Nutritional Control of the Fatty Acid Profile. Mike Dugan, Cletos Mapiye, Tyler Turner, Dave Rolland and Óscar López-Campos. Cattle are really wonders of nature. They live symbiotically with rumen microbes as pre-gastric fermenters.
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Nutritional Control of the Fatty Acid Profile Mike Dugan, Cletos Mapiye, Tyler Turner, Dave Rolland and Óscar López-Campos
Cattle are really wonders of nature. They live symbiotically with rumen microbes as pre-gastric fermenters.
If man could do the same we could eat barley to produce beer and never be hungry or thirsty again….
Rumen symbiosis Cellulose Volatile fatty acids Microbial PTN & ess. amino acids Low quality PTN & NPN (eg urea) PUFA Biohydrogenation intermediates?? Saturated fatty acids?? Polyunsaturated fatty acids (PUFA) Rumen outflow Diet
Biohydrog Hydrolysis SOAP Rumen bacteria Dietary PUFA Saturated Fat Firm Tallow PUFA Biohydrogenation = Detoxification
Ca+2 • There has been a major emphasis on increasing PUFA, mainly omega-3’s 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 there’s room for more effective methods. • Feeding ground flaxseed can increase omega-3’s (LaBrune et al. 2008, Nassu et al. 2011), and some physical processing can partly protect PUFAs (Maddock et al. 2008) but….
Levels of omega-3’s 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
CLA Omega-3 Vaccenic • In pigs and chickens biohydrogenation is very limited and omega-3 enrichements are more easily attained. • So in beef it’s likely wiser to focus on the complete fatty acid profile…omega-3’s and “good” biohydrogenation products…not found in pork or chicken.
3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 12 12 13 13 14 14 15 15 17 17 1 1 2 2 11 11 16 16 18 18 delta delta omega omega Linolenic acid (c9,c12,c15-18:3) Hydrogen Carbon Oxygen The major PUFA in feed include: Linoleic acid (c9,c12-18:2)
Biohydrogenation Step1: Isomerization 12 3 4 5 6 7 8 9 13 14 15 17 1 2 16 18 11 10 delta omega trans cis cis Changes linoleic acid to c9,t11-18:2 the main natural isomer of conjugated linoleic acid (CLA)
Biohydrogenation Step 2: Saturation 11 3 4 5 6 7 8 9 13 14 15 17 1 2 16 18 12 10 delta omega Rumenic Acid = c9,t11-CLA Vaccenic Acid = t11-18:1 Step 3: Complete saturation of double bonds with hydrogen to Stearic Acid
Animals can add some double bonds back to fatty acids 3 4 5 6 7 8 9 13 14 15 17 1 2 16 18 11 10 12 delta omega Vaccenic Acid = t11-18:1 Rumenic Acid = c9,t11-18:2 • Most CLA in beef is produced via desaturation of vaccenic acid (t11-18:1)
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.
3 4 5 6 7 8 9 10 12 13 14 15 17 1 2 11 16 18 delta omega Linoleic acid (c9,c12-18:2) • Under extensive conditions, the double at C12 is isomerized to C11 • Under intensive conditions, the double bond at C9 is isomerized to C10
Major Linoleic Biohydrogenation Products Extensive c9,c12-18:2 c9,c12-18:2 c9,t11-CLA c9,t11-CLA t11-18:1 t11-18:1 18:0 18:0 Tissue Rumen
Major Linoleic Biohydrogenation Products Intensive c9,c12-18:2 c9,c12-18:2 t10,c12-CLA t10,c12-CLA t10-18:1 t10-18:1 18:0 18:0 Tissue Rumen
3 4 5 6 7 8 9 10 12 13 14 15 17 1 2 11 16 18 delta omega Linolenic acid (c9,c12,c15-18:3) • Under extensive conditions, the double at C12 is isomerized to C11 • Under intensive conditions, the double bond at C12 is isomerized to C13
c9,t11 t11,c13 t11,c15 Major Linolenic Biohydrogenation Products Extensive c9,c12,c15-18:3 c9,c12,c15-18:3 c9,t11,c15 c9,t11,c15 c9,t11 t11,c13 t11,c15 t11-18:1 t11-18:1 c13-18:1 c13-18:1 c15-18:1 c15-18:1 18:0 18:0 Tissue Rumen
Major Linolenic Biohydrogenation Products Intensive c9,c12,c15-18:3 c9,c12,c15-18:3 c9,t11,c15-18:3 c9,t13,c15 c9,t13 t13,c15 c9,t13 t13,c15 ?? t13-18:1 t13-18:1 c15-18:1 c15-18:1 18:0 18:0 Tissue Rumen
Group A Group B PUFA→CLA→Trans Trans→Saturate 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.
Cattle Diets • Typically cattle diet ingredients contain only 3-4 major fatty acids and their GC chromatograms look like this:
Cattle Diets More in grains & oilseeds like Sunflower mVolts Linoleic Palmitic 18:2n-6 30 16:0 25 Oleic c9-18:1 20 More in forages & oilseeds like Flax 15 Linolenic 10 18:3n-3 c15-18:1/C19:0 UNKN-Peak 68.621 5 C18:0 c11-18:1 C14:0 C22:0 C24:0 C20:0 0 -3 20 30 40 50 60 Minutes
Typically in Beef fat – 3 to 4 major fatty acids mVolts Palmitic C16:0 Oleic 15 c9-18:1 Stearic 10 C18:0 C23:0 Linoleic Linolenic 5 C18:2n-6 C18:3n-3 0 -2 20 30 40 50 60 Minutes
c9-18:1 t11-18:1 C18:0 mVolts 1.50 1.25 1.00 t13-t14-/c6-c8-18:1 t7c9-/c9t11-/t8c10-18:2 t11c15-18:2 c9t13-/t8c12-18:2/UNKN-CIS 0.75 c11-18:1 c15-18:1/C19:0 C18:2n-6 0.50 C18:3n-3 UNKN-DI/CIS-Peak 35.917 t10-18:1 t16/c14-18:1 UNKN-DIENE-Peak 48.583 t12-18:1 UNKN-DI-Peak 37.593 t8c13-18:2 c12-18:1 UNKN-DI-Peak 47.224 c13-18:1 11-cyclohexyl-17:0 UNKN-CIS-Peak 46.367 0.25 t6-t8-18:1 t9-18:1 UNKN-DI-Peak 47.353 UNKN-DI-Peak 46.222 C12c15-18:2 c9c11-/t11c13-CLA c9c15-18:2 t7t9-t10t12-CLAall c11-20:1 t9c12-18:2 c9c11-CLA21:0 c9-20:1 C20:0 t4-18:1 c11c13-CLA c10t12-CLA t11t13-CLA c10c12-CLA c12t14-CLA c11t13-CLA t10c12-CLA t9c11-CLA t12t14-CLA C20:3n-9? C20:3n-6 C20:2n-6 0.00 C22:0 -0.25 -0.32 32.5 35.0 37.5 40.0 42.5 45.0 47.5 Minutes But when you zoom in > 80 fatty acids Trans-18:1 CLA’s Other BH
Which FAs to increase or decrease? • FA to increase: • The omega-3’s particularly long chain’s 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-3’s can’t be enriched that much in cattle, but a small increase was enough to allow for a novel food claim for milk!
Which FA to increase or decrease? • Also want increases in biohyhdrogenation products like rumenic acid (c9,t11-CLA) and vaccenic acid (t11-18:1). • CLA was shown in the late 1970’s by Mike Pariza’s 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 it’s own has been shown to reduce blood triglycerides in lab animals (Wang et al. 2008).
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.
Isn’t 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,t11-18:2 = rumenic acid). • Current emphasis is now on increasing both rumenic acid and vaccenic acid. • Earlier research, however, didn’t produce big net increases in CLA.
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?
Hideo Kojima “90% of what is considered impossible is, in fact, possible.” “The other 10% will become possible with the passage of time & technology.“
For example: Detailed analysis of biohydrogenation products is challenging. • Many products are closely related, and only differ in double bond position and don’t 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.
My ruminant research… • Started with wild Muskox. • They’re 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.
0.50 ** 0.45 Beef 0.40 Muskox 0.35 0.30 % of fatty acid methyl esters. 0.25 0.20 0.15 ** 0.10 ** 0.05 ** 0.00 t7,c9 c9,t11 t9,c11 t11,c13 Conjugated linoleic acid isomers • We compared it to beef finished on barley (85% DM) at the Lacombe Research Centre.
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-18:1 isomers
Partially hydrogenated vegetable oil Ruminant fats 70 60 50 40 % of total trans 18:1 isomers 30 20 10 0 t4/t5- t6,7,8- t9- t10- t11- t12- t13,14- t15- t16- Wolff et al 2000
Beef 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 18:1 isomers Dugan et al. 2007
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
70 Canadian Retail Survey backfat striploin muscle 60 50 40 % of total trans 18:1 isomers 30 20 10 0 t4/t5- t6,7,8- t9- t10- t11- t12- t13,14- t15- t16- Aldai et al. 2009
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).
70 60 50 40 % of total trans 18:1 isomers 30 20 10 0 t4/t5- t6,7,8- t9- t10- t11- t12- t13,14- t15- t16- Leheska et al. 2008 Striploin from: concentrate grass-fed
Reviewing the literature, our results were not unique as elevated t10-18:1 in beef had been found in the USA 20 year earlier (Wood 1983). • And dairy researchers were also aware of shifts toward t10-18:1 because feeding PUFA in high grain diets results in milk fat depression (Griinari et al. 1998).
Feeding barley at high levels creates a rumen environment supporting bacterial species biohydrogenating linoleic acid to t10-18:1 instead of t11-18:1 (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.
13% C18:1-9c 20 15 C18:1-10t C18:0 10 C18:1-13,14t C18:1-11t C18:1-12c C18:1-6-8t 5 C18:1-13c C18:1-9t C18:1-12t C18:1-5t C18:1-4t 0 -1 34 35 36 37 Minutes • 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 t10-18:1 to 18:0 (Troegleler-Meynadier et al. 2006)
Who cares about trans? • Foods with more than 0.2 g of trans fatty acids per serving are required to be labelled…except for foods of ruminant origin. • This is based on the assumption that ruminant trans fats are mostly vaccenic (t11-18:1) 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.
Who cares about trans? • On the plus side…our 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 hamburger…likely because cull cows are used in production(?).
Who cares about trans? • Problem: regulations only take into consideration total trans and don’t 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.
Controlling trans 10-18:1 • 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 10-18:1 in feedlot beef. • Added buffer (1.5% sodium sesquicarbonate) in a high barley diet improved the trans 11 to trans 10-18:1 ratio in beef, but the effect was lost over time (Aldai et al. 2010c).