STUDY ON EXTRACTION OF CHLOROPHYLL FROM SIKLWORM WASTE (FEACES BOMBYCUM)

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET INCLUDEPICTURE "http://www.vnua.edu.vn/vie/userfiles/image/gioithieu/logo-vnua.jpg" \* MERGEFORMATINET UNDERGRADUATE THESIS TITLE: STUDY ON EXTRACTION OF CHLOROPHYLL FROM SIKLWORM WASTE i COMMITMENT I hereby declare that all the information and data provided in this study are true, accurate, and not used in any other reports I assure all of the information in my study is referred at the end of my thesis and I have already give all of my respect and appreciation to every people helping me complete my thesis Hanoi, December 2018 Student Tran Thi Oanh ii ACKNOWLEDGEMENTS Firstly, I would like to express my gratitude to my supervisor Dr Bui Thi Thu Huong for providing me an opportunity to the final work in Vietnam National University of Agriculture and giving me all support which made me complete the project I would like to express my deepest gratitute to Dr Hoang Thi Bich and Dr Do Tien Lam As my teachers and supervisors, their guidance not only fully supported my study but also encouraged, inspired me to complete my thesis I am grateful to all of those with whom I have had the pleasure to work during this thesis semester Each of the members of Department of Protein Biochemistry has provided me extensive personal and professional guidance and taught me great lessons about both scientific research and life in general I would especially like to thank Assoc Prof Pham Hong Minh, the head of “Clean Technology and Materials Development Center”, who gave me permission to perform the necessary works and utilize the equipment in laboratory, Institute of Institute of Natural Products Chemistry (INPC) I have furthermore to thank M.S Tran Thi Hoai Van, M.S Vu Thi Thu Le, for their support, interest and valuable help under the thesis period Also, I am thankful to and fortunate enough to get constant encouragement, support and guidance from Dr Do Trung Sy, M.S Tran Huu Quang of Institute of Chemistry Concurrently, I would like to thank the topics of "Research on chlorophyll extract from silkworm excrement" and "Research on technological process of producing chlorophyll food additives (INS 140), copper chlorophyll (INS 141) and propylene glycol alginate (INS 405) from natural materials" that provided financial support so that I could complete this thesis It is also give my thankfulness to my family, to all of my friends, for sharing my difficulties, and giving me various used advices during the process of learning and studying Thank you very much! Hanoi, December 2018 Student Tran Thi Oanh iii TABLE OF CONTENT ACKNOWLEDGEMENTS .iii ABBREVIATIONS vii ABSTRACT viii INTRODUCTION LITERATURE OVERVIEW MATERIAL AND METHODS .21 RESULTS AND DISCUSSION 26 CONCLUSION AND PROPOSAL 46 REFERENCES .47 ADDENDUM 50 ADDENDUM 52 LIST OF FIGURE Figure 2.1 Standard absorbance spectra of chlorophyll a and b Figure 2.2 Molecular structure off chlorophyll a .6 Figure 2.3 Molecular structure of chlorophyll b Figure 2.4 Molecular tructure of chlorophyll d Figure 2.5 Molecular structure of Figure 2.6 Molecular structure of Figure 2.7 Spinach (Spinacia oleracea) .9 Figure 2.8 Alfalfa (Medicago sativa) 12 iv Figure 2.9 Mulberry leaves (Morus alba) 13 Figure 2.10 Spirulina algae (Spirulina plantensis) 13 Figure 2.11 Silkworms eat mulberry leaves 15 Figure 2.12 Silkworm excrement 15 Figure 2.13 Synthesis of the basic chlorin derivatives having reactive groups 17 Figure 3.14 Fresh silkworm excrement 21 Figure 4.15 Chlorophyll acetone extraction from various materials 26 Figure 4.16 Chlorophyll content and chlorophyll a ratio in some fresh materials 28 Figure 4.17 Chlorophyll content and chlorophyll a ratio in some dried materials 28 Figure 4.18 Experiment to test the level of mulching of silkworm excrement in some organic solvents 30 Figure 4.19 The solubility of the silkworm excrement in some organic solvents 31 Figure 4.20 Chlorophyll content in the silkworm excrement were affected by the extraction solvent 32 Figure 4.21 Effect of different NH4OH concentrations on chlorophyll extraction efficiency from silkworm excrement 34 Figure 4.22 Chlorophyll content in the silkworm excrement were affected by temperature 35 Figure 4.23 Effect of different material/solvent ratios on chlorophyll extraction efficiency from silkworm excrement 36 Figure 4.24 Effect of sample incubation time on chlorophyll extraction efficiency from silkworm excrement 38 Figure 4.25 Chlorophyll purification process from silkworm excrement 39 Figure 4.26 Chlorophyll purification process from silkworm excrement .40 Figure 4.27 Fragment did not contain chlorophyll 41 Figure 4.28 Fragment did not contain chlorophyll 41 Figure 4.29 Fragment contained chlorophyll (TA1) 41 v Figure 4.30 Technological process of extracting chlorophyll from silkworm excrement 43 LIST OF TABLE Table 4.1 Chlorophyll content of some materials 27 Table 4.2 Solubility and chlorophyll content of silkworm in some organic solvents 30 Table 4.3 Effect of different concentrations of some solvents on chlorophyll extraction efficiency from silkworm excrement 31 Table 4.4 Effect of different NH4OH concentrations on chlorophyll extraction efficiency from silkworm excrement 33 Table 4.5 Chlorophyll content in the silkworm excrement were affected by temperature 34 Table 4.6 Effect of different material/solvent ratios on chlorophyll extraction efficiency from silkworm excrement 36 Table 4.7 Effect of sample incubation time on chlorophyll extraction efficiency from silkworm excrement 37 vi ABBREVIATIONS TA Aceton extract of Bombycis Excrementum Chl a Chlorophyll a Chl b Chlorophyll b Chla+b Total Chlorophyll a and chlorophyll b GC-MS Gas chromatography mass spectiometry HPLC High Performance Liquid Chromatography TLC Thin layer chromatography PTLC Prepared thin-layer chromatography GPC Gel permeation chromatography vii ABSTRACT Silkworm excrement was a potential chlorophyll extraction material with the amount of chlorophyll obtained from fresh silkworm excrement is 0.568% (moisture content 32.71%), corresponding to 0.845% dry biomass The appropriate solvent for chlorophyll extraction was 90% acetone (NH4OH: Acetone = 1: 9), 25 oC extraction temperature, 2% material / solvent ratio, 24h extraction time The highest contained chlorophyll was 0.619%, in which the chla ratio was 63.81% The extraction of 100 g of fresh silkworm excrement with acetone procedured resulted in 2.94 g of chlorophyll precipitation After the chlorophyll precipitation was purified by GPC, obtained 0.7 g of partial purified chlorophyll with 70,58% of chlorophyll content (43,15% of chla and 27,43% of chlb) The technological process to extract and purify chlorophyll from silkworm excrement was developed that was stable and practical viii INTRODUCTION Present day, the deliberate or incidental addition of food colorants that were not allowed or used in excess of the prescribed dosage had been seriously affecting the food hygiene and safety, affecting to the health and psychological puzzling consumers Due to its superiority, it is not toxic to humans, ensuring food hygiene and safety than artificial synthetic pigment (coloring matter) Therefore, in the world today, we concentrate on studying the extraction of pigment groups from cheap natural materials or make use of by-products to collect pigment groups used as food colorants Chlorophyll is a natural pigment that is a component of photosynthesis of plants and photosynthesizing bacteria It is widely used in food, cosmetics, nanotechnology and cancer treatment Chlorophyll extract is extracted with turtles, spinat and some leaves used as food colorants, the trade name is Natural green or E140 used in the food and beverage industry Chlorophyllin is a safe natural, colorless substance that does not detect any toxicity to the human body Chlorophyll is not only a natural colorant that is safe for human health but also had outstanding biological properties for good health Chlorophyll had many different uses in medicine and industry In medicine, chlorophyll is used as a basic ingredient in diets and medicines Chlorophyll and derivatives were also used as photosensitizers to destroy cancer and anti-viral cells, wound healing and deodorant, blood sugar reduction for elderly patients Silkworm waste or silkworm excrement (Faeces Bombycum or Exerementum Bombycis) is a popular folk medicine, which had effect on curing glycosuria During digestion of silkworms, Chlorophyll is not digested and excreted in the digestive tract, so in the silkworm excrement, the chlorophyll content is higher than that of common plant materials, algae Therefore, the isolated process is simple, low cost chemical solvent and high economic efficiency, and could combine the intake of other precious compounds from the silkworm excrement In this study, we investigated the extraction of chlorophyll from silkworm, the first step of purification and building the technological process to extract chlorophyll from silkworm excrement in the pilot scale “Study on extraction of chlorophyll from silkworm waste” Purpose: Extraction and purification of chlorophyll from silkworm waste (silkworm excrement) Content: - Determination of chlorophyll content in some materials - Examination some factors (solvents, temperature, material/solvent ratio, extract - time) affect to chlorophyll extraction efficiency Particity purification of chlorophyll Bulding the technology process of chlorophyll extraction from silkworm excrement 4.3.2 Gel permeation chromatography – GPC with static phase sephadex LH -20 (Merck) Take g of acetone extract (TA) was subjected to column chromatograph using sephadex LH-20, the column was eluted with ethanol Examination of the small fragment by thin layer chromatography and coloring with vanillin-H 2SO4 reagent, then cumulated same fragments, obtained three major fragments TA1 → TA3 The TA1 fraction was subjected to column chromatograph using sephadex LH20, the column was eluted with ethanol to obtained compound chlorophyll TA1A (solids in amorphous, yellow-green) - TA1A fragment was separated by conventional silica gel prepared thin layer chromatography, solvent deployed n-butanol: ethanol: water (4: 1: 2) obtained chlorophyll a and chlorophyll b - The process is described in Figure 4.12: TA 1g Sephadex LH-20 TA1 TA2 0.57g Sephadex LH-20   TA1A 0.24 g PTL C   Chlorophyll a 0.147g Chlorophyll b 0.096g Figure 4.26 Chlorophyll purification process from silkworm excrement 40 TA3 The TA1 fraction was determined on a TLC plate; the control comparison was Chlorophyll a, b (Sigma) (Figures 4.10, 4.11 and 4.12) Figure 4.27 Fragment did not contain chlorophyll Figure 4.28 Fragment did not contain chlorophyll Chlorophyll a Figure 4.29 Fragment contained chlorophyll (TA1) 41 Remove the solvent, determine the content of chlorophyll a, b before and after separation Content (%) No Fragment Crude chlorophyll Chl a Chla+Chlb ratio Chla Chlb 10.70199 6.323179 17.02517 62.860 32.28922 23.06 55.34922 58.337 42.73757 27.8494 70.58697 60.546 Partial purified chlorophyll (silicagel column chromatography) Partial purified chlorophyll (Gel permeation chromatography) The extraction of 100 g of fresh silkworm excrement with acetone procedures resulted in 3.7 g of chlorophyll precipitation with 17.025 of chlorophyll content (10.7% of chla and 6.32% of chlb) After chlorophyll purification on silica gel column, we contained 0.58 g of partial purified chlorophyll which was 55.35% chlorophyll content (32.30% of chlorophyll a and 23.06% of chlorophyll b) In addition, after being purified by Gel Permeation Chromatography (GPC) with static phase sephadex LH-20 (Merck), we obtained higher results We obtained 0.89 g of purified chlorophyll extraction up to 70.58% (with chlorophyll a of 42.73% and chlorophyll b of 27.85%) From the above results, purification of chlorophyll from silkworm excrement by using Gel Permeation Chromatography (GPC) with static phase sephadex LH-20 (Merck), gave higher effective than using silica gel column chromatography: higher achieved chlorohyll content; sephadex LH-20 is reusable and eluting solvent is ethanol which was non-toxic Therefore, Sephadex LH-20 column purification with ethanol elution was preferred for subsequent studies 42 4.4 Development of technological process for extracting chlorophyll from silkworm excrement pilot scale Based on the investigation of some affecting factors and optimizing, we proposed the process of extracting chlorophyll from silkworm Silkworm excrement Remove impurities Grind Grinded silkworm excrement (power) Acetone:NH4OH 9:1 Material:solvent 2% 25oC, 24h Chlorophyll Extraction Filtration by Whatman N0 Supernatant PPT (Decarded) Distilled rotation Crude chlorophylls Sephadex LH-20 Ethanol solvent Chlorophylls contained Fragment Sephadex LH-20 Ethanol solvent Rich – chlorophylls Fragment Spray drying Partialy purified chlorophyll (power) Figure 4.30 Technological process of extracting chlorophyll from silkworm excrement 43 Silkworm excrement: + Fresh silkworm excrement should be cleaned and used immediately to avoid damage to the chlorophyll content + If the silkworm excrement were not used immediately after collection, they should be cleaned and stored in a low temperature, avoid light Grind: + For complete and effective extraction, we would grind the material together with the extraction solvent + Scale of silkworm, put into grinding tools Addition of acetone: 9: NH 4OH was added and the milling of the silkworm solution in the solvent was sufficient to reach a content of 2% Filtering: + After 24 hours at 25-30oC with a solvent/material ratio of 100: (v / w), take the filtered sample Use filter paper or filter cloth to filter the sample + Note that filtering was carried out in conditions that avoid light The filtered sample should also be stored in a dark colored instrument, limiting the metabolism of chlorophyll + Requirements: Crude chlorophyll would be clear and blue coloration Distilled rotation + The crude chlorophyll was obtained, after which it was made to dry would be distilled rotation + After the removal of the solvent, the chlorophyll residue was collected Purification: + Crude chlorophyll was chromatographed on sephadex LH-20 column with ethanol Examination of the small fragment by thin layer chromatography and coloring with vanillin-H2SO4 reagent, then cumulate same fragments, we obtained Chlorophylls contained Fragment + The Chlorophylls contained fragment was separated on Sephadex LH-20 column with a solvent of ethanol Spray drying: + Extraction after removal of solvent was sprayed on spray dryer 44 In 1992, Khalyfa also presented a technological process to extract and purify chlorophyll from spinach He extracted chlorophyll from fresh spinach leaves by use of methanol and acetone as two separate solvents The crude chlorophyll was partially purified by successive precipitation using a mixture of dioxane and water However, dioxane was a heterocyclic organic compound, classified as an ether, a toxic compound The partially purified chlorophyll was subjected to column chroma- tography with DEAE-Sepharose CL-6B Chlorophylls a and b were obtained by further purification of a Sepharose CL-6B column chromatography, which was column chromatography using agarose The recovery of chlorophyll fractions was determined, and the degree of their purity was demonstrated by spectrophotometric scanning and by HPLC analysis The extraction of 100 g of fresh spinach leaves with methanol and subsequent purification procedures resulted in 38 mg of chlorophyll a (30.1 % purity) and 13 mg of chlorophyll b (27.5% purity) Extraction with acetone and subsequent purification procedures resulted in 61 mg of chlorophyll a (44.5 % purity) and 29 mg of chlorophyll b (32.4% purity), respectively From the above results and this research (Khalyfa A., 1992), the offered technological process was quite positive Because used elute solvent was ethanol, which was non-toxic and easy to recover In addition, nesscesiry equipment was very simple and effective Particularly, partial purified chlorophyll was obtained higher efficiency The extraction of 100 g of fresh silkworm excrement with acetone and subsequent purification procedures resulted in 0.89 g of purified chlorophyll extraction up to 70.58% (with chlorophyll a of 42.73% and chlorophyll b of 27.85%) 45 CONCLUSION AND PROPOSAL 5.1 Conclusion - Silkworm excrement was a potential chlorophyll extraction material with the amount of chlorophyll obtained from fresh silkworm excrement is 0.568% - (moisture content 32.71%), corresponding to 0.845% dry biomass The appropriate solvent for chlorophyll extraction was 90% acetone (NH4OH: Acetone = 1: 9), 25oC extraction temperature, 2% material / solvent ratio, 24h extraction time The highest contained chlorophyll was 0.619%, in which the - chla ratio was 63.81% The most effective and safest way to purify chlorophyll was the use of gel permeation chromatography – GPC with static phase sephadex LH -20 (Merck) The extraction of 100 g of fresh silkworm excrement with acetone procedures resulted in 3.7 g of chlorophyll precipitation After the chlorophyll precipitation was purified by GPC, obtained 0.7 g of partial purified chlorophyll with - 70.58% of chlorophyll content (42.73% of chla and 27.85% of chlb) The technological process to extract and purify chlorophyll from silkworm 5.2 - excrement was developed that was stable and practical Proposal The process of extracting chlorophyll from the silkworm excrement into the - thesis was results in the first step For efficient extraction, it was necessary to optimize the technological process - of chlorophyll extract from silkworm excrement by experimental planning Water-soluble derivatives of chlorophyll should be created for higher application in food and industry 46 REFERENCES Vietnam references Nguyễn Thị Thanh Bình, Mai Văn Trì, Nguyễn Văn Hùng, Nguyễn Thành Minh (1999), Tách chiết ecdysteroids từ phân tằm, Tạp chí Sinh học, số 2, tập 21, tháng 6, 52-54 Vũ Ngọc Bội, Đặng Xuân Cường, Nguyễn Hoài Quốc (2015), Tối ưu hóa cơng đoạn chiết polyphenol, chlorophyll với hoạt tính chống oxy hóa từ măng tây, Tạp chí Khoa học Cơng nghệ Thủy sản, số 3,2015, 3-8 Ngô Xuân Cường (2012), Nghiên cứu công nghệ sản xuất chất màu thực phẩm từ chè, Đề tài thuộc dự án khoa học công nghệ nông nghiệp vốn vay adb, Viện khoa học kỹ thuật nông lâm nghiệp miền núi phía Bắc Bùi Thị Thu Hà (2002), Tác hại phẩm màu chất bảo quản thực 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CRC Press: Boca Raton, FL, 1991 37 Schwartz, S J.; Lorenzo, T V Chlorophylls in foods CRC Food Sci Nutr 1990,29, 11-17 38 UNESCO, (1966), Rep SCOR/UNESCO WG 17, UNESCO, Paris, Monogr Oceanogr Methodol, 1, 11 39 Uzakova D U., Kolesnik A A, Zherebin Yu L., Sarycheva I K (1987), CAROTENOIDS OF MULBERRY LEAVES AND OF SILKWORM EXCRETA, Plenum Publishing Corporation, UDC 547.979.7-8 40 Wisconsin: University of Wisconsin Press, 22 41 Wasmund N., Topp I., Schories D (2006), Optimising the storage and extraction of chlorophyll samples, OCEANOLOGIA, 48 (1), 125–144 42 Wasmund N., 1984, Probleme der spektrophotometrischen Chlorophyllbestimmung, Acta Hydroch Hydrob., 12, 255–272 43 Yoon Il, Jia Zhu Li and Young Key Shim (2013), Advance in Photosensitizers and Light Delivery for Photodynamic Therapy, Korean Society of Gastrointestinal Endoscopy, 46: 7-23 49 ADDENDUM Some Figures of practice and chlorophyll extraction at “Institute of natural products chemistry” 50 Figure Photometric measurements Figure Chlorophyll extraction in pilot scale Figure Gel permeation Figure Distilled rotation chromatography – GPC with static phase sephadex LH -20 51 Figure Spray drier ADDENDUM PUBLISHED RESEARCHES 52 53 54 ... of different concentrations of some solvents on chlorophyll extraction efficiency from silkworm excrement 31 Table 4.4 Effect of different NH4OH concentrations on chlorophyll extraction. .. step of purification and building the technological process to extract chlorophyll from silkworm excrement in the pilot scale Study on extraction of chlorophyll from silkworm waste Purpose: Extraction. .. material/solvent ratios on chlorophyll extraction efficiency from silkworm excrement 36 Table 4.7 Effect of sample incubation time on chlorophyll extraction efficiency from silkworm excrement