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- 1. Ganoderma lucidum Triterpene and Polysaccharide Extraction for Medicinal Applications Ramona Kahler, Jacob Simmons, Nicholas Tuttle BE 4380 https://www.google.com/search?biw=1920&bih=929&tbm=isch&sa=1&ei=10q- XMPoBqy40PEP7smk8A0&q=ganoderma+lucidum&oq=ganoderma+lucidum&gs_l=img.12..0j0i67j0l 2j0i67j0l5.76591.76775..80416...0.0..0.129.250.0j2......1....1..gws-wiz-img.BAf- oxWTMtc#imgrc=VqYCY8vFhZUblM: https://www.google.com/search?biw=1920&bih=929&tbm=isch&sa=1&ei=10q- XMPoBqy40PEP7smk8A0&q=ganoderma+lucidum&oq=ganoderma+lucidum&gs_l=img.12..0j0i67j0l 2j0i67j0l5.76591.76775..80416...0.0..0.129.250.0j2......1....1..gws-wiz-img.BAf- oxWTMtc#imgrc=VqYCY8vFhZUblM:
- 2. Problem: To Produce a Nutraceutical from Reishi Mushroom ❖ Definition of problem ➢ Reishi mushrooms must be highly processed to extract their medicinal compounds. Current Reishi products are expensive and of unreliably potency. ❖ Bioprocess goals ➢ Extract over 95% triterpenes and polysaccharides from reishi mushroom biomass. ❖ Structural goals ➢ Design entire system to be as efficient and sustainable as possible on an industrial scale ❖ Mechanical goals ➢ Optimize scale of machinery
- 3. Constraints and Considerations ❖ Designing for human consumption ➢ No harmful chemicals ➢ No contamination during process ➢ Product sterilization ❖ Economic constraints ➢ Startup and run cost vs profit of extracts ➢ Supply of mushrooms available ➢ Demand for the supplement ❖ Ultimate use ➢ No waste of natural materials https://www.google.com/search?q=ganoderma+lucidum+polysaccharides&source=lnms&tbm=isch&s a=X&ved=0ahUKEwjO_dWul-XhAhUHA6wKHZXXDgkQ_AUIDigB&biw=1920&bih=929#imgrc=- f36HygRPTsXyM:
- 4. Questions of User, Client, Designer ❖ User- Consumer ➢ What are the positive health benefits? ➢ How expensive is this product? ➢ Are there any negative side effects? ❖ Client- Biopharmaceutical Company ➢ How can I make a profit from this procedure? ➢ How long will it take to implement this project? ➢ How is this project unique? ❖ Design Team ➢ How to make the process most efficient? ➢ How to design a process with no waste? ➢ How often will the process be run?
- 5. Literature Review ❖ Health Benefits of Reishi Mushroom ➢ Long chain polysaccharides ■ Maintain healthy body weight ■ Increased antioxidant activity ■ Increase anti-inflammatory response ➢ Triterpenes ■ Lower total cholesterol and LDL-C ■ Lower total glyceride content ■ Modulate metabolic disorders
- 6. Literature Review ❖ Determining compound content- ➢ 112.82 and 31.5 mg/g- Nakagawa et al, 2018 ❖ Process of extraction ➢ Assuming mushrooms are pre-milled ❖ Polysaccharides ➢ Water soluble ➢ Varying temp- Optimal 80 degrees celsius 17.97g/100g ➢ Pressurize-Patents featuring high pressure- 3 MPa ➢ Time- typically done for 3-4 hours at temp ❖ Triterpenes ➢ Alcohol soluble ➢ Shorter extraction time
- 7. Consideration of Alternative Methods ❖ Alternative Extraction Methods Researched ➢ Used less safe materials like chloroform ➢ Would not be as easy to recycle our byproducts ➢ Required higher pressures and temperatures ➢ Utilized expensive and complicated equipment https://www.google.com/search?q=hazardous&source=lnms&tbm=isch&sa=X&ved=0ahUKEwi eyrCF5eThAhVSDq0KHZb8B4YQ_AUIDigB&biw=1920&bih=929#imgrc=-qs94r9OxXq8fM: https://www.google.com/search?q=hazardous&source=lnms&tbm=isch&sa=X&ved=0ahUKEwi eyrCF5eThAhVSDq0KHZb8B4YQ_AUIDigB&biw=1920&bih=929#imgrc=-qs94r9OxXq8fM: https://www.google.com/search?q=hazardous&source=lnms&tbm=isch&sa=X&ved=0ahUKEwi eyrCF5eThAhVSDq0KHZb8B4YQ_AUIDigB&biw=1920&bih=929#imgrc=-qs94r9OxXq8fM:
- 8. Synthesis of Design ❖ Governing Equations: ➢ S ➢ Kinetics of cell death at high temp. and pressure ❖ Initial Conditions: ➢ Mass comp. 8.06% polysaccharide, 2.17% triterpenes, 7.04 % misc. solubles ➢ 20 kg batches of pre-milled 1:15 biomass to solvent ❖ Two Stage Extraction procedure ❖ Stage 1 Operating Conditions: Water Extraction ➢ Blending/Storage: 80 C, 3 MPa, 1 hour (agitation, heating, pressurization) ❖ Stage 2 Operating Conditions: Ethanol Extraction ➢ Blending/Storage: 99.9% Ethanol, 30 mins., 40 C ❖ Both stages ➢ Stationary Screen: Remove 95% medicinal compounds, 50% misc. Solubles ➢ Tray Drying: Remove residual solvents
- 10. Sustainability Measures ❖ Zero on-site emissions ❖ Recycle water streams ❖ Recycle ethanol streams ❖ Waste biomass composted https://www.google.com/search?biw=1920&bih=929&tbm=isch&sa=1&ei=Y2q- XPylKMfcswWt2Yu4BQ&q=reishi+waste&oq=reishi+waste&gs_l=img.3...4302.10146..10432...10.0.. 0.120.1942.14j6......1....1..gws-wiz- img.......0j0i67j0i10i24j0i30j0i5i30j0i24j0i8i30.dhBS0buiY7w#imgrc=hFLwEo_jCG3yzM:
- 11. Timeline
- 12. Budget ❖ Prototype cost ➢ $732,000 Cs=C0* (Ss/S0)a where a is 0.6, Ss/S0=0.01 and C0 is $11.6 million ❖ Full scale cost ➢ Determined by SuperPro cost of processes ➢ Building cost: $11.6 million ➢ Operating cost: $4.53 million/year ❖ Product valuation ➢ Based on current market cost for reishi mushroom extract ➢ Approximately $7.50 per gram of extract ➢ 1.97 kg polysaccharide and triterpene yield per batch ➢ 5 batches per day= $26.9 million/year revenue ➢ Cost of product packaging and distribution not included
- 13. References/Patents References 1. Chen, Y., Chen, Q., Wang, Z., & Hua, C. (2019). Anti-Inflammatory and Hepatoprotective Effects of Ganoderma lucidum Polysaccharides against Carbon Tetrachloride-Induced Liver Injury in Kunming Mice. Pharmacology, 103(3- 4), 143-150. doi:10.1159/000493896 2. Liang, Z., Yuan, Z., Li, G., Fu, F., & Shan, Y. (2018). Hypolipidemic, Antioxidant, and Antiapoptotic Effects of Polysaccharides Extracted from Reishi Mushroom, Ganoderma lucidum (Leysser: Fr) Karst, in Mice Fed a High-Fat Diet. Journal of Medicinal Food,21(12), 1218-1227. doi:10.1089/jmf.2018.4182 3. Cheng, P., Phan, C., Sabaratnam, V., Abdullah, N., Abdulla, M. A., & Kuppusamy, U. R. (2013). Polysaccharides-Rich Extract of Ganoderma lucidum(M.A. Curtis:Fr.) P. Karst Accelerates Wound Healing in Streptozotocin-Induced Diabetic Rats. Evidence-Based Complementary and Alternative Medicine,2013, 1-9. doi:10.1155/2013/671252 4. Guo, W., Pan, Y., Li, L., Li, T., Liu, B., & Lv, X. (2018). Ethanol extract of Ganoderma lucidum ameliorates lipid metabolic disorders and modulates the gut microbiota composition in high-fat diet fed rats. Food & Function,9(6), 3419-3431. doi:10.1039/c8fo00836a 5. Nakagawa, T., Zhu, Q., Tamrakar, S., Amen, Y., Mori, Y., Suhara, H., . . . Shimizu, K. (2018). Changes in content of triterpenoids and polysaccharides in Ganoderma lingzhi at different growth stages. Journal of Natural Medicines,72(3), 734-744. doi:10.1007/s11418-018-1213-y 6. Nakach, M., Authelin, J., Perrin, M., & Lakkireddy, H. R. (2018). Comparison of high pressure homogenization and stirred bead milling for the production of nano-crystalline suspensions. International Journal of Pharmaceutics,547(1-2), 61-71. doi:10.1016/j.ijpharm.2018.05.042 7. Hu, H., Zhang, Z., Lei, Z., Yang, Y., & Sugiura, N. (2009). Comparative study of antioxidant activity and antiproliferative effect of hot water and ethanol extracts from the mushroom Inonotus obliquus. Journal of Bioscience and Bioengineering,107(1), 42-48. doi:10.1016/j.jbiosc.2008.09.004 8. Rhee, S. J., Cho, S. Y., Kim, K. M., Cha, D., & Park, H. (2008). A comparative study of analytical methods for alkali- soluble β-glucan in medicinal mushroom, Chaga (Inonotus obliquus). LWT - Food Science and Technology,41(3), 545- 549. doi:10.1016/j.lwt.2007.03.028 9. Uzel, R. A., & Yaman, Ü R. (2016). A novel method for food particle production using subcritical water extraction: Ganoderma mushroom as a case example. The Journal of Supercritical Fluids,111, 74-82. doi:10.1016/j.supflu.2016.01.021 10. Cör, D., Botić, T., Knez, Ž, Batista, U., Gregori, A., Pohleven, F., & Bončina, T. (2014). Two-stage extraction of antitumor, antioxidant and antiacetylcholinesterase compounds from Ganoderma lucidum fruiting body. The Journal of Supercritical Fluids,91, 53-60. doi:10.1016/j.supflu.2014.04.006 Patents 1. Junmin, F., & Zheng, C. (2018). U.S. Patent No. CN109096407A. Washington, DC: U.S. Patent and Trademark Office. 2. Chengzhong, W. (2014). U.S. Patent No. CN104257701A. Washington, DC: U.S. Patent and Trademark Office.
Editor's Notes
- Why did we pick fungi for our project They are without a doubt the most under researched and misunderstood piece of the enormous puzzle that we call our ecosystems There's an aversion towards studying fungi, that can be called myco or fungal phobia that results from the cultural stigma that mushrooms will either kill you or take you on a magic trip Eastern cultures don’t hold these stigmas They have cutlivated and comsumed gorumet mshrooms for food and medcine for centuries Modern research is finally proving the medicinal properties of these underappreciated natural artworks We pciked the rieshi mushroom because it grows widely and has the widest spectrum of proven bioactive compounds
- Mushroom mountain- local company grows reishi on logs and sawdust
- Low density lipoprotein- cholesterol (bad cholesterol)
- polysaccharides, proteins, and total phenolic content
- Supercritical carbon dioxide extraction is an alternative design method that we considered. This requires high temperatures and pressures increasing the energy and therefore money required to run the process. It also adds danger. Ultra-high pressure extraction would also be effective because it can drastically reduce the time of the procedure but was not chosen because it would require expensive and complicated machinery that we cannot test in superpro. Ultrasonic cell disruption with chloroform vapor extraction is also a possibility but was not seriously considered due to the highly complex procedure and due to the toxicity and hazardous nature of chloroform
- Miscellaneous solubles- vitamins, amino acids, minerals, lipids Our group decided to design a two-stage extraction process using hot water an alcohol. This extraction procedure was chosen because of its simplicity and lower cost. This method is also safer than most of the other extraction methods we researched. It was also considered that using a separate hot water extraction would make it easier to recycle our hot water. Analysis of information- Literature reviews showed multiple extraction processes can be used to obtain the polysaccharides and triterpenes from Ganoderma lucidum. These methods include hot water extraction, alcohol extraction, mixed hot water and alcohol extraction, supercritical carbon dioxide extraction, ultrasonic cell disruption coupled with chloroform vapor extraction, radiative extraction, ultra-high pressure solid phase column extraction. Hotter water extracts a higher percentage of polysaccharides. One key focus of the design methodology used was the focus on safe and reusable materials. Some research groups used more dangerous chemicals (chloroform, others???) in their experimental procedure. This procedure is able to get similar results without the risk of introducing health risks into a consumable product. These processes would also not be able to be recycled as efficiently, and would require more mass input.
- Extra notes: Polysaccharides and triterpenes can be successfully produced as a dry product from reishi mushroom biomass through a hot water extraction followed by an ethanol extraction. User- Consumer What are the positive health benefits? Lowers total triglycerides, lowers total cholesterol, lowers LDL-C, boosts immune system, anti-cancer properties, helps with fatigue, antioxidant abilities, How expensive is this product? Current market prices Triterpene- $12.78/gram, polysaccharide- $5.89/gram Are there any negative side effects? Upset stomach, can decrease blood sugar, no adverse effects on the liver or kidneys Client- Biopharmaceutical Company How can I make a profit from this procedure? Approximately $20,000 of product made from each 20 kg batch How long will it take to implement this project? Approximately a year How is this project unique? This design uses recycling of byproducts to reduce waste and cost Design Team How to make the process most efficient? Recycle water and ethanol, Overlapping unit operations to minimize batch time How to design a process with no waste? Byproducts get recycled for perpetual use, biomass is composted How often will the process be run? 5 batches per day, each 20 kg of milled biomass