FATTY ACID COMPOSITION, CHOLESTEROL-

Transcription

1 FATTY ACID COMPOSITION, CHOLESTEROL- AND INTRAMUSCULAR FAT- CONTENT OF LOIN MUSCLES FROM FINISHER PIGS FED SOY LECITHIN SUPPLEMENT 3A-112 Report prepared for the Co-operative Research Centre for an Internationally Competitive Pork Industry By Eric Ponnampalam, Gavin Rose, Matthew Kerr, David Allen and Ian McCauley Future Farming Systems Research Department of Primary Industries Victoria March 2011 This publication may be of assistance to you but the State of Victoria and its employees do not guarantee that the publication is without flaw of any kind or is wholly appropriate for your particular purposes and therefore disclaims all liability for any error, loss or other consequence which may arise from you relying on any information in this publication. Established and supported under the Australian Government s Cooperative Research Centres Program

2 Executive Summary This project was undertaken by the Victorian Department of Primary Industries to provide quantitative analysis of samples collected from an experiment undertaken by Pork CRC researchers in pigs fed a dietary supplement involving with soy lecithin. The analysis presented covers the fatty acid composition and cholesterol content of these samples. The analysis will be used in combination with other findings of the research study to better understand the dietary factors affecting pig production and pork meat quality. i

3 Table of Contents Executive Summary... i Introduction... 1 Methodology... 1 Outcomes... 1 Application of Research... 3 Conclusion... 4 Limitations/Risks... 4 Recommendations... 4 References... 4 Appendix 1: Method for fatty acid extraction and quantification... 5 Appendix 2: Method summary for determination of cholesterol in pork... 6 ii

4 Introduction An experiment was undertaken with pigs fed a diet supplemented with soy lecithin by Prof. Frank Dunshea at the University of Melbourne Dookie campus animal facilities. Pork muscle samples (longissimus dorsi) were collected from pigs at 24 h post-slaughter during this study. These muscle samples were transferred to the Department of Primary Industries, Werribee Centre, in November 2010 for the determination of fatty acid composition and cholesterol content. Samples were supplied with ID, but no information on treatment codes. DPI was to be responsible for the completion of the analytical work and provide results in Excel form with a description of the methodologies used for the analysis. Methodology Individual fatty acid content and the total composition were quantified by capillary Gas Chromatography (see Appendix 1 for details). Fatty acid peaks were identified using a reference standard (Supelco C4-C24 mix, Sigma Aldrich), which was run in each batch. Fatty acid and total fat results are expressed as mg/100 g of meat. The cholesterol content of the pork was determined using Gas Chromatography - Flame Ionization Detector (see Appendix for 2 for details). The concentration of cholesterol in samples was calculated from the quadratic regression of concentration against the ratio of cholesterol response to an internal standard response. Outcomes Results were tabulated in excel sheet format, with the results attributed to the sample ID as supplied. The electronic documents have been uploaded to Pork CRC imap management system. The details of individual fatty acid content and total fatty acid composition in pork are contained the Excel document - Fatty acid RESULTS-lecithin study.xls. The results are also presented in Table 1. Values for total fat do not include results of C4:0 as the uniformly high valued for this component are unusual. In general C4:0, C6:0 and C8:0 fatty acids are very small compared with other medium and long chain saturated fatty acids and monounsaturated fatty acids in meat (refer articles Enser et al. (1996); Haak et al. (2008) for details.). The observed level for C4:0 in the present study might have been due to highly volatile contaminants introduced during the preparation of the samples for analysis. The details of cholesterol content in pork are contained in the excel document - Lecithin study- cholesterol results.xls. The results are also presented in Table 2. 1

5 Table 1 - Fatty acid composition and total fat content (mg/100 g meat) Sample No C4:0 C6:0 C8:0 C10:0 C11:0 C12:0 C13:0 C14:0 C14:1 C15:0 C15:1 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1n9c+t C18:2n6t C18:2n6c C18:3n6 C18:3n3 # CLA-1 # CLA-2 # C18:4n3 C20:0 C20:1n9 C20:2n6 C21:0 C20:3n6 C20:4n6 C20:3n3 C22:0 C20:5n3 C22:1n9 C22:2n6 C23:0 C22:3n6 C22:4n6 C24:0 C22:5n3 C24:1n9 C22:6n3 total muscle fat

6 Table 2 - Cholesterol content of meat samples (mg/100 g meat) muscle cholesterol sample No mg/kg mg/100g Application of Research The results presented in this report will be used along with other measurements in scientific publications and industry articles related to the effect of soy lecithin on pig production and meat quality. 3

7 Conclusion Analysis of meat samples has been conducted to determine the amounts of fatty acids, total fat and cholesterol. Limitations/Risks These results are to be used in combination with the appropriate codes to allow interpretation with respect to the treatments used in the study. Recommendations None References L. Haak, S. De Smet, D. Fremaut, K. Van Walleghem and K. Raes. (2008). Fatty acid profile and oxidative stability of pork as influenced by duration and time of dietary linseed or fish oil supplementation.,j Anim Sci., 86: M. Enser, K. Hallett, B. Hewitt, G.A.J. Fursey and J.D. Wood. (1996). Fatty acid content and composition of UK beef and lamb muscle in relation to production system and implications for human nutrition. Meat Sci., 42, Ponnampalam, E.N., Warner, R.D., Kitessa, S., McDonagh, M.B., Pethick, D.W., Allen, D., Hopkins, D.L., Influence of finishing systems and sampling site on fatty acid composition and retail shelf-life of lamb. Anim. Prod. Sci. 50,

8 Appendix 1: Method for fatty acid extraction and quantification Muscle samples were freeze-dried and a homogeneous sample of 0.6 g of ground material was used for the determination of fatty acid composition using a rapid modified procedure developed from the method reported by O Fallon et al One hundred µl of nonadecanoic acid methyl ester (C19: 0, Sigma Aldrich, Castle Hill, NSW, Australia) was added to muscle samples as an internal standard dissolved in chloroform (10 mg C19 : 0/mL CHCl 3 ). The contents were hydrolysed using 0.7 ml of 10 N KOH in water and 5.3 ml of methanol to form free fatty acids. After mixing well with vortex, the contents were incubated at 55 C for 1.5 h with vigorous mixing at 20-min intervals and then cooled to room temperature using tap water. Upon cooling, the contents were mixed with 0.6 ml of 24 N sulfuric acid in water and mixing, incubation and cooling process proceeded as above. After cooling the tubes to room temperature, the fatty acid methyl ester (FAME) was separated with 1 ml of hexane solvent by mixing for 5 min and centrifuging at 2000g for 10 min. Two-hundred µl of hexane containing FAME was collected into a GC vial and fatty acid fractions were quantified by capillary GC (HP INNOWAX 60 m 0.25 mm, 0.5 micron, Agilent J & W Scientific, Santa Clara, CA, USA). Fatty acid peaks were identified using a reference standard (Supelco C4-C24 mix, Sigma Aldrich), which was run in each batch. Fatty acid levels in the muscles are reported in mg/100 g meat as this relates to nutrition information for the labelling of foods according to Food Standards Australia & New Zealand (FSANZ). Reference: Ponnampalam, E.N., Warner, R.D., Kitessa, S., McDonagh, M.B., Pethick, D.W., Allen, D., Hopkins, D.L., Influence of finishing systems and sampling site on fatty acid composition and retail shelf-life of lamb. Anim. Prod. Sci. 50,

9 Appendix 2: Method summary for determination of cholesterol in pork Extraction: 1. Weigh 2g of sample into a 150 ml QF (Quickfit ) Erlenmeyer flask and record the mass to the nearest 0.01 g. Include an homogenised luncheon meat control sample and a spiked recovery of cholesterol in each batch. 2. Add 40 ml special methylated spirits to the flask and add 10 ml of 100% KOH 3. Connect the flask to a condenser and reflux on a stirrer/hotplate for 30 minutes with magnetic stirring. 4. Remove the flask from the condenser and cool rapidly in an ice bath. 5. Remove the stirrer from the flask and rinse any residue on the stirrer into the flask with a small amount of water followed by a small amount of special methylated spirits. 6. Transfer the contents of the flask quantitatively to a 250 ml separating funnel using 50 ml of water followed by 10 ml of special methylated spirits then 50mL of hexane. Shake the flask vigorously for 2 minutes. 7. Allow phases to separate. If emulsions have formed, they may be broken by adding approximately 5 ml of special methylated spirits and swirling the flask gently, or alternatively by centrifuging the solution. 8. Transfer the lower aqueous fraction to another separating funnel and reextract with 20 ml of hexane. Remove hexane layer and combine with original hexane extract. 9. Re-extract aqueous fraction with a further 20 ml of hexane, and combine with previous hexane extracts. 10. Wash the combined hexane extracts with 3 x 100 ml of water. 11. The washed hexane is then transferred to a dry 100 ml volumetric flask containing sufficient anhydrous sodium sulphate to dry the extract and made to the mark with hexane and mixed. 12. Transfer a 10 ml, aliquot to a graduated 15 ml tube and add 100 µl of internal standard solution (500 mg/l 5α-cholestane). 13. Reduce sample to 2 ml under a flow of nitrogen and transfer 1mL to a GC vial. 14. Make up four GC calibration standards of cholesterol 10, 20, 50 & 100 mg/l containing internal standard solution at a matching level to the sample solutions. 6

10 GC-FID method: Instrument Agilent 6890 GC-FID with 7673 autosampler OVEN Initial temp: 220 o C (On) Maximum temp: 325 o C Initial time: 4.00 min Equilibration time: 0.50 min Ramps: No. :Rate: Final temp :Final time 1 :20.00 o C/min:280 o C:5.00 min 2 0.0(Off) Post temp: 0 o C Post time: 0.00 min Run time: min INLET (SPLIT/SPLITLESS) Mode: Split Initial temp: 250 o C (On) Pressure: 19.1 psi (On) Split ratio: 20:1 Split flow: 50.0 ml/min Total flow: 54.1 ml/min Gas saver: On Saver flow: 15.0 ml/min Saver time: 2.00 min Gas type: Helium COLUMN Capillary Column Model Number: JW Methyl siloxane (50% Phenyl) DB-17 Max temperature: 300 o C Nominal length: 15.0 m Nominal diameter: µm Nominal film thickness: µm Mode: constant flow Initial flow: 2.5 ml/min Nominal init pressure: 19.1 psi Average velocity: 69 cm/sec DETECTOR (FID) Temperature: 300 'C (On) Hydrogen flow: 40.0 ml/min (On) Air flow: ml/min (On) Mode: Constant makeup flow Makeup flow: 45.0 ml/min (On) Makeup Gas Type: Nitrogen The concentration of cholesterol in samples was calculated from the quadratic regression of concentration against the ratio of cholesterol response (peak area) to internal standard response (peak area). 7