Züchtungskunde, 80, (5) S. 420 – 427, 2008, ISSN 0044-5401 © Eugen Ulmer KG, Stuttgart

Effect of supplementing isomerised poppy seed oil with high concentration of linoleic isomer T10,C12 and C9,T11 on fat level in sheep milk and its fatty acids profile R. Bodkowski1, Boz ˙ena Patkowska-Sokol⁄ a1, Wiesl⁄ awa Walisiewicz-Niedbalska2

Introduction An intensified interest in chemical composition of dietary lipides, and mainly of their fatty acids profile has been observed in recent years. Results of the research indicate that a high consumption of fats rich in saturated fatty acids leads to increase in cholesterol level and its LDL fraction, thus showing the strong atherogenic activity. These acids may also negatively influence the blood coagulation system and thus might effect the incidence rates of certain tumors. Unfortunately, most lipids of animal origin, including milk fats, are characterised by fatty acids profiles unfavorable from a health point of view, i.e. high content of saturated fatty acids and low of unsaturated ones. A number of genetic and environmental factors influence the content of fat and its fatty acids composition in milk. Also fatty acids themselves may to some extent influence and regulate this process. CLA is a common term describing the mixture of positional (8 and 10, 9 and 11, 10 and 12 or 11 and 13) and geometric (cis and trans) isomers of octadecenoic acid C18:2, where double bonds are isolated by only one single bond (Eulitz et al,. 1999). The main mechanism of their creation is an isomerisation of linoleic and linolenic acids that takes place in the alimentary tract of ruminants – synthesis in mammary gland and in adipose tissues from trans-11 C18:1 acid take place with the participation of ∆9 desaturase. Antimutagenic and anticarcinogenic properties of CLA have been recognized the best so far (Ip et al., 1999). However, the results of the latest research indicate that these dienes may also influence the lipid balance by reducing fat content in the body mass (Park et al,. 1999) and inhibiting its synthesis in the mammary gland. Having the above mentioned in mind, research was conducted aiming to reduce the fat content in sheep milk and to favorably influence its fatty acid profile by addition of isomerised poppy seed oil enriched with conjugated dienes of linoleic acid of trans10,cis-12 and cis-9,trans-11 configuration to the diet of milking sheep.

1 Material and methods Gaining and determination the fatty acids content of poppy seed oil Poppy seed oil was pressed on an expeller from a low-morphine poppy seed strain – “Michal⁄ ko”. Fatty acids content was determined using capillary gas chromatography method on Hewlett Packard II apparatus with flame-ionization detector (FID) and 50 meters long capillary column CP Sil 88.

1 Institute of Animal Breeding, Wroclaw University of Environmental and Life Sciences, ul. Chel/mo`nskiego 38c, 51-630 Wroclaw, Poland [email protected] 2 Industrial Chemistry Research Institute, ul. Rydygiera 8 , 01-793 Warsaw, Poland

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Conditions of separation: temperature of a column – 170°C, dosimeter – 200°C, of detector – 250°C, carrier gas – helium. Methyl esters were obtained according to AOCS Official Methods Ce2-66. Analysis was conducted in agreement with AOCS Official Methods Ce 1f-96. Qualitative identification was done by a comparison of retention times of analysed components with standards. The mixture of methyl esters of fatty acids was used as standards (Supelco Company). As an internal standard heptadecanoic acid C17 (Fluka Company) was applied.

Synthesis of conjugated dienes of linoleic acid cis-9, trans-11 and trans-10, cis-12 from cis-9, trans-12 C18:2 acid. Initial substrate: Low-morphine poppy seed oil Isomerisation conditions: temperature – 180°C, pressure – normal; reaction environment – distilled glycerine of 99 % purity, molar ratio of reagents: 1 mole of oil + 60 moles of glycerine + 7.4 moles of NaOH; reaction time – 3 hours Method of conducting the reaction: Glycerine was placed in a reactor, warmed to a temperature of 50 – 60°C and NaOH was introduced. The whole mixture continued to be warmed to a temperature of about 140°C and consistently mixed until hydroxide dissolution. Next, the oil was introduced, and the temperature of reaction was raised to 180°C. The process was carried out for 2 hours under these conditions. At the end of the reaction and cooling the reactor content to below 100°C, water was added in ratio 1:1 in order to dilute the created soap solution. Next, sulphuric acid was added to the reactor in order to acidify soaps to the form of free fatty acids. The acidification process was conducted in a temperature of 70 – 80°C for half an hour. The termination of an acidification process was determined by measurement of excess mineral acid in the water layer. After the complete acidification of soaps, the reaction mixture was moved to a separator where the water layer was separated, and a layer of fatty acids was washed with hot water (about 90°C) to obtain the neutral reaction of washed water. After complete washing, acids were dried under sodium sulphate. The resulting product was analysed. Analytical methods: Identification of fatty acids was done using capillary gas chromatography method on Hewlett Packard II apparatus with flame-ionization detector (FID) and 50 meters long capillary column CP Sil 88. Conditions of separation: temperature of a column – 17°C, dosimeter – 200°C, of detector – 250°C, carrier gas – helium. Methyl esters were obtained according to AOCS Ce2-66. Quality identification was conducted by a comparison of retention times of analysed components with standards. CLA isomers by Sigma Company were used as standards. Identification of positional isomers of main compounds was done using GC/MS method applying the specific fragmentation of fatty derivatives with 2-amino-2-methylo-1propanol (DMOX). Separation of methyl esters trans and cis of fatty acids was conducted using thin-layer chromatography TLC-Ag+. Collected bands of methyl esters corresponding to trans and cis fractions were transformed into their derivatives with 4,4dimethylooxazoline. Obtained DMOX derivatives were analysed using the GC/MS method. Isomerisation of poppy seed oil and determination of fatty acids content were conducted in an Industrial Chemistry Research Institute in Warsaw.

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The application of isomerised poppy seed oil in milking sheep feeding In the second part of the experiment, isomerised poppy seed oil (IPSO) enriched with conjugated dienes of linoleic acid t10,c12 and c9,t11, was spread using nozzle spraying method on humic-mineral carrier – “Humokarbowit”. The amount of sprayed oil was 18 % of the carrier. 30 randomly chosen ewes of the Friesian breed aged 3–5 years and in their 3rd month of lactation were used. All animals were fed based on indoor system. Feeding was established according to standards for lactating ewes based on feed mixture for calves and lambs and meadow hay. Animals had an access to water and salt licks. The experiment covered two randomly created groups, 15 heads in each (control and experimental). During the experiment, ewes of the experimental group in addition to their basic feed were given “Humokarbowit” with isomerised poppy seed oil in an amount of 50 g per head and day, while ewes of the control group were given “Humokarbowit” only in the same quantity. The amount of the supplement in the experimental group was established so that the effective daily dose of trans-10, cis-12 isomer was 3 g per head and day. According to methodical assumptions, milk for analysis of fat content and fatty acids composition was collected three times: on the day of the beginning of the experiment and after 7 and 14 days. Fatty acids content was determined using capillary gas chromatography method using PU 4410 apparatus (Philips Company) with flame-ionisation detector (FID) and 105 m long capillary column Rtx2330. Conditions of separation: Initial isotherm – 160 OC (30 min) – 3°C/min. to 180°C – 17 min. in temp. 180°C, for 5 min. to 210°C – 20 min. in temp. 210°C. Other conditions: temperature of a column – 160°C, temperature of detector – 230°C, temperature of spraying chamber – 220°C, carrier gas – Hel 80 PSI. Qualitative identification was conducted by comparison of retention times of obtained peaks with retention times of standards (Sigma Company). To discriminate significance of differences between analysed parameters statistical software SAS (1996) was used.

2 Results As a result of cold pressing 51 % of low-morphine oil of favorable, as regards synthesis of linoleic acid isomers (CLA), fatty acids content was obtained from poppy seeds. Oil was characterized by high concentration of linoleic acid C18:2 (about 73.1 %) and low content of linolenic acid C18:3 (about 0.5 %) and saturated acids C16-18 (about 12 %) (Table 1). However, it did not contain, like other unprocessed plant oils, conjugated dienes of linoleic acid (CLA). The process of alkaline isomerisation caused that two conjugated dienes of linoleic acid of cis-9, trans-11 and trans-10, cis-12 configuration in amount of 32.2 % and 34.3 %, respectively, of all fatty acids pools were created from linoleic acid C18:2 cis-9, trans-12 included in poppy seed oil. Except CLA, isomerised poppy seed oil contained saturated acid: palmitic C16:0 and stearic C18:0, and unsaturated oleic C18:1, linoleic C18:2 and linolenic C18:3 and its isomers (Table 1). In a second part of the experiment, isomerised poppy seed oil enriched in trans-10,cis12c and cis-9,trans-11 dienes was added to feeding dose of milking sheep. An effect of above supplement application was a decrease in fat content in milk of 27 per cent after 7 days of application, and of 33 per cent (P ≤ 0.01) after 14 days (Figure 1). Beside the reduction of the fat content in milk, addition of isomerised poppy seed oil caused also a favorable modification of its fatty acids content (Table 2). After 7 and 14 days of supplementation, there was a decrease in concentration of short-chain saturated fatty acids C8-13:0 in milk fat of 16.9 per cent (P ≤ 0.05) and 19.7 per cent (P ≤ 0.01), respectively, miristic acid C14:0 of 6.5 per cent and 12.9 per cent

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Table 1. Fatty acids content of poppy seed oil before and after isomerisation (%). Fettsäurengehalt von Mohnöl vor und nach Isomerisation Fatty acids C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 9c,t11 C18:2 isomer 10t,c12 C18:2 isomer C18:3 C18:3 isomers C20:1

Fatty acids content [%] Before isomerisation

After isomerisation

trace 10.2 0.1 1.8 14.2 73.1 0.5 trace

trace 9.2 < 0.1 2.1 14.9 6.6 32.2 343 0.5 < 0.1 -

Table 2. Fatty acids profile of milk fat of sheep receiving an addition of isomerised poppy seed oil (%) Fettsäurenprofil von Schafmilch nach Zusatz von isomerisiertem Mohnöl im Futter Fatty acid %

Supplement of isomerised poppy oil Control After 7 days

After 14 days

C8-13:0 14:0 14:1 15:0 16:0 16:1 17:0 18:0 18:1 trans- 9 18:1 trans- 11 18:1 other trans isomers 18:1 cis-9 18:2 trans 18:2 cis-9, cis-12 18:2 cis-9, trans-11 CLA 18:2 trans-10, cis-12 CLA C18:2 other isomers CLA C18:3 cis-9, cis-12, cis-15 > 20

15.1Aa

12.6b

11.0a 1.1 1.2 26.5a 2.4 0.7Aa 10.5A 0.2A 1.0A 0.9A 22.9a 0.3Aa 2.7a 0.4A 0.03A 0.04 0.9 1.3

10.2 1.3 1.3 24.0b 2.6 0.9b 13.7B 0.2B 4.0Ba 2.5Ba 19.5b 0.5Ab 3.2b 0.5B 0.1B 0.04 0.9 1.3

12.2B 9.5b 1.5 1.2 23.3b 2.7 0.9B 14.5B 0.3B 5.1Bb 2.1Bb 19.1b 1.0B 2.9 0.6C 0.1C 0.04 0.9 1.2

∑ SFA ∑ MUFA ∑ PUFA

65.6 28.6 4.8a

63.4 30.1 5.4

62.4 30.9 6.0b

Total trans FA Total CLA C9,t11CLA/18:1 t11 a,b; A,B

3.3A 0.4A 0.4

8.3Ba 0.7Ba 0.1

– differences significant on a level of P ≤ 0.05 and P ≤ 0.01

9.7Cb 0.8Cb 0.1

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Fig. 1. Fat content in sheep milk Fettgehalt in Schafmilch (P ≤ 0.05), respectively, palmitic acid C16:0 of 9.5 and 12 per cent (P ≤ 0.05), respectively, and oleic acid of 14.7 and 16.6 per cent (P ≤ 0.05), respectively (Table 2). Also the ratio of C18:2 c9,t11 to C18:1 t11 decreased almost 3-fold (Table 2). Moreover, the tendency towards a decrease in total content of saturated fatty acids was observed (Table 2). The increase in vaccenic acid t-11 C18:1 content of 306 and 411 per cent (P ≤ 0.01), respectively, c9,t11 and t10,c12 CLA isomers of 51 and 79 per cent (P ≤ 0.01), 238 and 355 per cent (P ≤ 0.01), respectively, trans isomers of unsaturated fatty acids of 155 and 198 per cent (P ≤ 0.01), polyunsaturated fatty acids of 11.7 and 23.4 (P ≤ 0.05) per cent and stearic acid C18:0 of 30 and 38 per cent (P ≤ 0.01) was noted in sheep milk (Table 2).

3 Discussion The fatty acids profile of initial substrate significantly influences the amount of conjugated dienes of linoleic acid trans-10,cis-12 and cis-9,trans-11 synthesized in an isomerisation process. Its high usefulness is determined mainly by the high content of linoleic acid C18:2 cis-9,trans-12 that is a main substrate in a process of these isomers synthesis, and a low concentration of linolenic acid C18:3 cis-9,cis-12,cis-15, that also is subjected to isomerisation process creating positional and geometric isomers of unknown biological properties. Also the content of saturated fatty acids may be of importance, however they may be removed from the final product by extraction from urea, increasing this way the concentration of desirable isomers in fatty acids pool (this method was not applied in the present research). The process of poppy seed oil isomerisation caused changes in an arrangement of double bonds in chains of unsaturated fatty acids (positional) and the changes in radical arrangements with respect to the axis of double bond (geometrical). As a result, two conjugated dienes of linoleic acid cis-9, trans-11 and trans-10, cis-12, that naturally are not present in poppy seed oil and possess quite different features than linoleic acid, were created. Since isomerised poppy seed oil was in an oily form, it was spread on a carrier with a high sorptive capacity and antioxidative properties in order to obtain an easier application in sheep feeding. In the present study “Humokarbowit” – preparation of a proper biostimulating and preventive activity, containing mainly natural humic-mineral substances and used in poultry, pigs, cattle and sheep feeding, was applied (Dobrza´ nski and Tronina 1999).

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It is known for a long time that certain kinds of diets cause a distinct decrease in fat content in milk (MFD). The biological mechanism of this process has not been unequivocally and fully explained so far. To explain that phenomenon, Bauman and Griinari (2003) proposed the “biohydrogenation theory”. They hypothesize that in some dietary conditions biohydrogenation in the rumen is altered, and some fatty acids, that are strong inhibitors of milk fat synthesis, are created as intermediates. According to that hypothesis, Baumgart et al. (2000) showed that such a strong inhibitor of milk fat synthesis is trans-10 cis-12 CLA isomer. The activity of lipogenic enzymes may also be inhibited, however to a considerably smaller degree, by trans C18:1 isomers (Piperova et al. 2000). In the present study, the feed of milking sheep was enriched with isomerised poppy seed oil containing a strong inhibitor of milk fat synthesis – trans-10 cis-12 CLA isomer. The amount of supplement was established so that the effective dose of t10,c12 isomer was 3 g per head and day. The use of the above mentioned supplement caused a decrease in fat content in sheep milk, depending on the time of application between 27 and 33. Observed MFD effect was probably caused by trans-10, cis-12 CLA isomer included in isomerised poppy seed oil, and by vaccenic acid C18:1 trans-11 created during changes in the rumen, that inhibited an activity of lipogenic enzymes. This may be confirmed by a decrease in the content of short-chain saturated fatty acids in milk, that are synthesized de novo just directly in the mammary gland. The decrease of fat content in cows’ milk from 25 % to 50 % as a result of CLA application directly to the abomasum in a protected form was observed also by other authors (Giesy et al. 2002; Loor and Herbein 1998). Isomerised poppy seed oil, besides the reduction of fat level in milk, favorably influenced its fatty acids profile. A significant increase in biologically active components of profitable activity, like conjugated dienes of linoleic acid c9,t11 and t10,c12, and vaccenic acid was observed, and a decrease in a content of acids of atherogenic activity, i.e. short-chain fatty acids and palmitic acid. Observed changes in fatty acids content of milk fat are probably due to a high supply of CLA and changes they are subject to in the rumen. A definite majority of conjugated dienes, undergoes biohydrogenation processes with participation of rumen microorganisms, first to vaccenic acid and then to stearic acid, and in that form they are transferred to the milk. However, part of them in unchanged form pass to further parts of alimentary tract where they are absorbed and then incorporated. Moreover, vaccenic acid is used in the mammary gland as a substrate for endogenous synthesis of CLA with participation of ∆9 desaturase. Also the results of other works (Moore et al., 2004; Perfield et al., 2004) prove the favorable influence of CLA addition on composition of fatty acids in milk fat. As a consequence of the present study, the ability to reduce fat content in milk and its enrichment in biologically active compounds of favorable activity should be recognized as the most important ability of trans10, cis-12 CLA isomer from a dietary point of view.

Summary The influence of an addition of isomerised poppy seed oil on fat content in sheep milk and fatty acids composition was assessed in the present study. As a result of the isomerisation process of linoleic acid C18:2 cis-9, trans-12 in poppy seed oil, the synthesis of its conjugated dienes trans-10, cis-12 and cis-9, trans-11 in amount 34.3 % and 32.2 %, respectively, took place. Addition of this oil to the feed of milking sheep caused a decrease in fat concentration in milk by 26 to 33 per cent. It also modified fatty acids composition causing an increase in concentration of conjugated dienes of linoleic acid c9,t11 and t10,c12, vaccenic acid and trans isomers of unsaturated fatty acids, and a decrease in short-chain saturated fatty acids content.

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Key words: Sheep milk, isomerised poppy seed oil, CLA, milk fat, fatty acids profile The present study was carried out in the framework of the Research Project 3 T09B 130 29 financed by the Ministry of Science and Higher Education.

References Bauman D. E., and J. M. Griinarii. 2003. Nutritional regulation of milk fat synthesis. Ann. Rev. Nutr. 23, 203–207. Baumgard L. H., B. A. Corl, D. A. Wyder, A. Saebo and D. E. Bauman. 2000. Identification of the conjugated linoleic acid izomer that inhibits milk fat synthesis. Am. J. Physiol. 278, 179–184. Dobrza´ nski Z., and S. Tronina. 1999. Proecological humin preparation for livestock. Zesz. Nauk. AR Wrocl⁄ aw 361, 65–71. Eulitz K., M. P. Yurawecz, N. Sehat, J. Fritsche, J. A. G. Roach, M. M. Mossoba, J. K. G. Kramer, R. O. Adolf and Y. Ku. 1999. Preparation, separation and confirmation of the eight geometrical cis/trans conjugated linoleic acid isomers 8,10 – through 11,13 – 18:2. Lipids 34, 873–877. Giesy J. G., M. A. McGuire, B. Shafii, and T. W. Hanson. 2002. Effect of dose of calcium salt of conjugated linoleic acid (CLA) on percentage and fatty acid content of milk fat in midlactation Holstein cows. J. Dairy Sci. 85, 2023–2029. Ip, C., S. Banni, E. Angioni, G. Carta, J. McGinley, H. J. Thompson, D. Barbano and D. Bauman. 1999. Conjugated linoleic acid enriched butter fat alters mammary gland morphogenesis and reduces cancer risk in rats. J. Nutr. 129, 2135–2142. Loor, J. J. and J. H. Herbein. 1998. Exogenous conjugated linoloiec acid isomers reduce bovine milk fat concentration and yield by inhibiting de novo fatty acid synthesis. J. Nutr. 128, 2411–2419. Moore C. E., H. C. Hafliger III, O. B. Mendivil, S. R. Sanders, D. E. Bauman, and L. H. Baumgard. 2004. Increasing amount of conjugated linoleic acid (CLA) progressively reduces milk fat synthesis immediately postpartum. J. Dairy Sci. 87, 1886–1895. Park Y., J. M. Storkson, K. J. Albright, W. Liu and M. W. Pariza. 1999. Evidence that the trans-10, cis-12 isomer of conjugated linoleic acid induces body composition changes in mice. Lipids, 34, 235–241. Perfield II J. W., A. Sœbø, and D. E. Bauman. 2004. Use of conjugated linoleic acid (CLA) enrichments to examine the effects of trans-8, cis-10 CLA, and cis-11, trans-13 CLA on milk-fat syntheis. J. Dairy Sci. 87, 1196–1202. Piperova L. S., B. B. Teter, I. Bruckental, J. Sampugna, and S. E. Mills. 2000. Mammary lipogenic enzyme activity, trans fatty and conjugated linoleic acids are altered in lacting dairy cows fed a milk fat depressing diet. J. Nutr. 130, 2658–2674.

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Einfluss der Suplementierung mit isisomerisiertem Mohnöl, erhöhter Konzentration der Linolsäure t10, c12 und c9, t11, auf Fettgehalt und Profil der Fettsäuren in der Schafmilch Von R. Bodkowski, Boz ˙ena Patkowska-Sokol⁄ a, Wiesl⁄ awa Walisiewicz-Niedbalska Im Experiment wurde das Futter der Milchschafe durch einen Zusatz von isomerem Mohnöl ergänzt, das in der Synthese um konjugierte Diene der Linolsäure trans-10, cis-12 und cis-9, trans- 11 in einer Menge von 34,3 % und 32,2 % angereichert wurde. Die Anwendung dieser Ergänzung führte zu einer Senkung des Fettgehaltes in der Schafmilch um 27 – 33 Prozent und gleichzeitig zu einer günstigen Modifizierung der Zusammensetzung der Fettsäuren. So kam es einerseits zu einer Steigerung der biologisch aktiven Fettsäure CLA und von Vaccensäure, anderenseits zu einer Senkung der aterogenischen Säuren (kurzkettige gesättigte und Palmitinsäure). Schlüsselwörter: Schafmilch, isomerisiertes Mohnöl, CLA, Milchfett, Fettsäureprofil