MS,

In vitro susceptibility of plasma/tissue(homogenate) to lipoperoxidation evaluated by measuring the kinetic production of conjugated dienes (CD) indicating i) the degree of resistance to lipoperoxidation (Lag phase), ii) the lipoperoxidation velocity (Tx max) iii) the maximal amount of produced CD (Q max).





1717 / 15 / 15

Effets of maturation and packaging treatments on beef lipoperoxidation (Gobert et al, 2008 and 2009)





Packaging treatments and beef peroxidation and colour

Effect of linseed and antioxidants given to cows in the finishing period

(Gobert et al , 2009, Parafita et al, 2008)





Packaging treatments and beef peroxidationEffect of linseed and antioxidants and of emotional stress just prior

slaughter (Durand et al , 2009)





Breeding and technological factors and beef lipoperoxidation

Beef lipoperoxidation Increased with the contact of meat with O2 during packaging

stimulated in beef from cows given linseed-supplemented diet

Prevention of beef against lipoperoxidation most effective from animals given dietary vit E +

PERP than with vit E alone

especially in beef packaged with O2

Emotional and physical stress of animals highly increased beef lipoperoxidation,

especially in beef packaged with O2 from cows given linseed-supplemented diet

Beef colour Beef colour inversely varied with lipoperoxidation

Oxidation of beef myoglobin (into metmyoglobin) generally increased with beef packaging

Partial prevention of myoglobin oxidation only with beef from animals given vit E + PERP

1 inra-urh, research centre of clermont fd/theix, france,

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