1. Identification of unsaturated fatty acid from discarded Goya seeds. Okinawa National College of Technology Bioresources engineering * Momoko Mashiyama, Yuka Shingaki, Eisuke Kuraya and Kei Hirayama

2. Our research addressed for added extra value to the unused Goya seeds that containing high amount of polyunsaturated fatty acid. Purpose

3. Unripe green Goya ・ largely cultivated in Okinawa. ・ can harvest from spring to autumn in Okinawa. ・ containing full of vitamin C. ・ well consumed by people in Okinawa. Back ground However the seeds are discarded all the time. Is it really useless ?

4. Back ground Harvest of Goya are about 25,000 ton in Japan. [1] [1] Ministry of Agriculture, Forestry and Fisheries, Research for production of local specialty vegetables. Annual statistic reports on 2008. Large quantity of Goya seeds about 500 ton have been discarded.

5. Methods ① Thin layer chromatography Flow controller Carrier gas Column Sample injector Column oven Mass spectrometry ③ GC-MS analysis Visualized by Iodine Experimental flow ② ・ Soxhlet extraction ・ Methyl esterification ① To find out plants hold rich polyunsaturated fatty acid. ② To prepare crude seeds oil ③ Analyzed by GC-MS.

6. ・ TLC plate : silica gel (whatman) ・ Solvent : hexane: diethyl ether: acetic acid (80:20:1, V/V) ・ Development for fatty acid: visualized by Iodine. Method ① Conditions for thin layer chromatography

7. Preparation of crude Goya seeds oil. ・ seeds were separated from Goya fruit. ・ dried at 50 ℃ for 24 hours. ・ homogenized by blender for 30 sec. Method ② Vertical angle of Goya seed Goya seed

8. ・ Soxhlet extraction crude oil from unripe green Goya seeds. ・ Methyl esterification n-Heptadecanoic acid was using as a internal standard. Fatty acid methyl ester was analyzed by GC-MS. Method ②

9. Column DB-23 （ Agilent Technologies ） (50 ％‐ cyanopropyl)-methyl polysiloxane ID 0.25mm×30m Film thickness 0.25µm The analytical conditions analyzing fatty acid methyl ester JET 240 ℃ DET 240 ℃ Split10 Total flow15.0 ml/min Linear velocity40.0 cm /s ① 50 ℃,1min ② 175 ℃,25 ℃ /min ③ 230 ℃,4 ℃ /min, 5.25min (HOLD) Method ③ GC-17A （ Shimadzu Corporation ） GC-MS analytical conditions

10. free fatty acid 1 2 3 4 1. Black rice 2. Goya seed 3. Oleic acid 4. Linoleic acid Thin layer chromatography of plant oil triglyceride We analyzed about 30 kinds of plants inhabited in Okinawa Island. Picture Showing triglyceride from black rice and Goya seed. Unripe Goya hold high amount of polyunsaturated fatty acid. Therefore, we focused on analyzing unsaturated fatty acid from Goya seeds. Results

11. Five fatty acids were detected from unripe green Goya seeds. These fatty acids were identified by mass spectrogram for using typical unsaturated and saturated fatty acid standards. Goya seeds oil analyzed by GC-MS Results

12. Stearic acid Total ion chromatogram of fatty acid from Goya seeds oil by GC-MS. Two saturated and three unsaturated fatty acids were detected. Unknown polyunsaturated fatty acid （ No.5 ） intensity Unknown polyunsaturated fatty acid （ No.5 ） 0.5 1.5 1.0 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0 ×10 7

13. No.5 fatty acid51.5% Linoleic acid 9.6% Oleic acid6.8% Stearic acid28.1% Palmitic acid4.0% Ratio of fatty acid contained in the Goya seeds. Results 67.9% Unsaturated 32.1% Saturated No.5 fatty acid was polyunsaturated fatty acid and which account for over half of all fatty acids.

14. α- linolenate methyl ester standard No.5 polyunsaturated fatty acid Results Mass spectrum No.5 polyunsaturated fatty acid has similar mass spectrum patern with α- linolenate except peak height for 292.

15. Data from total ion chromatogram, no.5 polyunsaturated fatty acid has 20 carbon. Results

16. Predicted structure for no.5 polyunsaturated fatty acid The position of double bond in this polyunsaturated fatty acid was determined by fragment ion of m/z 292. Results

17. ・ Five fatty acid were detected. →Goya seeds hold three unsaturated fatty acids. ・ Peak no.5 polyunsaturated fatty acid has account for over half of all fatty acids. Conclusion

18. Polyunsaturated fatty acid … has various functionalities. For Example Reduce … risk against for cardiovascular disease and for blood clotting in the brain. Conclusion

19. 1.Discarded resource of seeds from unripe green Goya might be able to use for functional food or supplements. 2. Reduce 500 ton of waste every year. Contribute to environmental conservation. Conclusion ？

20. 1 ． Identifying peak no.5 of unknown polyunsaturated fatty acid. 2 ． Evaluate its bio-functionality. Future work Find out for the potential of bio-functional value to the useless Goya seeds!

21. Making the new bio-functional products from Goya seeds. ・ activate economy in Okinawa. ・ contribute to environmental conservation. Final goal in our research