Analytical Approach for Checking the Compliance of Fats and Oils Against a Possible Regulated Limit for Industrial Trans Fatty Acids

Ref. Ares(2018)3313247 - 22/06/2018

EUROPEAN COMMISSION DIRECTORATE-GENERAL JOINT RESEARCH CENTRE Directorate F - Health, Consumer & Reference Materials (Geel) Fraud Detection & Prevention

Analytical approach for checking the compliance of fats and oils against a possible regulated limit for industrial trans fatty acids

This report shall be read in conjunction with an earlier report submitted by DG JRC to DG SANTE (ARES(2016)6994854).

Retieseweg 111, 2440 Geel, Belgium. Telephone: (+32-14) 571 316, email: [email protected]

Background

Evidence from epidemiological as well as controlled human intervention studies indicates that consumption of diets containing elevated levels of trans fatty acids (TFA) increases the risk of coronary heart disease. The European Food Safety Authority (EFSA) concluded that TFA intakes should be as low as is possible within the context of a nutritionally adequate diet [1]. A further conclusion drawn by EFSA was that the available evidence is insufficient to establish whether there is a difference between ruminant and industrial trans fatty acids consumed in equivalent amounts on the risk of coronary heart disease. In the Report form the Commission to the European Parliament and the Council regarding trans fats in foods and in the overall diet of the Union population [2] several options are explored to limit the amount of TFA in the EU food supply. Introducing a legal limit was seen as the most appropriate policy measure to achieve an effective reduction of industrial TFA (iTFA). They are formed in high amounts by partial hydrogenation and to a much lesser degree by physical raffination of edible oils. Regulating ruminant TFA (rTFA) is not feasible as TFA are formed naturally in relatively stable proportions in ruminant fats, and cannot be avoided in ruminant products, which contribute essential nutrients in the EU diet. In case the presence of iTFA in food products will be regulated, reliable analytical tools are needed to control and enforce legislation.

Terms of Reference

DG JRC has been requested by DG SANTE to devise an analytical approach that allows the estimation of the amount of iTFA in food products containing mixtures of partially hydrogenated oils and ruminant fats (dairy fat or beef tallow). The methods shall permit the control of an envisaged limit of 2 g iTFA/100 g fat contained in the food product.

Trans fatty acids

Regulation (EC) No 1169/2011 defines ‘trans fat’ as fatty acids with at least one non-conjugated (namely interrupted by at least one methylene group) carbon-carbon double bond in the trans configuration [3]. Therefore, this definition includes all mono- and poly-unsaturated fatty acids of edible oils and fats, but excludes fatty acids whose double-bond system is conjugated (not- interrupted by at least one methylene group).

TFA in edible fats and oils result from either industrial processing (hydrogenation and/or physical refining) or biohydrogenation of fatty acids in the stomach of ruminant animals (milk and body fat of cows, sheep, goat). Industrial processing and biohydrogenation of unsaturated fats and oils results in different profiles of the generated TFA, while, with few exceptions, the physico-chemical nature of the TFA generated by those two pathways do not differ at all. Differences in the TFA profiles between partial hydrogenated vegetable oils and ruminant fats

relate primarily to the distribution of positional isomers of trans octacedenoic acid (18:1)1 and the exclusive occurrence of conjugated fatty acids (CFA) in ruminant fats, the main isomer being c9,t11-18:2 (conjugated linoleic acid, CLA). Trans-vaccenic acid (t11-18:1) is the predominant monounsaturated FA (MUFA) of ruminant fats (35-55 % of trans MUFA), while in partially hydrogenated fats and oils the distribution of positional isomers of trans MUFA follows a bell- shaped curve centred on t9/t10/t11 isomers of 18:1 [4].

Quantities of TFA in industrially processed fats and oils range from around 1 % for physically refined vegetable oils, mostly trans isomers of linoleic (18:2) and linolenic acid (18:3), up to 50 % for partially hydrogenated oils (PHO), mostly trans isomers of oleic acid (18:1).

Vegetable oils are partially hydrogenated to modify their functional properties with two main objectives: (i) to improve oxidative stability to make oils more suitable for frying of food and (ii) to change physical properties (texture, plasticity) for the production of margarine and shortenings. The latter are used in bakery products (Danish pastry, cakes, cookies, cream fillings, and frostings), savoury snacks (crackers, biscuits, and popcorn), confectionary (candy bars, chewing gum), instant foods, and stock cubes. PHO are also used as carriers of certain food additives (flavours and colours) and as processing aid (pan release agent); contributions from those minor uses to dietary intake are, however, minimal.

Ruminant fats, predominantly bovine milk fat (MF), are another source of dietary TFA. Based on the analysis of more than 2000 MF samples obtained from 14 EU Member States, Precht and Molkentin [5] estimated a mean total TFA content in MF of 4.92 % (range 1.29 %-7.17 %), of 1.76 % (0.35 %-4.46 %) for t11-18:1, and of 0.76 % (0.10 %-1.89 %) for c9, t11-18:2. The c9, t11- 18:2 content was strongly correlated to the t11-18:1 and the total TFA content (r=0.97). The total TFA amount as well as the distribution of positional trans isomers of FA in ruminant fats is very variable and depends on the type of feeding regimen [6]. The distribution of the TFA concentrations does not follow a Gaussian distribution but is bimodal meaning that, in general, the TFA content during pasture feeding is higher than during barn feeding. For this reason arbitrary values to characterise the total TFA content of MF are more appropriate than the arithmetic mean or percentiles, which require that the data follow a Gaussian distribution. Table 1 (Annex) provides an overview of recently published TFA data of MF. Noteworthy is the fact that many of the published data contained in Table 1 result from experiments designed to demonstrate the influence of certain feeding regimes on MF composition and does not reflect routine practices. However, the data also demonstrate that even under such extreme conditions the total amount of TFA in milk fat is less than 10 g/100 g. The TRANSFAIR Study estimated the TFA content in commercialised dairy products from 14 European countries; the amounts ranged between 3.2 and 6.2% of fatty acids [7].

On the basis of the available data it is reasonable to use a content of 6 g TFA/100 g MF for source apportionment of TFA contained in a mixture of ruminant and industrial fats. It is a rather conservative estimate, which will not disadvantage food business operators, while still be effective in protecting public health.

1 The carbon chain length of a FA and its degree of unsaturation are denoted by xx:y; the geometrical configuration of a double bond is denoted by c (cis) or t (trans); the position of the double bond is denoted by a number counting from the carboxyl group (e.g. 9c, 12t-18:2).

Principles of the approach for checking the compliance of fats and oils against a possible regulated limit for industrial trans fatty acids

Whereas several collaboratively tested analytical methods for the determination of total and individual TFA in edible fats and oils exist, methods to differentiate them according to their origin (iTFA or rTFA) are scarce. It is not possible to chemically differentiate if an individual TFA originates from an industrially processed oil/fat or from ruminant fat. Therefore, it has to be well understood that the proposed approach approximates the amount of iTFA in a mixture where rTFA co-occur, but it does not allow a quantification delivering accurate results with an associated uncertainty.

The proposed approach builds on the use of

 the amounts (g/100 g fat) of butyric acid (4:0), total TFA (sum of fatty acids with at least one non-conjugated carbon-carbon double bond in the trans configuration, usually the trans-isomers of hexadecenoic acid (t16:1), octadecenoic acid (t18:1), octadecadienoic acid (t18:2) and octadecatrienoic acid (t18:3)), and of c9, t11-18:2, as well as the proportion (%) of trans-vaccenic acid (t11-18:1) relative to the sum of t18:1,  determined by a collaboratively tested analytical method (AOAC 2012.13 | ISO 16958:2015 | IDF 231:2015 [8]), which is based on capillary gas-liquid chromatography with flame-ionisation detection,  subjected to a decision making algorithm (decision tree) to identify whether the fat contains: o less than a regulated limit for TFA (e.g. less than 2 g/100 g fat), o only iTFA in amounts exceeding the limit, o only rTFA in amounts exceeding the limit, o a mixture of iTFA and rTFA in amounts exceeding the limit.

In case butyric acid is present next to iTFA and rTFA, the amount of butyric acid is used to, firstly, approximate the amount of MF in the mixture and, secondly, the amount of rTFA originating from MF (Equation 1). rTFA [g/100 g] = (Butyric acid [g/100 g] * 29.4* 6)/100 Equation 1

The factor 29.4 is used to convert the measured amount of butyric acid to MF based on an average content of 3.4 g butyric acid/100 g MF [9]; furthermore, it is assumed that MF contains 6 g TFA /100 g.

In the rare event that butyric acid is not present while c9, t11-18:2 is present next to iTFA and rTFA, the amount of c9, t11-18:2 is used to approximate the amount of rTFA originating from bovine body fat (tallow) in the mixture (Equation 2). rTFA [g/100 g] = (c9, t11-18:2 [g/100 g])/0.15 Equation 2

As the TFA concentration in tallow is similar to MF, a factor of 0.15, which approximates the relation of total TFA to c9, t11-18:2 for MF, is used to estimate the amount of total TFA in tallow.

Finally, subtraction of the amount of rTFA from total TFA gives the amount of iTFA (Equation 3): iTFA [g/100 g] = total TFA – rTFA Equation 3

N.B. Zero replaces negative values in Equation 3.

Work instruction for checking the compliance of fats and oils against a possible regulated limit for industrial trans fatty acids

1. Scope

This procedure serves to estimate the content of industrial trans fatty acids (iTFA) in samples containing a blend of ruminant fat and industrially processed fats or oils. The method described shall be applied if the individual fats/oils contained in the blend are not available/accessible; otherwise, the contribution of each of the blended fats to total trans fatty acids (TFA) shall be estimated via the analysis of the pure fats. N.B. The procedure does not allow to accurately determine the amount of TFA attributable to industrially processed fats or oils. It provides an estimate which is based on empirical factors for correcting the total amount of analytically determined TFA by the contribution resulting from ruminant fats (milk fat or tallow).

2. Principle

The method comprises the determination of butyric acid (4:0), total TFA (sum of fatty acids with at least one non-conjugated carbon-carbon double bond in the trans configuration, usually the trans-isomers of hexadecenoic acid (t16:1), octadecenoic acid (t18:1), octadecadienoic acid (t18:2) and octadecatrienoic acid (t18:3)), and conjugated linoleic acid (c9, t11-18:2), as well as the proportion (%) of trans-vaccenic acid (t11-18:1) relative to the sum of t18:1, by gas chromatography with flame ionisation detection using AOAC 2012.13 | ISO 16958:2015 | IDF 231:2015 [8] or another standardised method with similar performance characteristics. A correction of the TFA content by the TFA content stemming from ruminant fat (rTFA) is then applied. The contribution of rTFA to the total TFA content of the blend is either estimated via the butyric acid content if the blend contains milk fat or via conjugated linoleic acid if the blend contains ruminant body fat (tallow). The content of trans fatty acids is expressed as gram per 100 gram fat.

3. Definitions

Trans fatty acids: fatty acids with at least one non-conjugated (namely interrupted by at least one methylene group) carbon-carbon double bond in the trans configuration iTFA: total trans fatty acids contained in industrially processed fats/oils, expressed as gram per 100 gram of fat/oil rTFA: total trans fatty acids contained in ruminant fats (milk fat or body fat), expressed as gram per 100 gram of fat tTFA: total trans fatty acids is the sum of the contents of isomers of non-conjugated trans fatty acids of hexadecenoic acid (t16:1), octadecenoic acid (t18:1), octadecadienoic acid (t18:2) and octadecatrienoic acid (t18:3), expressed as gram per 100 gram of fat.

CLA: conjugated linoleic acid; for the purpose of this document, CLA is the cis-9, trans-11 isomer of linoleic acid (c9, t11-18:2)

4. Sample preparation and analysis

The ingredient list of the tested food shall be checked whether ruminant fats (milk fat and/or tallow) have been used. If their amounts are specified, this has to be recorded for cross- checking the plausibility of the obtained testing results. Depending on the analytical method used for the determination of FAME by GLC-FID, fat might be transesterified directly in the test sample, or after the extraction of a representative portion of fat. Only internationally accepted methods shall be used for the determination of the content of TFA and of butyric acid in the test sample. AOAC 2012.13 | ISO 16958:2015 | IDF 231:2015 [8] shall be used for the determination of the TFA content in different food matrices; other suitable methods such as AOAC 996.06 [10] and AOCS Ce 1j-07 [11] may be used if it can be proven that they deliver equivalent results. Figure 1 (Annex) presents the analytical workflow for the estimation of iTFA in mixtures of industrial fat and ruminant fat.

5. Estimation of the content of industrial TFA in mixtures of ruminant and industrial fats

In case butyric acid is present next to iTFA and rTFA, the amount of butyric acid is used to approximate the amount of MF in the mixture (Equation 1). rTFA [g/100 g] = (Butyric acid [g/100 g] × 29.4 × 6)/100 Equation 1

The factor 29.4 is used to convert the measured amount of butyric acid to MF based on an average content of 3.4 g butyric acid/100 g MF; furthermore, it is assumed that MF contains 6 g TFA /100 g.

As the TFA concentration in tallow is similar to MF, a factor of 0.15, which approximates the relation of total TFA to c9, t11-18:2 for MF is used to estimate the amount of total TFA in tallow.

Finally, subtraction of the amount of rTFA from total TFA gives the amount of iTFA (Equation 3): iTFA [g/100 g] = total TFA – rTFA Equation 3

N.B. Zero replaces negative values in Equation 3.

References

1. European Food Safety Authority: Scientific Opinion on Dietary Reference Values for fats, including saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, trans fatty acids, and cholesterol. EFSA Journal 2010; 8(3):1461. 2. Report form the Commission to the European Parliament and the Council regarding trans fats in foods and in the overall diet of the Union population. COM(2015) 619 final. 3. Regulation (EU) No 1169/2011 of the European Parliament and of the Council of 25 October 2011 on the provision of food information to consumers. 4. Aldai N., de Renobales M., Barron L.J.R., Kramer J.K.G.: What are the trans fatty acids issues in foods after discontinuation of industrially produced trans fats? Ruminant products, vegetable oils, and synthetic supplements. European Journal of Lipid Science and Technology 115 (2013) 1378-1401. 5. Precht D., Molkentin J.: Frequency distribution of conjugated linoleic and trans fatty acid contents in European bovine milk fats. Milchwissenschaft 55 (2000) 687-691. 6. Tyburczy C., Mossoba M.M., Rader J.I.: Determination of trans fat in edible oils: current official methods and overview of recent developments. Analytical and Bioanalytical Chemistry 405 (2013) 5759–5772. 7. Aro A, Antoine J.M., Pizzoferrato L., Reykdal O., van Poppel G. Trans fatty acids in dairy and meat products from 14 European countries: The TRANSFAIR Study. Journal of Food Composition and Analysis 11 (1998) 150-160. 8. ISO 16958:2015 Milk, milk products, infant formula and adult nutritionals — Determination of fatty acids composition — Capillary gas chromatographic method. International Organization for Standardization, Geneva, CH. 9. Molkentin J., Precht D. Representative determination of the butyric acid content in European milk fats. Milchwissenschaft 52 (1997) 82-85. 10. AOAC Official Method 996.06 Fat (Total, Saturated, and Unsaturated) in Foods – Hydrolytic Extraction Gas Chromatographic Method. AOAC International, Rockville, MD, USA 11. AOCS Official Method AOCS Official Method Ce 1j-07 Determination of cis-, trans-, saturated, monounsaturated, and polyunsaturated fatty acids in extracted fats by capillary GLC. American Oil Chemists' Society, IL, USA.

Annex

Figure 1: Work-flow for checking the compliance of fats and oils against a possible regulated limit of industrial trans fatty acids contained in oils and fats

Pre-packaged food

Check label declaration for use of animal and/or partially hydrogenated fats

Extract fat (if necessary)

Determine fatty acid profile using standardised method V E R I

Compliant with F

YES Total TFA < 2 g/100 Y proposed limit

TFA of Contains 9c, 11t-18:2 NO industrial origin

YES Not compliant with proposed limit

Contains TFA of TFA of < 7 g/100 g total TFA and ruminant and ruminant YES > 0.10 g/100 g CLA and NO industrial origin > 35 % 11t-C18:1 of total origin trans-C18:1

Industrial TFA = Compliant with YES total TFA – 1.76*BA, YES Butyric acid (BA) present proposed limit If < 2 g/100 g

NO NO

Industrial TFA = Not compliant with NO total TFA – 6.67*CLA, proposed limit If < 2 g/100 g

YES

Compliant with proposed limit

Table 1. Literature overview regarding the TFA content of milk fat

CLA CLA Sampling total tTFA min max CLA CLA Samples C4:0 t-16:1 t-18:1 t-18:2 c9,t11/t- c9,t11/C18:1 Comment year TFA SD tTFA tTFA c9,t11 c9,t11/tTFA C18:1 t11

Feeding regime: indoor group (maize silage), pasture group (pasture during summer, silage 0.3- 0.19- Jahreis et al.1 36 bulk % FAME 0.4-0.5 1.8-3.4 0.5-0.7 3.0-5.0 0.11-0.16 0.25+0.07391) during other seasons) and ecological group 0.81) 0.23 (grazing during summer, silage during other seasons); 1)Σc9,t11C18:2 and t9,c11-C18:2

Precht, 1756 DE samples; influence barn/pasteure feeding; 1995 % FA 3.6 0.82) 0.44x+0.05 Molkentin2 individual 2)g FA/100g fat

136 g Molkentin, European FA/100g 3.4 na na na na Precht3 milk fat samples

3) function for correlation between CLA and 8 Jahreis et al.4 % FAME 1.0 0.318x+0.1413) t11-C18:1 covers different species, not all individual ruminants

Precht, ~2000 1995 % FA 3.7 1.1 4.9 1.5 8.7 0.8 Samples are from EU14 Molkentin 5 individual

Seasonal variations, highest CLA values Lock, 433 1997- 0.19- % FAME 2.0-3.1 2.6-4.5 0.6-1.7 0.19-0.61 pasture feeding early summer, lowest CLA Garnsworthy6 individual 2000 0.61 values in Oct-Dec

Lindmark % FA 1995- Composition od Swedish dairy milk; 4)not all t- Mansson et 63 bulk (weighted 4.7 0.1 2.2 0.2 2.54) 0.3 2.2 2.9 na na na na 1996 FAs considered in tTFA value al.7 means)

Retieseweg 111, 2440 Geel, Belgium. Telephone: (+32-14) 571 316, email: [email protected]

CLA CLA Sampling total tTFA min max CLA CLA Samples C4:0 t-16:1 t-18:1 t-18:2 c9,t11/t- c9,t11/C18:1 Comment year TFA SD tTFA tTFA c9,t11 c9,t11/tTFA C18:1 t11

correlation between CLA and tTFA, fed basis: g maize silage, lucerne hay, and maize grains; Secchiari et 32 0.35- 0.17- FA/100g 3.2-3.8 1.8-3.0 0.51-0.63 supplemented by full fat extruded soy bean, al.8 individual 0.5 0.21 fat full fat linseed, soybean meal coated with palm oil soap, or olive oil soap

64 supplementation of feed with different Brzoska9 % FA 0.8-1.1 individual vegetable oils, no control group

16 2,7- 1.9- 0.16- supplementation of low/high percentage Loor et al.10 % FA 0.1-2.8 0.6-2.5 0.19-0.34 0.41-0.61 individual 12.1 12.8 0.23 concentrate feed with linseed oil

Couvreur et 32 0.20- 2003 % FA 3.8-4.3 2.4-5.9 2.6-6.5 0.5-1.6 0.18-0.26 0.56-0.35 corn silage replaced by fresh grass (spring) al.11 individual 0.28

% FA Lindmark 28 bulk 2001 (weighted 4.4 0.4 2.1 0.2 2.7 0.7 3.9 0.4 0.15 0.19 significant seasonal variation, Mansson12 means)

Canadian dairy products, average of Mendis et 41 2006- % FA 3.9 5.5 0.7 4.2 7.4 0.5 0.09 0.13 0.39 combined values for cream, butter, milk, al.13 individual 2007 cheese;

supplementation of control group feed with different vegetable oils (rapeseed, sunflower, 64 5.4- 0.17- Rego et al.14 2006 % FA 4.5-8.7 1.1-1.6 0.14-0.22 0.42-0.48x and linseed oil), control group diet: 20 h individual 10.8 0.26 grazing pasture + 5 kg corn based concentrate.

bulk milk samples covering US milk O'Donnel15 228 bulk 2008 % FA 4.1 3.2 0.55 0.17 0.37x production, ratio CLA c9,t11/t11-C18:1

CLA CLA Sampling total tTFA min max CLA CLA Samples C4:0 t-16:1 t-18:1 t-18:2 c9,t11/t- c9,t11/C18:1 Comment year TFA SD tTFA tTFA c9,t11 c9,t11/tTFA C18:1 t11

Rouille, 2008- 0.23- Highest value: pasture feeding early summer; 85 bulk % FA 3.7-3.9 2.4-3.4 3.3-4.9 1.1 2.8 6.8 0.6-1.2 0.17-0.25 0.456x-0.047 Montourcy 16 2009 0.36 in winter

23 2007- Kuhnt et al.17 % FAME na 2.7 3.2 2.3 4.6 0.9 0.38 0.79x+0.79 data for butter individual 2009

Commercial milk samples, conventional 88 2006- 1.2- Butler et al.18 % FA 1.9 0.6-0.7 versus organic production, 5) value represents individual 2008 1.65) only t11-C18:1

mean value of milk from Fr, No, Sl, and Sk; 6) Chassaing et g FA/100 360 bulk 2008 3.86) 1.86) 8.76) tTFA values include conjugated linoleic acid al.19 g fat contents

Bada Algom 14 % FA 4.95) 1.75 0.35x+0.21 5) value represents only t11-C18:1 et al.20 individual

bulk milk samples from conventional and Schwendel et 2010- g FA/100 2.3- 160 bulk 2.8-3.0 0.9-1.6 organic farms; 5) value represents only t11- al.21 2013 g fat 5.35) C18:1

correlation between CLA and tTFA; data from 296 0.18- Spatny22 2006 % FA 4.2-4.4 2.8-3.1 0.6-0.7 0.39-0.41 conventional, rbST-free, and organic milk individual 0.25 production

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5 Precht, D. and J. Molkentin, Frequency distributions of conjugated linoleic acid and trans fatty acid contents in European bovine milk fats. Milchwissenschaft = Milk science international, 2000. 55(12): p. 687-691. 6 Lock, A.L. and P.C. Garnsworthy, 9-desaturase activity in dairy cows. Livestock Production Science, 2003. 79(1): p. 47-59. 7 Lindmark-Månsson, H., R. Fondén, and H.-E. Pettersson, Composition of Swedish dairy milk. International Dairy Journal, 2003. 13(6): p. 409-425. 8 Secchiari, P., et al., Effect of kind of dietary fat on the quality of milk fat from Italian Friesian cows. Livestock Production Science, 2003. 83(1): p. 43-52. 9 Brzoska, F., Effect of dietary vegetable oils on milk yield, composition and CLA isomer profile in milk from dairy cows. Journal of Animal and Feed Sciences, 2005. 14(3): p. 445-459. 10 Loor, J.J., et al., Relationship Among Trans and Conjugated Fatty Acids and Bovine Milk Fat Yield Due to Dietary Concentrate and Linseed Oil. Journal of Dairy Science, 2005. 88(2): p. 726-740. 11 Couvreur, S., et al., The linear relationship between the proportion of fresh grass in the cow diet, milk fatty acid composition, and butter properties. Journal of Dairy Science, 2006. 89(6): p. 1956-1969. 12 Lindmark-Månsson, H., Fatty acids in bovine milk fat. Food Nutr Res, 2008. 52. 13 Mendis, S., C. Cruz-Hernandez, and W.M.N. Ratnayake, Fatty acid profile of Canadian dairy products with special attention to the trans-octadecenoic acid and conjugated linoleic acid isomers. Journal of AOAC International, 2008. 91(4): p. 811-819. 14 Rego, O.A., et al., Rumen biohydrogenation-derived fatty acids in milk fat from grazing dairy cows supplemented with rapeseed, sunflower, or linseed oils. Journal of Dairy Science. 92(9): p. 4530-4540. 15 O'Donnel, A.M., Milk fatty acids: retail milk fat composition and efforts to naturally enhance bioactive fatty acids in milk for the benefit of human health, in Dissertation. 2010, Cornell University. 16 Rouîlle, B. and M. Montourcy, Influence de quelques sytèmes d'alimentation sur la composition en acides gras du lait de vache en France. 2010. p. 33. 17 Kuhnt, K., et al., Trans fatty acid isomers and the trans-9/trans-11 index in fat containing foods. European Journal of Lipid Science and Technology, 2011. 113(10): p. 1281-1292. 18 Butler, G., et al., Fat composition of organic and conventional retail milk in northeast England. Journal of Dairy Science, 2011. 94(1): p. 24-36. 19 Chassaing, C., et al., Mineral, vitamin A and fat composition of bulk milk related to European production conditions throughout the year. Dairy Science and Technology, 2016. 96(5): p. 715-733. 20 Bada Algom, O., et al., Comparison of milk fatty acid profiles measured on Kouri cows near Lake Chad and on dairy cattle as reported by meta-analytical data. Tropical Animal Health and Production, 2017. 49(5): p. 915-921. 21 Schwendel, B.H., et al., Pasture feeding conventional cows removes differences between organic and conventionally produced milk. Food Chemistry, 2017. 229(Supplement C): p. 805-813. 22 Spatny, K.P., Survey of retail milk: comparison of the fatty acid composition of conventional milk and milk labelled as "rbST-free" and "organic". 2009, Cornell University: Research Honors Program 2009. p. 32.