Nutritional Control of the Fatty Acid Profile

1
Nutritional Control of the Fatty Acid Profile

• Mike Dugan, Cletos Mapiye, Tyler Turner,
• Dave Rolland and Óscar López-Campos

2

• Cattle are really wonders of nature. They live
  symbiotically with rumen microbes as pre-gastric
  fermenters.

3

• If man could do the same we could eat barley to
  produce beer and never be hungry or thirsty
  again.

4
Rumen symbiosis
Rumen outflow
Diet
5
PUFA Biohydrogenation Detoxification
6

• There has been a major emphasis on increasing
  PUFA, mainly omega-3s in animal products
• Protecting PUFA has been attempted by
• Making Ca salts or fatty acid amides
• Encapsulating in protein and treating with
  formaldehyde is a bit more efficient
• But all treatments add expense and theres room
  for more effective methods.
• Feeding ground flaxseed can increase omega-3s
  (LaBrune et al. 2008, Nassu et al. 2011), and
  some physical processing can partly protect PUFAs
  (Maddock et al. 2008) but.

7

• Levels of omega-3s in steak have not been enough
  to reach source claim requirements in Canada (300
  mg per serving)
• But regular ground beef can reach this level when
  feeding flax

8

• In pigs and chickens biohydrogenation is very
  limited and omega-3 enrichements are more easily
  attained.
• So in beef its likely wiser to focus on the
  complete fatty acid profileomega-3s and good
  biohydrogenation productsnot found in pork or
  chicken.

9
The major PUFA in feed include

• Linoleic acid (c9,c12-182)

10
Biohydrogenation
Step1 Isomerization
12
1
2
3
4
5
6
7
8
9
14
15
16
17
18
13
10
11
delta
omega
cis
trans
cis
Changes linoleic acid to c9,t11-182 the main
natural isomer of conjugated linoleic acid (CLA)
11
Biohydrogenation
Step 2 Saturation
1
2
3
4
5
6
7
8
14
15
16
17
18
11
13
9
12
10
delta
omega
Vaccenic Acid t11-181
Rumenic Acid c9,t11-CLA
Step 3 Complete saturation of double bonds with
hydrogen to Stearic Acid
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• Animals can add some double bonds back to fatty
  acids

1
2
3
4
5
6
7
8
14
15
16
17
18
13
9
11
10
12
delta
omega
Vaccenic Acid t11-181
Rumenic Acid c9,t11-182

• Most CLA in beef is produced via desaturation of
  vaccenic acid (t11-181)

13
Biohydrogenation

• The type and level of biohydrogenation
  intermediates passing from the rumen is greatly
  influenced by the diet.
• In general, the intermediates are influenced by
  how intensive (concentrate based) or extensive
  (forage based) the production system.
• It can also be modified by buffers, antibiotics,
  vitamin E, tannins etc.

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Linoleic acid (c9,c12-182)

• Under extensive conditions, the double at C12 is
  isomerized to C11
• Under intensive conditions, the double bond at C9
  is isomerized to C10

15
Major Linoleic Biohydrogenation Products
Extensive
c9,c12-182
c9,c12-182
c9,t11-CLA
c9,t11-CLA
t11-181
t11-181
180
180
Tissue
Rumen
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Major Linoleic Biohydrogenation Products
Intensive
c9,c12-182
c9,c12-182
t10,c12-CLA
t10,c12-CLA
t10-181
t10-181
180
180
Tissue
Rumen
17

• Under extensive conditions, the double at C12 is
  isomerized to C11
• Under intensive conditions, the double bond at
  C12 is isomerized to C13

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Major Linolenic Biohydrogenation Products
Extensive
c9,c12,c15-183
c9,c12,c15-183
c9,t11,c15
c9,t11,c15
c9,t11 t11,c13 t11,c15
t11-181
t11-181
c13-181
c13-181
c15-181
c15-181
180
180
Tissue
Rumen
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Major Linolenic Biohydrogenation Products
Intensive
c9,c12,c15-183
c9,c12,c15-183
c9,t11,c15-183
c9,t13,c15
c9,t13 t13,c15

c9,t13 t13,c15
??
t13-181
t13-181
c15-181
c15-181
180
180
Tissue
Rumen
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Complicated?
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Biohydrogenation

• Typically, bacteria that convert PUFA to CLA and
  trans fatty acids (Group A) are different from
  species that convert trans to saturated fatty
  acids (Group B).
• Finding ways to control these groups is important
  in producing high levels of the good
  biohydrogenation intermediates.

22
Cattle Diets

• Typically cattle diet ingredients contain only
  3-4 major fatty acids and their GC chromatograms
  look like this

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Cattle Diets
More in grains oilseeds like Sunflower
mVolts
Linoleic
Palmitic
182n-6
30
160
25
Oleic
c9-181
20
More in forages oilseeds like Flax
15
Linolenic
10
183n-3
c15-181/C190
UNKN-Peak 68.621
5
C180
c11-181
C140
C220
C240
C200
0
-3
20
30
40
50
60
Minutes
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Typically in Beef fat 3 to 4 major fatty acids
mVolts
Palmitic
C160
Oleic
15
c9-181
Stearic
10
C180
C230
Linoleic
Linolenic
5
C182n-6
C183n-3
0
-2
20
30
40
50
60
Minutes
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But when you zoom in gt 80 fatty acids
Trans-181
CLAs
Other BH
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Which FAs to increase or decrease?

• FA to increase
• The omega-3s particularly long chains like EPA,
  DPA and DHA for their heart health,
  anti-inflamatory and other effects.
• EPA and DHA are highly concentrated in cold water
  fish, and DPA is found to a greater extent in
  beef
• Typically LC-omega-3s cant be enriched that
  much in cattle, but a small increase was enough
  to allow for a novel food claim for milk!

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Which FA to increase or decrease?

• Also want increases in biohyhdrogenation products
  like rumenic acid (c9,t11-CLA) and vaccenic acid
  (t11-181).
• CLA was shown in the late 1970s by Mike Parizas
  group in Wisconsin to have anti-cancer
  properties, and additional healthful properties
  have been demonstrated using animal models (ex.
  for asthma, inflammatory diseases etc).
• Vaccenic acid is the precursor for rumenic acid,
  and on its own has been shown to reduce blood
  triglycerides in lab animals (Wang et al. 2008).

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Which FA to increase or decrease?

• FA to decrease
• Saturated fatty acids with less than 18 carbons
  trans fatty acids other than vaccenic acid as
  they are associated with unhealthy changes in
  blood cholesterol levels .
• Gray area
• Work is currently underway to look at effects of
  other BH products, which can accumulate
  particularly when feeding linolenic acid.

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Isnt this old news?

• The fact that CLA is found in beef has been known
  for some time.
• Original interest was on trying to increase the
  main natural isomer of CLA (c9,t11-182 rumenic
  acid).
• Current emphasis is now on increasing both
  rumenic acid and vaccenic acid.
• Earlier research, however, didnt produce big net
  increases in CLA.

30
Diet Country CLA concentration ( of fat)
Unknown Canada 0.10.3
Barley (800 g/kg diet) Canada 0.170.18
Grass silage and concentrate UK 0.320.8
Maize (820 g/kg diet) United States 0.390.49
Unknown United States 0.290.43
Unknown United States 0.170.55
Grain United States 0.51
Concentrate Japan 0.34
Grass United States 0.74
Grass (?) Australia 0.231.25
Grass Ireland 0.371.08
Grass and sunflower oil Ireland 1.76
Unknown Germany 0.121.2
Corn extruded soybeans United States 0.660.78
Range United States 0.350.56
Feedlot United States 0.290.32
Feedlot soybeans United States 0.320.36
Maloney et al. 2008
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Why bother?

• And if you have a 6 oz steak, 4 fat, 1 CLA in
  the fat, your steak contains 68 mg of CLA or
  0.068 g of CLA.
• This is 40-50x less than what Ip et al. (1994)
  estimated would be required to show an effect on
  breast cancer.
• So why should we keep going up this road?

32
Hideo Kojima
90 of what is considered impossible is, in
fact, possible.
The other 10 will become possible with the
passage of time technology.
33

• For example Detailed analysis of
  biohydrogenation products is challenging.
• Many products are closely related, and only
  differ in double bond position and dont separate
  well using older methods.
• They required longer and more polar GC columns
  (100 m) and the use of Ag-HPLC, and some
  expertise to figure out which peaks are which.

34
From Precht and Molkentin 1995
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My ruminant research

• Started with wild Muskox.
• Theyre harvested for their fine under-wool
  called qiviut.
• Muskox were harvested in the NWT, and we worked
  on meat toughness problems related to fast
  chilling.
• Animals grazed on the tundra of Banks Island, and
  meat was well marbled.

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• We compared it to beef finished on barley (85
  DM) at the Lacombe Research Centre.

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Muskox vs Beef

• We also looked at the trans fatty acid profile.
• For humans, a big source of trans fats has been
  from partially hydrogenated vegetable oil in the
  form of margarine.
• These can have a broad spectrum of trans-181
  isomers

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70
60
50
40
of total trans 181 isomers
30
20
10
0
t4/t5- t6,7,8- t9- t10-
t11- t12- t13,14- t15- t16-
Wolff et al 2000
39
2.50
2.00
Muskox
1.50
of fatty acid methyl esters.
1.00
0.50
0.00
t6-t8-
t9-
t10-
t11-
t12-
t13/t14-
t15-
t16-
trans
181 isomers
Dugan et al. 2007
40
Muskox vs Beef

• We were a little surprised at the results.
• And we wanted to see what the composition of beef
  fat would be at retail

41
70
Canadian Retail Survey backfat
striploin muscle
60
50
40
of total trans 181 isomers
30
20
10
0
t4/t5- t6,7,8- t9- t10-
t11- t12- t13,14- t15- t16-
Aldai et al. 2009
42
Muskox vs Beef

• We were a little surprized at the results.
• And we wanted to see what the composition of beef
  fat would be at retail and in the packing plant.
• Our results were similar a USA survey comparing
  grain and forage finished animals (Leheska et
  al., 2008).

43
Striploin from concentrate grass-fed
44

• Reviewing the literature, our results were not
  unique as elevated t10-181 in beef had been
  found in the USA 20 year earlier (Wood 1983).
• And dairy researchers were also aware of shifts
  toward t10-181 because feeding PUFA in high
  grain diets results in milk fat depression
  (Griinari et al. 1998).

45

• Feeding barley at high levels creates a rumen
  environment supporting bacterial species
  biohydrogenating linoleic acid to t10-181
  instead of t11-181 (vaccenic acid).
• This relates to the high content of rapidly
  fermentable starch and in part to lower rumen pH.
• And with low levels of vaccenic acid in beef, CLA
  levels have also been low.

46

• Further problems arise if you add a source of
  linoleic acid (ex. sunflower seed or soybean
  oil) to a high barley diet
• Because low pH can inhibit the last step in
  biohydrogenation from t10-181 to 180
  (Troegleler-Meynadier et al. 2006)

47
Who cares about trans?

• Foods with more than 0.2 g of trans fatty acids
  per serving are required to be labelledexcept
  for foods of ruminant origin.
• This is based on the assumption that ruminant
  trans fats are mostly vaccenic (t11-181) and
  rumenic acids (c9,t11-CLA).
• Regulations for trans labelling are, however,
  currently being reviewed in the USA and Canada
  will likely be following suit.

48
Who cares about trans?

• On the plus sideour survey indicated only 10 of
  striploin steaks would require a label (Aldai el
  al. 2009).
• But higher fat products would exceed levels
  needed for labelling.
• However, our survey showed vaccenic acid was the
  most concentrated trans fatty acid in
  hamburgerlikely because cull cows are used in
  production(?).

49
Who cares about trans?

• Problem regulations only take into consideration
  total trans and dont differentiate between
  good and bad trans fatty acids.
• Irrespective of labelling issues, from a
  marketing and trade perspective, having beef with
  a healthy trans fatty profile should certainly
  be a priority.

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Controlling trans 10-181

• It would be impractical to forage finish all the
  beef produced in Alberta, and we wanted to see
  what could be done to control trans 10-181 in
  feedlot beef.
• Added buffer (1.5 sodium sesquicarbonate) in a
  high barley diet improved the trans 11 to trans
  10-181 ratio in beef, but the effect was lost
  over time (Aldai et al. 2010c).

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Controlling trans 10-181

• We also substituted wheat dried distillers grains
  plus solubles (DDGS) for barley.
• This reduced dietary starch and increased
  protein, fibre and oil which could all be helpful
  in producing more trans 11- than trans 10-181

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Controlling trans 10-181

• Adding vitamin E to the diet can also have a
  positive effect.
• We fed either 1051 vs 451 IU of vitamin E in a
  barley /hay (72/22) diet for 90 days and found

54
Controlling trans 10-181
Juarez et al. (2009)
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Matrix interactions are also important

• Duckett et al. (2002) fed a conventional corn
  diet (80 grain in DM), a high oil corn diet or a
  conventional corn plus oil up to the level of the
  high oil corn diet.

• They measured fatty acids flowing from the rumen.
  

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20
Conventional Corn
18
16
High Oil Corn
14
Conventional Corn Corn Oil
12
10
grams flowing from rumen per day..
8
6
4
2
0
t9-181
t10-181
t11-181
t12-181
trans-181 isomers
57
More research is required

• To explain and control large between animal
  variation that can occur when feeding the same
  diet.
• To expand studies on grain types, processing and
  matrix interactions.
• To study additional biohydrogenation modifiers
  and the use of combined strategies for reducing
  trans 10-181.

58
Increasing Vaccenic and Rumenic Acids

• Beyond decreasing trans 10-181, theres still
  the challenge to increase vaccenic and rumenic
  acids to meaningful levels.
• Keys are to
• create a rumen environment favoring the right
  group A bacteria
• supplying a source of PUFA for biohydrogenation
• preventing complete biohydrogenation to 180 by
  group B bacteria.
• Creating a good rumen environment can be done by
  increasing the right type of forage in the diet.

59
Increasing Vaccenic and Rumenic Acids

• We fed 15 flax to cull cows in a 5050 forage
  concentrate diet (DM basis), with either grass
  hay or barley silage as forage sources for 140
  days (Nassu et al. 2011).

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Over time in Backfat (He et al. submitted)
Hay
Silage
Silage Flax
Hay Flax
2.5
c
2.0
1.5
b
VA,
b
ab
1.0
b
b
b
c
a
a
a
0.5
a
b
a
a
a
0.0
1.6
c
b
1.2
CLA,
c
a
0.8
c
b
b
c
b
a
b
a
0.4
a
b
a
a
0.0
0 wk (initial)
6 wk
12 wk
20 wk (end)
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Increasing Vaccenic and Rumenic Acids

• We also found bumping up the forage to 70 hay
  plus 15 flax (substituted for barley) for 250
  days netted further improvements in heifers
  (Block et al. in preparation)

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Increasing Vaccenic and Rumenic Acids

• As mentioned there can be quite a bit of between
  animal variation. In the Block study, one animal
  had 12 vaccenic acid!!
• To compensate
• One strategy we can use is to analyze blood early
  in the feeding period and remove animals that
  wont produce high levels of vaccenic and rumenic
  acids.
• Were also working on an NIR method for analysing
  fat depots for sorting purposes (Prieto et al.
  2011).

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Increasing Vaccenic and Rumenic Acids

• Another possibility is mining different fat
  depots, as some have more vaccenic and others
  more rumenic acid.
• Kidney fat example from a trial underway..

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Meaningful levels

• We believe we are getting to levels of
  biohydrogenation products which may have some
  health effects and will be working with Dr.
  Spencer Proctor at the U of A to test these.
• Keys to getting these levels are
• The right type and level of forage (gt50 hay?).
• A source of PUFA for biohydrogenation.
• A high enough level of PUFA which may help
  inhibit complete biohydrogenation to 180 (maybe
  linolenic better than linoleic?)
• A longer feeding period/slower growth rate which
  could mean less fat synthesis from VFAs and less
  dilution of vaccenic and rumenic acids.
• There are still possibilities for boosting or
  stabilizing vaccenic and rumenic acid by feeding
  vitamin E, fish oils and other biohydrogenation
  modifiers.

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• Nutrition can control beef fatty acid profiles
• But the degree of control is based on a complex
  interaction.

66

• And understanding the interaction will put us on
  the road to the impossible 10

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Acknowledgements

• Dr. Noelia Aldai a PDF in my lab for 3 years who
  worked on many of these projects.
• For studies conducted at Lacombe, financial
  support from AAFC Peer Review Program, the Beef
  Cattle Research Council and the Alberta Livestock
  and Meat Agency.

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Total trans-181, 10t-181 and 11t-181 in the
fat of retail sirloin steaks (Canada USA
combined)
Relative total beef lipids
Beef samples