INFLUENCE OF RAPE SEEDS THERMAL PROCESSING ON THE LIMITATION OF LYPOLYSIS PROCESSES AND BIOHYDROGENATION OF UNSATURATED FATTY ACIDS IN RUMEN (PART I)

Acta Sci. Pol., Medicina Veterinaria 9(3) 2010, 3-10 INFLUENCE OF RAPE SEEDS THERMAL PROCESSING ON THE LIMITATION OF LYPOLYSIS PROCESSES AND BIOHYDROGENATION OF UNSATURATED FATTY ACIDS IN RUMEN (PART I) Robert Bodkowski, Bożena Patkowska-Sokoła, Wojciech Zawadzki Wrocław University of Environmental and Life Sciences 1 Abstract. The aim of the present work was to assess the efficiency of thermal treatment of rape seeds in the reduction of its fat lypolysis in rumen and unsaturated fatty acids biohydrogenation. The studies were conducted using in sacco method with polyamide sacs on 6 rams of Merino breed with simple fistula of rumen. The protection of caked rape seeds involved its thermal processing at temperature 120 o C for 30 minutes. Raw and thermally processed rape seeds were incubated in rumen for 2, 4, 8, 16 and 24 hours and thermal decomposition of air dry mass, raw fat, fatty acids with various degree of saturation and particular fatty acids were studied. On the basis of the obtained results, it was found that thermal processing of rape seeds resulted in significant reduction of occurring in rumen lypolysis of their fat and biohydrogenation of unsaturated fatty acids (mono- as well as polyunsaturated). Moreover, it was found that in the seeds that were processed with thermal method, monounsaturated fatty acids were protected better than polyunsaturated ones. Key words: rumen, rape seeds, thermal protection, fat and unsaturated fatty acids INTRODUCTION Fats of animal origin contain mainly saturated fatty acids that favor the development of various diseases referred as civilization ones [Ziemlański and Budzyńska-Topolowska 1991]. In the case of monogastric animals, the advantageous modification of fatty acids profile (increase in the content of unsaturated fatty acids and decrease in saturated ones) can be obtained by an enrichment of their diet with vegetable fat rich in unsaturated fatty acids [Ajuyah et al. 1991, Chachułowa et al. 1997, Morgan et al. 1992]. Corresponding author Adres do korespondencji: Robert Bodkowski, Institute of Animal Breeding, Wrocław University of Environmental and Life Sciences, ul. Chełmońskiego 38C, 50-630 Wrocław, e-mail: robert.bodkowski@up.wroc.pl

4 R. Bodkowski i in. However, the modification of fatty acids composition in ruminants is more difficult, due to a specific structure of their digestive tract and microflora present in their stomachs. Exceeding the fat level in diet of ruminants above 5 7%, disadvantageously influences metabolism and digestion in rumen [Grummer 1988]. Moreover, reducing environment of rumen causes that the majority of unsaturated fatty acids are subject to hydrogenation process in rumen [Mills et al. 1970]. One of the methods to avoid reduction of the quantity of fat in the diet for ruminants as well as protection of unsaturated fatty acids from transformation in rumen, is the application of fat in the protected form. The aim of the present work was to assess the effect of thermal processing of caked rape seeds on the reduction of the processes of their fat lypolysis and biohydrogenation of unsaturated fatty acids in rumen. MATERIAL AND METHODS In the preliminary studies on rape seeds of 00 species, the analyses of the content of dry matter, total protein and raw fat [Ładoński and Gospodarek 1986] as well as determination of fatty acids profile were conducted. In order to perform chromatographic determination, caked samples of rape seeds were esterified twice with Folch mixture (chloroform in methanol in the ratio 2:1). Lipid fraction of fat isolated in this way, after filtration through anhydrous sodium sulfate, was subject to hydrolysis with 0.5 mol/dm 3 of KOH and esterification with 14% BF 3 in the presence of methanol. In the next stage, methyl esters of fatty acids in the quantity 1μl were transferred to gas chromatograph PU4410 (Philips) with flame-ionic detector (FID) and 105 m long capillary column Rtx-2330 at Industrial Chemistry Research Institute, Warsaw, Poland. In the experimental part at the laboratory of Institute of Animal Breeding, Wrocław University of Environmental and Life Sciences, caked rape seeds of 00 species were processed thermally. The protective agent was temperature of 120 o C for 30 minutes. In the prepared seeds, the content of the dry matter, total protein and raw fat and the composition of fatty acids was determined again. In the second part of the study, decomposition of air dry matter (PSM) and raw fat (TS) as well as mono- and polyunsaturated fatty acids in both raw and prepared seeds was assessed. The studies were conducted with in sacco method with the application of polyamide sacs on 6 rams of Polish Merino breed with simple fistula to the rumen. In order to perform the study, 4 g of raw and protected with thermal method caked rape seeds were introduced to the rumen and incubated for 2, 4, 8, 16 and 24 hours. The experiment was conducted in the following system: 1 sac x 6 animals, that is in 6 repetitions for each type of the material studied and incubation time, according to the procedure given by Ørskov and McDonald [1979]. Due to insufficient quantity of the material studied that remained after incubation, in order to perform further analyses, the samples were simultaneously incubated and raw fat (in two repetitions) and the fatty acids composition were determined. Extraction of fat and chromatographic analyses of fatty acids composition were carried out at Industrial Chemistry Research Institute, Warsaw, Poland. Acta Sci. Pol.

Influence of rape seeds thermal processing... 5 To assess the significance of differences between analysed parameters of PSM, TS and fatty acids, SAS (1996) statistical software was used. Due to very low levels of the analyzed parameters (obtained by longer incubation times in the rumen), all the results presented in tables were 100-times multiplied. RESULTS Table 1 presents the chemical composition of rape seeds and their fatty acids profile. The content of total protein in rape seeds was 19%, and of raw fat 46 %. About 89% of all fatty acids in rape seeds consisted of unsaturated fatty acids, and 11% of saturated fatty acids. In the group of unsaturated fatty acids, monounsaturated fatty acids dominated 71%, mainly oleic acid C 18:1. Thermal processing that rape seeds were subject to did not influence significantly the content of dry matter, total protein and raw fat as well as fatty acids composition (data not published). Table 1. The composition of raw rape seeds [%] Tabela 1. Skład nasion rzepaku Specification Wyszczególnienie Dry matter Sucha masa Protein Białko Raw fat Tłuszcz surowy Fatty acids Kwasy tłuszczowe: C 8:0 C 10:0 C 12:0 C 14:0 C 16:0 C 16:1 C 17:0 C 18:0 C 18:1 C 18:2 C 18:3 C 20:0 C 20:1 C 22:0 C 22:1 Content [%] Zawartość 94.80 19.20 46.10 0.10 0.10 0.10 1.00 5.60 0.40 0.30 2.40 59.50 18.40 6.80 0.70 3.00 0.60 0.90 The results concerning the rate of decomposition of raw and thermally processed rape seeds and lypolysis of their fat in rumen are presented in table 2. Decomposition of fat in raw rape seeds after 2 hours of incubation was 67%; after 4 hours 77%; after 8 hours 90%, however after 16 and 24 hours almost 100%. In the case of processed seeds, decomposition of fat was as follows: 44%; 56%; 74%; 85% and 89%. The differences detected in the rate of lypolysis of raw fats and thermally prepared rape seeds were statistically highly significant. Medicina Veterinaria 9(3) 2010

6 R. Bodkowski i in. Table 2. Decomposition of dry matter and fat in raw and thermally processed rape seeds in rumen of sheep [g] Tabela 2. Rozkład w żwaczu suchej masy oraz tłuszczu w surowych i poddanych obróbce termicznej nasionach rzepaku Specification Wyszczególnienie Dry matter Sucha masa Raw fat Tłuszcz surowy Type of seeds Rodzaj nasion Raw Surowe Protected Chronione Raw Surowe Protected Chronione Time of seeds incubation in rumen [hours] Czas inkubacji nasion w żwaczu [godz.] 0 2 4 8 16 24 400.0 400.0 184.6 188.2 150.9 A 103.5 A 47.6 A 21.2 A 11.7 A 240.0 A 194.5 B 166.0 B 107.9 B 96.8 B 60.6 A 42.4 A 19.3 A 1.7 A 0.9 A 104.6 B 82.7 B 49.8 B 27.3 B 21.4 B A,B difference significant on the level of P 0.01 A,B różnice istotne na poziomie P 0,01 Table 3 presents the results concerning the degree of unsaturated fatty acids decomposition. After 2, 4, 8, 16 and 24 hours of incubation, decomposition of unsaturated fatty acids in raw and processed rape seeds was 67 and 45%; 77 and 56%; 90 and 74%; 99 and 86%, 99 and 89%, respectively (all differences were statistically highly significant). Table 3. Decomposition of fatty acids with different degree of saturation of raw and processed thermally rape seeds in rumen [g] Tabela 3. Rozkład w żwaczu kwasów tłuszczowych o różnym stopniu nasycenia w surowych i poddanych termicznej obróbce nasionach rzepaku Type of seeds Rodzaj nasion Incubation time [hours] Czas inkubacji [godz.] 0 163.6 167.3 Unsaturated Nienasycone Fatty acids [g)] Kwasy tłuszczowe Monounsaturated Jednonienasycone 117.1 118.0 2 53.6 A 40.9 A 12,7 A 91.9 B 70.6 B 21,2 B 4 37.5 A 73.5 B 28.7 A 56.6 B 8,6 A Polyunsaturated Wielonienasycone 46,5 49,2 16,9 B 8 16.4 A 13.2 A 3,2 A 43.1 B 34.0 B 9,1 B 16 1.4 A 1.7 A 0,1 A 23.2 B 20.2 B 3,0 B 24 1.3 A 1.2 A 0,08 A 18.9 B 16.8 B 2,1 B A, B differences significant on the level of P 0.01 A, B różnice istotne na poziomie P 0,01 Acta Sci. Pol.

Influence of rape seeds thermal processing... 7 The process of rape seeds heating also reduced the rate of single unsaturated fatty acids biohydrogenation in rumen. In the case of seeds that were prepared with thermal method, the process of biohydrogenation of oleic C 18:1 (fig. 1), linoleic C 18:2 (fig. 2) and linolenic C 18:3 (Fig. 3) acids was significantly slower than raw seeds. 120 109,7111,1 100 80 65,7 B 52,8 B (g) 60 40 38,3 A 26,6 A 31,3 B 18,7 B 15,7 B 20 12 A 1,1 A 0,9 A 0 0 2 4 8 16 24 Incubation time [hours] Czas inkubacji [godz.] raw rape seeds surowe nasiona rzepaku thermally processed rape seeds nasiona rzepaku poddane obróbce termicznej Fig. 1. Decomposition of oleic acid C18:1 in raw and thermally processed rape seeds after incubation in the rumen [g] Rys. 1. Rozkład kwasu oleinowego C18:1 w surowych i poddanych obróbce termicznej nasionach rzepaku po inkubacji w żwaczu (g) 35 30 25 20 15 10 5 0 15,4 B 9,4 A 6,3 B 33,9 32,8 12,5 B 6,4 A 2,4 A 0,2 A 2,3 B 0,1 A 1,6 B 0 2 4 8 16 24 Incubaction time [hours] Czas inkubacji [godz.] raw rape seeds surowe nasiona rzepaku thermally processed rape seeds nasiona rzepaku poddane obróbce termicznej Fig. 2. Decomposition of linoleic acid C18:2 in raw and thermally processed rape seeds after incubation in the rumen [g] Rys. 2. Rozkład kwasu linolowego C18:2 w surowych i poddanych obróbce termicznej nasionach rzepaku po inkubacji w żwaczu Medicina Veterinaria 9(3) 2010

8 R. Bodkowski i in. (g) 3,2 A 2,3 A 2,7 B 14 12,5 12 11,2 10 8 5,8 B 6 4,4 B 4 2 0,9 A 0,03 A 0,7 B 0,02 A 0,5 B 0 0 2 4 8 16 24 Incubaction time [hours] Czas inkubacji [godz.] raw rape seeds surowe nasiona rzepaku thermally processed rape seeds nasiona rzepaku poddane obróbce termicznej Fig. 3. Decomposition of linoleic acid C18:3 in raw and processed thermally rape seeds after incubation in the rumen (g) Rys. 3. Rozkład kwasu linolenowego C18:3 w surowych i poddanych obróbce termicznej nasionach rzepaku po inkubacji w żwaczu (g) DISCUSSION In the present study, the effect of rape seeds thermal processing on the reduction of some transformations that occur in their fat in rumen of ruminants was investigated. The obtained results enabled to assess that the process of rape seeds thermal processing significantly reduced the rate and the extent of lypolysis of fat and biohydrogenation of unsaturated fatty acids in the rumen. The application of high temperature (120 o C for 30 min) resulted in partial denaturation of protein of rape seeds which decreased their susceptibility to proteolysis and deamination in the rumen. Due to the increase of the resistance of protein integuments on microbiological proteolysis of fat molecules in non-disintegrated form, the seeds passed through the rumen (ph 6 7) into stomach, where more acidic environment (ph 2 3) caused hydrolysis of protein integument and release of fat. For this reason, enzymatic decomposition of fat to fatty acids occurred not in the rumen but in small intestine. The efficiency of thermal method in the protection of fat from other seeds of oil plants was also demonstrated by other authors [Kowalski et al. 1995, Popiołek 1999]. Interesting results for the protection of fat in plants were also obtained by hydrothermal processing that involved heating with water vapor [Kozłowska et al. 1987]. The efficient way of protection of fat is also providing its in the form of calcium or magnesium salts [Kraszewski et al. 1993, Sklan et al. 1990]. In the case of liquid plant oils, a method of protection is also encapsulation in hardened gelatin capsules resistant to decomposition in the rumen [Edmondson et al. 1974, Patkowska-Sokoła and Bodkowski 1995, Patkowska-Sokoła et al. 1994]. Acta Sci. Pol.

Influence of rape seeds thermal processing... 9 CONCLUSIONS 1. Thermal processing of rape seeds significantly reduced the rate and extent of fat lypolysis and biohydrogenation of unsaturated fatty acids in rumen of sheep. 2. In the seeds that were subject to thermal processing, monounsaturated and polyunsaturated fatty acids were protected to the larger extent against the process of biohydrogenation. 3. Due to heating treatment of rape seeds, a part of fatty acids, i.e. oleic C 18:1, linoleic C 18:2 and linolenic C 18:3, passed through the rumen to stomach in an unchanged form. REFERENCES Ajuyah A.O., Lee K.H., Harding R.T., Sim J.S., 1991. Changes in the yield and in the fatty acids composition of whole carcass and selected meat portions of broiler chickens fed full fat oilseed. Poulty Sci., 70, 2304 2314. Chachułowa J., Sawosz E., Sokół J.L., Skomiał J., 1997. Wpływ stosowania nasion rzepaku w mieszankach dla tuczników na skład chemiczny mięsa. Konf. Nauk. Współczesne zasady żywienia świń. Jabłonna, 3 4 czerwca 1997, 130 133. Edmondson L.F., Yoncoskie R.A., Rainey N.H., Douglas F.W., 1974. Feeding encapsulated oils to increase the polyunsaturation in milk and meat fat. J. Am. Oil Chem. Soc., 51 (3), 72 78. Grummer R.R., 1988. Influence oil fatty acids on ruminal fermentation and nutrient digestability. J. Dairy Sci., 71, 117 123. Kowalski Z.M., Marszałek A., Micek P., 1995. Wpływ temperatury na rozkład białka w żwaczu. XXV Sesja Naukowa KŻZ KNZ PAN Koncentraty białkowe i energetyczne w mieszankach treściwych i dawkach pokarmowych. Poznań 8 9 listopada 1995, 139 142. Kozłowska H., Rotkiewicz D., Kozłowski M., Faruga A., Mikulski D., 1987. Nutritional value of rapeseed meals obtained from hydrothermally treated seeds. Proc. 7 th Intern. Rapeseed Congress. Poznań 11 14 maja 1987, 1668 1673. Kraszewski J., Wawrzyńczak S., Bielak F., 1993. Przydatność dodatku preparatu tłuszczowego Erafet w postaci mydeł wapniowych i magnezowych w żywieniu krów wysokomlecznych. Rocz. Nauk. Zoot., 20 (2), 183 191. Ładoński W., Gospodarek T., 1986. Podstawowe metody analityczne produktów żywnościowych. Warszawa Wrocław. Mills S.C., Scott T.W., Russel G.R., Smith H.M., 1970. Hydrogenation of C18: unsaturated fatty acids by pure cultures of rumen microccocus. Australian J. Biol. Sci., 23, 1109 1115. Morgan C.A., Noble R.C., Cocchi M., McCartney R., 1992. Manipulation of the fatty acids composition of pig meat lipids by dietary means. J. Sci. Food Agric., 58, 357 368. Ørskov E.R., McDonald I., 1979. The estimation of protein degrability in rumen from incubation measurements weighted according to rate of passage. J. Anim. Sci., 92, 499 503. Patkowska-Sokoła B., Bodkowski R., 1995. Wpływ podawania utwardzonych żelatynowych kapsułek z olejami roślinnymi na skład kwasowy tłuszczu jagnięcego. Konf. Perspektywy hodowli zwierząt w Polsce. Wrocław 18 19 września 1995, 2, 113 116. Patkowska-Sokoła B., Bodkowski R., Dobrzański Z., Bodak E., 1994. Improve of lamb meat quality through supplementing of diet with protected plant oils. 8 th Intern. Congress of Animal Hygiene, St. Paul Minnesota USA. 12 16 sierpnia 1994, 46 51. Popiołek R., 1999. Wzbogacanie diety owiec nasionami roślin oleistych jako metoda modyfikacji składu kwasów tłuszczowych mleka i sera owczego. Praca doktorska AR Wrocław. Medicina Veterinaria 9(3) 2010

10 R. Bodkowski i in. Sklan D., Nedar L., Arieli A., 1990. Effect of feeding different levels of fatty acid or calcium soaps of fatty acids on digestion and metabolisable energy in sheep. Anim. Prod., 15, 93 98. Ziemlański Ś., Budzyńska-Topolowska J., 1991. Tłuszcze pożywienia i lipidy ustrojowe. Wydawnictwo Naukowe PWN, Warszawa. WPŁYW TERMICZNEJ OBRÓBKI NASION RZEPAKU NA ZAHAMOWANIE PROCESU LIPOLIZY TŁUSZCZU I BIOUWODOROWANIA NIENASYCONYCH KWASÓW TŁUSZCZOWYCH W ŻWACZU (CZĘŚĆ I) Streszczenie. Celem pracy była ocena wpływu zabiegu ogrzewania nasion rzepaku na ograniczenie procesów lipolizy tłuszczu oraz biouwodorowania nienasyconych kwasów tłuszczowych w żwaczu. Badania przeprowadzono metodą in sacco, z użyciem poliamidowych woreczków, na 6 tryczkach rasy merynos z przetokami prostymi do żwacza. Zabieg termicznej ochrony ześrutowanych nasion rzepaku polegał na ich ogrzewaniu w temp. 120 o C przez 30 min. Surowe i preparowane termiczną metodą nasiona rzepaku inkubowano w żwaczu w czasie 2, 4, 8, 16 oraz 24 godzin i oznaczano rozkład: powietrznie suchej masy, tłuszczu surowego, grup kwasów tłuszczowych o różnym stopniu nasycenia oraz poszczególnych kwasów tłuszczowych. Na podstawie uzyskanych wyników stwierdzono, że termiczna obróbka nasion rzepaku statystycznie istotnie ograniczyła proces lipolizy tłuszczu w żwaczu oraz biouwodorowanie nienasyconych kwasów tłuszczowych (zarówno jedno-, jak i wielonienasyconych). Ponadto zaobserwowano, że kwasy tłuszczowe jednonienasycone w większym stopniu chronione były przed biouwodorowaniem niż wielonienasycone. Słowa kluczowe: żwacz, nasiona rzepaku, ochrona termiczna, tłuszcz i nienasycone kwasy tłuszczowe Accepted for print Zaakceptowano do druku: 30.09.2010 For citation Do cytowania: Bodkowskie R., Patkowska-Sokoła B., Zawadzki W., 2010. Influence of rape seeds thermal processing on the limitation of lypolysis process and biohydrogenation of unsaturated fatty acids in rumen (part I). Acta Sci. Pol. Med. Vet., 9(3). Acta Sci. Pol.