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Eudrilus Eugeniae THE DETERMINATION OF PROTEIN AND CERTAIN ESSENTIAL ELEMENTS IN EARTHWORM EUDRILUS EUGENIAE FED WITH DIFFERENT KINDS OF ORGANIC LITTERS Malliga Thongkheaw A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in Chemistry Suranaree University of Technology Academic Year 2010 การหาปริมาณโปรตีนและธาตุที่จาเป็นบางชนิด ในไส้เดอื นสายพนั ธ์ุ ยูดริลลสั ยูจนี ิอี ที่เลี้ยงด้วยขยะอินทรีย์ต่างชนิด นางสาวมัลลิกา ทองเขียว วทิ ยานิพนธ์นีเ้ ป็ นส่วนหนึ่งของการศึกษาตามหลกั สูตรปริญญาวทิ ยาศาสตรมหาบัณฑิต สาขาวิชาเคมี มหาวิทยาลัยเทคโนโลยีสุรนารี ปีการศึกษา 2553 มัลลิกา ทองเขียว : การหาปริมาณโปรตีนและธาตุที่จาเป็นบางชนิด ในไส้เดือนสายพันธุ์ ยูดริลลัส ยูจีนิอี ที่เล้ียงดว้ ยขยะอินทรียต์ า่ งชนิด (THE DETERMINATION OF PROTEIN AND CERTAIN ESSENTIAL ELEMENTS IN EARTHWORM EUDRILUS EUGENIAE FED WITH DIFFERENT KINDS OF ORGANIC LITTERS) อาจารย์ที่ปรึกษา : ผชู้ ่วยศาสตราจารย ์ ดร.ตริตาภรณ์ ชูศรี, 91 หน้า. การศึกษาน้ีมีวตั ถุประสงค ์ เพื่อหาปริมาณโปรตีนและธาตุที่จาเป็นบางชนิดในไส้เดือน ที่เล้ียง ดว้ ยขยะอินทรียต์ ่างชนิดกนั เพื่อเป็นข้อมูลในการนาไส้เดือนไปใช้เป็นอาหารสัตว์ในอนาคต ในการ ทดลองได้ใช้ไส้เดือนสายพันธุ์ยูดริลลัส ยูจินีอี ซึ่งเป็นสายพันธุ์ที่สามารถขยายพันธุ์ได้ดีและโตเร็ว สามารถย่อยขยะอินทรียไ์ ด้ดี ได้เปรียบเทียบปริมาณโปรตีนและปริมาณธาตุที่จาเป็นบางชนิดใน ไส้เดือนที่เล้ียงดว้ ยขยะอินทรียต์ ่างชนิดกนั ไดแ้ ก่ กากถว่ั เหลือง ใบสะเดา กล้วยสุก และลังกระดาษ กบั ไส้เดือนที่ไม่ให้อาหารอื่นเพ่ิมเติมซ่ึงเป็นตวั ควบคุม ผลการวิเคราะห์พบว่า ไส้เดือนมีโปรตีน 55.37-64.59 เปอร์เซ็นต์ ซ่ึงใกลเ้ คียงกบั ปริมาณโปรตีนที่มีอยูใ่ นเน้ือผง ผลการวิเคราะห์ธาตุปริมาณ หลักที่จาเป็น ในหน่วยเปอร์เซ็นต์ มีดงั น้ี แคลเซียม 0.52-0.65 คลอรีน 0.75-0.97 โพแทสเซียม 1.24-1.42 แมกนีเซียม 0.24-0.42 โซเดียม 0.44-0.50 ฟอสฟอรัส 0.24-0.27 กา มะถนั 0.21-0.27 และธาตุ ปริมาณนอ้ ยที่จา เป็นในหน่วยมิลลิกรัมต่อกิโลกรัม คือ เหล็ก 2,116-3,231 ทองแดง 46.3-52.5 สังกะสี 180.2-192.7 แมงกานีส 35.4-94.7 และโคบอลต์ 1.16-4.96 ผลการศึกษาทา ใหส้ รุปไดว้ า่ ไส้เดือนอาจใช้ เป็นแหล่งโปรตีนเสริมส าหรับอาหารสัตว์ได้เนื่องจากมีปริมาณโปรตีนค่อนขา้ งสูงและยังมีปริมาณ ธาตุที่จา เป็นตอ่ ชีวติ ในปริมาณที่ใกลเ้ คียงหรือมากกวา่ ที่มีอยใู่ นอาหารสัตวช์ นิดอื่น สาขาวิชาเคมี ลายมือชื่อนักศึกษา ปีการศึกษา 2553 ลายมือชื่ออาจารย์ที่ปรึกษา MALLIGA THONGKHEAW : THE DETERMINATION OF PROTEIN AND CERTAIN ESSENTIAL ELEMENTS IN EARTHWORM EUDRILUS EUGENIAE FED WITH DIFFERENT KINDS OF ORGANIC LITTERS. THESIS ADVISOR : ASST. PROF. TRITAPORN CHOOSRI, Ph.D. 91 PP. EARTHWORM EUDRILUS EUGENIAE/PROTEIN/ESSENTIAL ELEMENTS/ ORGANIC LITTERS The objective of this study was to determine the protein and certain essential elements contents in earthworms fed with different kinds of organic litters. The information would be useful for the utilization of earthworm as animal feed in the future. Earthworm Eudrilus eugeniae was used in the experiment because it has high rate of reproduction, grows extremely fast and is good in decomposing organic wastes. The protein and certain essential elements contents in earthworms fed with different kinds of organic wastes: soybean waste, neem leaves, ripe banana, and carton, were compared with the control group which was fed with nothing. The percentages of protein in earthworm were found to be in the range 55.37-64.59, comparable with meat powder meal. The results for the analyses of major essential elements contents of the earthworm in percentages were: Ca 0.52-0.65, Cl 0.75-0.97, K 1.24-1.42, Mg 0.24-0.42, Na 0.44- 0.50, P 0.24-0.27, and S 0.21-0.27. The trace essential elements in mg/kg were: Fe 2,116-3,231, Cu 46.3-52.5, Zn 180.2-192.7, Mn 35.4-94.7, and Co 1.16-4.96. III The results showed that earthworms could be the protein source for animal feeds since the protein contents were moderately high, and the essential elements contents were quite comparable or better than the other feeds. School of Chemistry Student’s Signature Academic Year 2010 Advisor’s Signature ACKNOWLEDGEMENTS The successful of the thesis is due to the help received from many people whom I want to express my acknowledgements. To my thesis advisor, Assistant Professor Dr. Tritaporn Choosri who gave me the opportunity to increase my knowledge and always support me when I had problems in my study and my research work. I also would like to thank her for the financial support on my experiments. I would like to thank the members of my thesis committee, Assistant Professor Dr. Kulwadee Rangsriwatananon, Associate Professor Dr. Jatuporn Wittayakun, and Assistant Professor Dr. Sanchai Prayoonpokarach. I also would like to thank all the lecturers of the School of Chemistry for their good attitude and useful advice, staffs of the Center for Scientific and Technological Equipment for their assistance and suggestion on the use of the instruments. I would like to acknowledge Suranaree University of Technology for the financial support during May 2007 to October 2007 through the financial support for good graduate student program. Finally, I would like to thank my parents and my sisters for their understandings, encouragements and supports during my study. Thank you to Miss Supissa Thongkheaw, who provided me some of the reagents used in my research. Malliga Thongkheaw CONTENTS Page ABSTRACT IN THAI...………………………………………………………………….I ABSTRACT IN ENGLISH..…………………………………………………………… II ACKNOWLEDGEMENTS…………………………………………………………….III CONTENTS…………………………………………………………………………….IV LIST OF TABLES ……………………………………………………………………VIII LIST OF FIGURES …………………………………………………………………….IX LIST OF ABBREVIATIONS………………………………………………………….XII CHAPTER I INTRODUCTION……………………………………………………………… 1 1.1 Background of the problem and significance of the study...…………….. 3 1.2 Research objectives……………………………………………….………6 1.3 Research hypotheses…………………………...………………………....6 1.4 Scope and limitation of the study……...……………………………….... 6 1.5 Expected results...………………………………………………………... 7 II LITERATURE REVIEWS……………………………………………………. 8 2.1 Earthworm as a source of protein …………………………………….…..8 2.2 Researches on E. eugeniae earthworm ……………………………….…11 2.3 Protein and essential elements……………………………………….…..15 2.3.1 Protein…………………………………………………….……..15 2.3.2 Essential elements………………………………………….……16 V CONTENTS (Continued) Page 2.4 Techniques used for the analyses………………………………………. 18 2.4.1 Atomic absorption spectroscopy……………………………….. 18 2.4.2 Atomic emission spectroscopy………………………………….19 2.4.3 CHNS analyzer………………………………………………….20 2.4.4 UV-Vis Spectroscopy…………………………...………………22 2.4.5 Vanadomolybdophosphoric acid method……………………….22 2.4.6 Kjeldahl method………………………………………………... 23 2.4.7 Volhard method…………………………………………………25 III MATERIALS AND METHODS ……………………………………......….. 26 3.1 Earthworm collection and breeding……………………………………..26 3.1.1 Preparation of organic litters for earthworm breeding…………. 26 3.2 Sample names………………………………………….………………..28 3.3 Cleaning of the glasswares and plasticwares……………………………28 3.4 Samples preparation for the analyses………………..…………………. 28 3.4.1 Preparation of earthworm……………………….………………28 3.4.2 Preparation of organic litters…………………….……………... 31 3.5 Protein analyses………………………………………...……………….31 3.6 Elemental analyses……………………………………………………... 36 3.6.1 Elemental analyses by atomic absorption spectroscopy technique………………………………………………………...36 3.6.1.1 Determination of calcium …………………………….39 VI CONTENTS (Continued) Page 3.6.1.2 Determination of cobalt……………….………………40 3.6.1.3 Determination of copper………………………………41 3.6.1.4 Determination of iron………………….……………... 42 3.6.1.5 Determination of potassium………...………………... 43 3.6.1.6 Determination of magnesium………………………… 44 3.6.1.7 Determination of manganese………………………….45 3.6.1.8 Determination of zinc ………………………………...46 3.6.1.9 Determination of sodium …………..…………………47 3.6.2 Determination of chloride by Volhard method………………… 48 3.6.3 Determination of phosphorus by Vanadomolybdophosphoric acid method………………………..…………………………… 51 3.6.4 Determination of sulfur by CHNS analyzer……………………. 53 3.7 The study on the effect of organic wastes on the biomass and the population of earthworms……………………………………………….55 VI RESULTS AND DISCUSSION……………………………………………… 57 4.1 Protein analyses ………………………………………………………...57 4.2 Elemental analyses …………………………………………………….. 61 4.2.1 Major essential elements……………………………………….. 62 4.2.2 Trace essential elements ………………………………………..70 4.3 The study on the effect of organic wastes on the biomass and the population of earthworms……………………………………………….78 VII CONTENTS (Continued) Page V CONCLUSIONS.................................................................................................8 0 REFERENCES ................................................................................................................ 82 CURRICULUM VITAE...................................................................................................90 LIST OF TABLES Table Page 2.1 Protein and water contents of the earthworms from the study of Lawrence and Millar (1945)………………………………………………………………….8. 2.2 Protein contents of some animal feeds…………………………….………….…16 2.3 Major and trace essential elements in some animal species………………….….17 4.1 Protein and nitrogen contents of the earthworms fed with different kinds of organic litters.……………………………………………………………….…...58 4.2 Protein and nitrogen contents of the organic litters used as feeds……………… 58 4.3 Protein contents in earthworm samples from other studies…………………….. 61 4.4 Major essential elements contents of the earthworms fed with different kinds of organic litters…………………………....…………………….……….……...63 4.5 Major essential elements contents of the organic litters used as feeds…………. 63 4.6 Major essential elements contents of the earthworms from
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