BCFA
BCFA
BCFA
Author manuscript
Br J Nutr. Author manuscript; available in PMC 2015 August 28.
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Abstract
Branched chain fatty acids (BCFA) are bioactive food components that constitute about 2% of
fatty acids in cow’s milk fat. Little systematic information on the BCFA content of other foods is
available to estimate dietary intakes. We report BCFA distribution and content of fresh and
processed foods representing the major foods of Americans and estimate BCFA intake. BCFA are
primarily components of dairy and ruminant foods, and were absent from chicken, pork, and
salmon. Dairy and beef delivered most of the 500 mg per day mean intake; in comparison, intake
of the widely studied long chain polyunsaturates eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA) is estimated to average 100 mg per day. Common adjustments in
diet can double BCFA daily intake. The fermented foods sauerkraut and miso had appreciable
fractions of BCFA but overall are low fat foods providing very small amounts in the diet, and
other fermented foods did not contain BCFA as might have been expected from microbial
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exposure. These data support the quantitative importance of BCFA delivered primarily from dairy
and beef and highlight the need for research into their health effects.
Introduction
Branched chain fatty acids (BCFA) are primarily saturated fatty acids (FA) with a methyl
branch or more on the carbon chain. BCFA are categorized as mono-, di-, or multi-methyl
BCFA. In monomethyl BCFA, the predominant branching is near the terminal end of the
carbon chain. FA terminating with an isopropyl or isobutyl group are referred to as iso-or
anteiso- BCFA, respectively (Figure 1). BCFA modulate the biophysical properties of
membranes in a manner similar to that of cis double bonds: both interfere with the ability of
saturated FA to pack tightly to form rigid, high melting point extended structures, and thus
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BCFA are major components of bacterial membranes across many genera and species. They
are particularly prominent in Bacilli, constituting 95% of the FA in many species of Bacilli
and Lactobacilli1. In some Bifidobacterium strains a single BCFA can be as high as
24%wt 2. In bacteria that exhibit a large range of BCFA concentrations, they may influence
phenotype in potentially important ways. For instance, when present in the environment they
are readily taken up by the pathogen Pseudomonas aeruginosa and dramatically reduce
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BCFA are rare in internal human tissues, but they are present in high concentrations in skin
and in vernix caseosa, the unique waxy white substance coating the skin of term newborns,
where their concentrations are about 29%wt 4, including BCFA with branches in positions
other than the iso and anteiso positions, and also including dimethyl BCFA.
We reported previously 4 that BCFA are constituents of the healthy term newborn infant’s
gut and that the human alimentary canal selectively metabolizes BCFA, suggesting that
BCFA play specific role in the gut. Consistent with this hypothesis, our recent neonatal rat
study showed that substitution of 20%wt of fat as BCFA altered the gastrointestinal (GI)
microbial ecology toward organisms that use BCFA and reduced the incidence of
necrotizing enterocolitis5. Others showed BCFA to induce apoptosis in human breast cancer
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cells, and to inhibit tumor growth in cultured cells and in a mouse model 6;7. These data all
point to previously neglected nutritional properties of BCFA that may be important for
development and maintenance of microbiota, enterocyte health, skin, and possibly other
functions.
Information on BCFA intake in the human diet is scant. We recently reported the profile and
concentrations of BCFA in a representative sampling of retail milk in the United States.
Using these measurements with reports of others for beef BCFA, we estimated BCFA intake
from cheese and beef products in the American diet to be about 400 mg/day. However, these
calculations were based on retail milk and did not take into account changes in BCFA
profiles or increase or loss in total BCFA, due to, for instance, fermentation or processing 8.
Here we present BCFA analysis in foods prominently featured in the American diet, and
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from our measurements estimate the nutritional contribution of BCFA from these various
foods in the American diet using USDA Economic Research Service intake data.
Experimental Procedures
Sampling
Food samples were purchased from local supermarkets in Ithaca, New York, USA. Because
previous reports of BCFA content focus on dairy and meats, we chose a cross section from
these groups and additionally added fermented foods. Particular foods were chosen based on
consideration of the prevalence of consumption within respective food groups according to
USDA Economic Research Service, Loss-Adjusted Food Availability data. 9
The following foods from the dairy food group were analyzed: Whole milk cheese (Bovine-
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milk: cheddar, low-moisture mozzarella, provolone, Swiss, cottage, ricotta, cream; two
samples of ovine (sheep) milk-derived cheese (blue, romano); one sample of goat-derived),
plain and Greek yogurt, and ice cream. We also analyzed dairy-based foods from the added
fat food group such as sour cream, light cream and butter. However, these foods were
merged with the dairy food group to simplify the analysis.
Foods were chosen for the prevalence of consumption in the dairy and protein food groups
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Fermented food products such as sauerkraut and miso (soy), tofu, kimchi, and tempeh were
included in the analysis because the addition of bacterial/fungal during the fermentation
process of these products may influence the presence of BCFA. Chocolate bars were also
chosen to be included in the analysis as a fat and dairy containing snack. Samples with high
content of moisture such as yogurt were placed in a centrifugal evaporator (Savant
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SpeedVac, Thermo Fisher), for at least 24 hours before analysis. Samples were stored in
−80°C until processed.
FAME identities were determined by electron ionization (EI) mass spectrometry (MS),
chemical ionization, and electron ionization tandem mass spectrometry (EI-MS/MS) as
described previously 14 using a Varian Star 3400 GC coupled to a Varian Saturn 2000 ion
trap MS, with identities verified by GC retention times. For our protocols with GC-FID
analysis we estimate the limit of quantification is about 0.15 ng FAME for a 1 μL injection.
Here we prepared approximately 6 μg FAME mixture per 1 μL injection. These
concentrations lead to limit of quantification for individual FAME of about 0.003%w/w of
total FAME.
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fat food category accounted for 10 of 14g (sour cream, cream cheese, and light cream). We
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present results for these dairy-based added fat foods together with the dairy food group.
Table 1 provides detailed information on BCFA distribution in dairy (Table 1a), protein
(Table 1b), and in fermented food products (Table 1c). BCFA were detected in dairy and
beef products. Small amounts of BCFA were also detected in canned tuna and in the
fermented foods miso and sauerkraut. BCFA were below detection limits in the following
foods: Ground turkey, ham, bacon, pork sausages, chicken thighs, chicken breasts, eggs,
almonds, tofu, tempeh, fresh salmon.
The total BCFA concentrations of BCFA in the various dairy products in our study ranged
between 1.37%wt for light cream cheese and 2.73%wt for sheep cheese (Table 1a). Sheep
and goat milk have long been known for their higher BCFA concentrations, especially when
fed a barley-rich diet 15. The relatively higher levels of BCFA in sheep and goat cheeses
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show that BCFA concentration in dairy products is influenced by which ruminant species
milk is used. In addition, differences in cheese composition can be affected by many factors,
such as the breed of cow, feeding practice, and the microbial activity in milk and cheese,
especially during ripening 16. Lower concentrations of BCFA in the original milk used in the
production of these cow’s milk-based products is the most likely explanation for differences
in BCFA among these dairy products, particularly for low-moisture mozzarella, where the
BCFA concentrations are lower compared to provolone (1.4%wt vs 1.9%wt), despite
sharing a similar production process.
Figure 2 shows BCFA distribution in retail cow’s milk 8, and in the dairy products as a
group which derive the bulk of their BCFA from cow’s milk. Similar to fluid milk 8, BCFA
in dairy products included BCFA from both iso- and anteiso-type and contained BCFA with
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chain length from 14 to 17 carbons, with very few iso-18:0. In addition, the odd chain,
anteiso-BCFA comprise at least half of the total BCFA in the cow’s milk-based dairy
products. Our BCFA profile is in line with BCFA distribution published previously for
European dairy products 17; 18.
Because introduction of bacteria is an integral part of the production of most dairy products
and because bacteria can be a source of BCFA -- in addition to the inherent presence of
BCFA in fluid milk -- we speculated that the total BCFA content and distribution may be
different between fluid milk and dairy products, with a tendency/trend to be higher in the
dairy products. Surprisingly, the collective mean levels of total BCFA, anteiso-17:0, sum of
BCFA longer than 16 carbons, and the sum of anteiso-BCFA in cow’s milk-based dairy
products were significantly lower than the mean BCFA levels of these aggregated
parameters in retail milk. These differences are driven by the higher levels of anteiso-17:0 in
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retail milk compared to the collective mean of cow’s milk-based dairy product. The cows’
diet can strongly affect the levels of FA and BCFA in milk 16, and lower levels of BCFA in
the milk-based dairy products observed in our study implies that bacterial BCFA may not
have the same prominent effect on BCFA levels in dairy products as diet and production
processes. Furthermore, it is possible that the milk BCFA used for some of the dairy
products contained somewhat lower BCFA levels than average retail milk, masking possible
contributions of bacterial BCFA to the final product. Alternatively, a lack of BCFA in the
bacterial cultures introduced in the production of dairy foods could explain why BCFA
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concentrations in cow’s milk-based dairy products did not increase compared to fluid milk.
A literature search for the FA composition in known dairy cultures, such as Streptococcus
thermophilus and Lactobacillus bulgaricus, did not yield reports on BCFA concentrations in
these strains 2; 19; 20; 21 with the exception of one report on the presence of 0.7%wt
anteiso-15:0 in Lactobacillus bulgaricus. 2. However, the bacterial culture in making
cheese, for example, is important for the texture and flavor of different cheeses 22, and thus
it is reasonable to assume that different dairy product have different mixtures of bacterial
cultures. These cultures can have both BCFA-containing bacteria strains along with non-
BCFA-containing bacteria strains, and the proportions between them may affect the
bacterial contribution of BCFA to the different dairy products.
In addition to dairy products, we also analyzed the most highly consumed foods from the
protein food group according to the USDA Economic Research Service, Loss-Adjusted
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Food Availability data.9 Table 1b shows the BCFA concentrations in foods from the protein
group. Commercial and pastured ground beef had total BCFA concentrations of 1.60%wt,
and 1.89%wt; In a recent survey of retail beef in Canada 23 BCFA concentrations ranged
between 1.25–1.82%wt depending on the season and the cut, consistent with our results.
Similar to cow’s milk, BCFA concentrations in beef can be affected by the cattle’s diet, by
the breed 24; 25, and by the cut of meat within a given breed 23. Finally, we note that
production practice such as organic vs conventional dairy products, and wild vs farmed
seafood, may all have an influence on BCFA.
BCFA concentrations were below detection limits in poultry and pork products, as would be
expected if the primary origin of BCFA in meats is from rumen bacteria. These observations
are in line with a previous report on FA concentrations in lard, chicken fat, mutton tallow,
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and beef tallow, using a GC×GC-Time of Flight mass spectrometer 26. A wide variety of
BCFA were detected in beef and mutton tallow but not in chicken fat, and a small amount of
anteiso-17:0 was reported in lard.
Previous reports indicate the presence of small amounts of BCFA in fish 18. Because the
current 2010 Dietary Guidelines for Americans recommend consumption of two servings of
4oz each of seafood per week, we investigated the presence of BCFA in canned tuna and
salmon, which, among Americans, are the second- and third-most consumed types of
seafood (after shrimp), according to National Oceanic and Atmospheric Administration
statistics 27. In addition, these fish are considered to be cold-water fish.
Because BCFA modulate membrane properties at low temperatures 1; 28, we speculated that
BCFA may be in their membranes. Our data show BCFA levels in salmon were below
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detection limits, and very small amounts of iso-17:0 (0.14%wt) were detected in canned
tuna. Low concentrations of BCFA in fresh tuna and in salmon were reported by others 18,
and iso-BCFA in canned tuna is in agreement with the same authors’ observation that the
dominant BCFA in fish are odd-numbered-carbon iso-BCFA.
Taken together, the above results indicate that BCFA are found in high concentrations in
ruminant products, but not in poultry and at zero or low amounts in the fish we examined.
Considering that the foods in our study were the most commonly consumed foods in the US
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diet, our data indicate that the main source of BCFA in the dairy and protein food groups
originate from ruminant products. In addition, BCFA in cow’s milk-based dairy products
were similar or lower compared to retail cow’s milk.
BCFA were also detected in fermented products such as sauerkraut and miso (Table 1c).
These products undergo fermentation with various fungi and bacteria, both of which may
serve as the source for BCFA in these products. Miso processing uses the fungus Aspergillus
oryzae, a variety of bacterial strains such as Enterococcus and Pediococcus strains, as well
as Lactobacillus and Weissella strains 29. In the current study, miso contained total BCFA
concentrations of 0.54%wt. These included a small amount of iso-15:0 (0.08%wt); the rest
(0.46%wt, Table 1c) was contributed by a unique BCFA with a putative assignment of 2-
methyl hexadecanoic acid. This rare BCFA was also present at lower concentration in
sauerkraut (0.14%wt, Table 1c). Park et al. reported the presence of Weisella in sauerkraut,
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which may explain the presence of this BCFA in sauerkraut and miso 30. A paper on the FA
distribution in Weissela strains did not report BCFA 31.
BCFA were not detected in other fermented food products, such as kimchi or tempeh,
however. Kimchi, for example, contains many different bacterial genera 32, and thus it is
likely that BCFA present in some strains were diluted to below detection limits by
overwhelming amounts of bacteria with normal FA. In addition, various processing methods
may also influence bacterial ecology in kimchi 32 and the detection of BCFA in other
fermented food products. We did not attempt here to do an exhaustive sampling of
fermented foods.
Small amount of BCFA (0.15%wt) were detected in milk chocolate. The two BCFA
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detected in chocolate were both with 17 carbons, iso-17:0 (0.07%wt), and anteiso-17:0
(0.08%wt) (data not shown). The ratio between iso-17:0 and anteiso-17:0 in chocolate (1:1)
is different from their ratio in milk (1:2 or more). This and the absence of anteiso-15:0, a
predominant BCFA in milk, implies that the milk ingredient in chocolate is not solely
responsible for the presence of BCFA in chocolate, and there may be another source for
these two BCFA in chocolate.
the present study and for which per capita consumption data are greater than 1 g/d are shown
in Table 2. The contribution of the selected foods in Table 2 to the BCFA consumption by
Americans reaches about 492 mg per capita per day. Americans consume about 317 mg
BCFA from dairy products, and about 170mg from beef. Chocolate contributes about 6 mg
BCFA per day. BCFA are 0.6%wt and 1.9%wt from the total daily intake of 76.8g fat and
the 25.5g saturated fat, respectively, for Americans two years and older.
Notable examples of foods excluded from Table 1 are goat cheese, which contains relatively
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large amounts of fat (27%) and BCFA (2.2%wt) but has no per capita consumption
information, miso (per capita consumption is not tabulated), and sauerkraut because its per
capita consumption is very small (<0.4g/day) and fat contribution is negligible.
The estimated per capita intake of BCFA from the dairy and protein sources of 492 mg/d is
greater than the 100 mg average daily consumption of the docosahexaenoic acid (DHA) and
eicosapentaenoic acid (EPA) reported in a survey of 8604 Americans between 1999 and
2000 and by women of child-bearing age, based on NHANES III data 33; 34. The per capita
estimated intake of BCFA is higher than the recommendation for DHA and EPA intake for
pregnant and lactating women 34. Therefore the consumption of BCFA from dairy and
protein food is almost five times the consumption of DHA and EPA in the US (Figure 3). As
a reference, DHA intake of 200 mg per day, and 300 mg combined DHA+EPA, is
recommended for pregnant and lactating women to support infant development by the Food
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For children ages 2–11 years, the mean intake of beef is 43.5 g/d.36 Children who consume
28 g (1 oz) of cow’s milk-based cheese with an average of 25% fat, and 43.5 g/d of cooked
beef with an average of 18% fat, both with average of 1.8%wt BCFA, consume about 267
mg BCFA per day. Addition of 1 cup of whole milk (156 mg BCFA) increases their daily
BCFA intake to 423 mg. For comparison, the mean intake of DHA and arachidonic acid
(ARA) in Canadian children, ages 4–7 years, was recently estimated to be 37 mg and 57mg
per day 37; both combined are almost about one fifth of the daily BCFA consumption from
milk, cheese and beef.
Taken together, the calculations shown above imply that BCFA are being consumed in
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substantial amounts by most non-vegans, during different life stages, and their consumption
exceeds the consumption of bioactive FA.
Our data indicate that BCFA intake can exceed the estimated per capita consumption of 492
mg/d by at least two-fold with common intakes of popular foods. For example, daily
consumption of 150 g (5.3 oz) of cooked ground beef (18% fat, with 1.8%wt BCFA on
average), two cups (473 ml) of whole milk (3.25%fat, 2%wt BCFA), and 57 g (2 oz) of
cow’s milk-based cheese (25% fat, 1.8%wt BCFA on average) contains about 1050 mg
BCFA. If the cows’ milk cheese was replaced with sheep cheese (31% fat and 2.7%wt
BCFA), then the meal BCFA would exceed 1700 mg BCFA, or three fold the average
intake.
The 2010 Dietary Guidelines for Americans 38 recommend that Americans consume low-fat
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ruminant products in order to lower consumption of saturated fat and to reduce risk for
metabolic diseases. Our results indicate that a higher intake of BCFA is due to the
consumption of animal fat and more specifically foods of ruminant origin, with the leading
foods in the American diet being dairy and beef. A recent meta-analysis of prospective
cohort studies failed to find a significant relation between dietary saturated fat intake and
cardiovascular disease (CVD) 39. Others showed 40 that increased consumption of high-fat
dairy products did not significantly increase the odds ratio for obesity, abnormal glucose
homeostasis, dyslipidemia, elevated blood pressure, or insulin resistance. In fact, the odds
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ratios for these disorders were lower with increasing intake of both reduced-fat and high-fat
dairy products, and inverse associations were observed for both reduced-fat and high-fat
dairy products. Another study reported a non-significant trend toward lower risk for first
myocardial infarction (MI) with consumption of high-fat dairy compared to low-fat dairy 41.
Another meta-analysis found no association between animal fat intake and colorectal
cancer 42. Others showed 43 that intake of saturated fat from an animal source was not
significantly associated with risk of colorectal cancer in women; in that study, consumption
of red meat was negatively associated with risk of colorectal cancer. These studies highlight
evolving science on saturated fat intake and disease risks, indicating that the effect of
saturated fat or animal fat consumption on metabolic diseases and cancer is complex.
BCFA are normal constituents in the gut from very early age, and they are present in the gut
throughout the human life cycle. Previous studies show that BCFA are not inert components
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of the GI tract and are metabolized by the enterocytes. BCFA have beneficial role against
inflammation in the premature intestine, alter the microbiota, and increase expression of
anti-inflammatory cytokines in an animal model. These studies show that BCFA may have a
beneficial effect on proper gut functions, thus, their intake in the population becomes
relevant. The present study is the first to investigate a wide range of products consumed by
the US population. Our data shows that the consumption of BCFA per capita per day is
substantial, and is above the consumption of bioactive n-3 FA such as EPA and DHA. The
prominence of BCFA in the US food supply and their bioactivity strongly suggest that
BCFA health effects should be studied.
Acknowledgments
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This work was supported by NIH grants T32 HD007331 and R21 HD064604.
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Figure 1.
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Figure 2.
Description of individual BCFA, total BCFA, and chain length distribution of BCFA (mean
± SD) in fluid milk versus dairy products. Fluid milk data was obtained from our previous
publication 8
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Figure 3.
Daily BCFA intake (mg/d) versus intake of bioactive FA DHA and EPA, all calculated from
USDA intake data. See text for details.
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Table 1a
Total and individual BCFA concentrations in various foods within the dairy category
Blue
Sheep Goat Ice Swiss Provolone American Ricotta Cottage Greek Cheddar Sour Cream Mozzarella Light
Cheese Milk Yogurt Butter
Cheese Cheese Cream Cheese Cheese Cheese Cheese Cheese Yogurt Cheese Cream Cheese Cheese cream
(sheep)
Ran-Ressler et al.
BCFA, %wt
TOTAL BCFA 2.73 2.21 2.11 2.08 2.05 2.05 2.01 1.92 1.91 1.90 1.84 1.83 1.75 1.70 1.52 1.51 1.41 1.37
iso-14:0 0.22 0.07 0.18 0.14 0.13 0.15 0.12 0.14 0.13 0.13 0.17 0.12 0.13 0.11 0.05 <0.003 0.12 <0.003
iso-15:0 0.42 0.02 0.24 0.33 0.13 0.17 0.15 0.17 0.19 0.13 0.01 0.13 0.14 0.18 0.11 0.13 0.14 <0.003
anteiso-15:0 0.88 0.38 0.54 0.42 0.56 0.57 0.62 0.55 0.64 0.56 0.63 0.57 0.63 0.56 0.46 0.57 0.57 0.49
iso-16:0 0.37 1.18 0.38 0.46 0.31 0.31 0.29 0.29 0.38 0.30 0.34 0.27 0.30 0.33 0.24 <0.003 0.15 0.24
iso-17:0 0.15 0.05 0.26 0.17 0.26 0.26 0.25 0.30 0.20 0.24 0.31 0.18 0.16 0.18 0.30 0.21 0.14 0.27
anteiso-17:0 0.61 0.51 0.51 0.57 0.61 0.58 0.59 0.58 0.37 0.54 0.38 0.56 0.56 0.33 0.36 0.60 0.29 0.37
iso-18:0 0.09 <0.003 <0.003 <0.003 0.04 <0.003 0.04 <0.003 <0.003 <0.003 <0.003 <0.003 <0.003 <0.003 <0.003 <0.003 <0.003 <0.003
Sum BCFA≤16 C 1.89 1.65 1.34 1.35 1.13 1.20 1.17 1.15 0.70 1.12 1.15 1.09 1.03 1.18 0.86 0.70 0.97 0.73
Sum BCFA>16C 0.84 0.56 0.77 0.74 0.91 0.85 0.84 0.89 0.57 0.78 0.69 0.74 0.72 0.52 0.66 0.81 0.43 0.64
Sum iso-BCFA 1.24 1.32 1.06 1.10 0.87 0.89 0.85 0.90 0.90 0.80 0.83 0.70 0.71 0.80 0.70 0.34 0.54 0.51
Sum anteiso-BCFA 1.49 0.89 1.05 0.98 1.17 1.16 1.20 1.13 1.01 1.11 1.01 1.13 1.04 0.90 0.82 1.17 0.86 0.87
Table 1b
Total and individual BCFA concentrations in beef and tuna. Other foods in the protein group tested were
Author Manuscript
ground turkey, chicken breasts, chicken thighs, pork sausages, ham, bacon, fresh salmon, eggs, almonds; all
had less than 0.003%wt BCFA.
BCFA. %wt
Table 1c
Total and individual BCFA concentrations in sauerkraut and miso. Other fermented foods tested were tofu,
Author Manuscript
Sauerkraut Miso
BCFA. %wt
Table 2
Estimated intake per capita of BCFA from dairy and protein food group in the US.
Food Food consumed (g/d)# Fat, %# Fat consumed (g/d) BCFA (%) BCFA consumed (mg/d)
Beef (cooked) 53.9 18 9.7 1.8 169.8
Ran-Ressler et al.
#
Data are from USDA loss-adjusted food bioavailability data system <http://www.ers.usda.gov/data-products/food-availability-(per-capita)-data-system.aspx> and on fat content from USDA National
Nutrient Database for Standard Reference, Release 25, http://www.ars.usda.gov/Services/docs.htm?docid=8964, and USDA-ARS 2012. Nutrient Intakes from Food: Mean Amounts Consumed per
Individual, by Gender and Age, What We Eat in America, NHANES 2009–2010. Available: www.ars.usda.gov/ba/bhnrc/fsrg. Both databases accessed December 2012.
$
Per capita consumption information obtained from the following sources:
• - Sheep cheese consumption from reference 44
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^
Chocolate consumption in the US was taken from Seligson et al 45.
For estimation of intake, the following were calculated and used: Yogurts: mean BCFA in the two major styles (plain and Greek yogurts); Beef: mean BCFA levels in retail and small farm pasture-fed beef;
Tuna: Mean fat concentrations in tuna canned in oil and tuna canned in water.
Ran-Ressler et al.