Cultivated Edible Mushrooms as a Safe Source for
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Alyssa Finch MOISD Math/Science/Technology Center 15760 190th Ave. Big Rapids, MI 49307 Introduction One of the big concerns of America is “going green.” Organic foods and medicines are being developed everyday. Three mushroom species are being tested to determine the Lipid Peroxidation (LPO) inhibition rate, the percent inhibition rate of Cyclooxygenas (COX) enzymes and the composition compounds in the mycelium to potentially be used as a form of treatment or prevention. Lentinula edodes (Shiitake) Pleurotus ostreatus (Oyster) Agrocybe aegerita (Black Poplar) Purpose The active components of mushrooms can potentially ascribe to: antiviral, antimicrobial, anti-inflammatory, and anti-diabetic (Jong and Birmingham, 1993) properties; anticancer properties(Borchers et al, 1999; Mizuno et al, 1995; Smith et al, 2002); hypoglycemic and hypocholesterolemic properties, and they are effective in the treatment of hypertension. Materials Labconco Purifier Delta Class II Biosafety Cabinet Ethanol Small Glass Beaker Fisherbrand Stainless-Steel No. 11 Scalpel Blade Flame Fast Bunsen Burner Lab-Tex Sterile Petri Plates (100 mm X 15 mm) Malt Yeast Agar Potato Dextrose Agar Liquid Inoculum Polypropylene Spawn Growth Jars Tuttnauer Model 3870 Benchtop Autoclave Sterile Glass Pipettes Zip Lock Plastic Bags Precision High Capacity Mechanical Convection Incubator Parafilm Waring Two-Speed Stainless Steel Blender Inoculation of Petri Plates Sterilized Hood with Ethanol and UV light. Attached Bunsen Burner. Prepare Small Glass Beaker with Ethanol for sterilizing of the Scalpel blade. Gathered full grown controlled strains of Shiitake, Oyster, and Black Poplar mushroom mycelium. Prepare a stack of 6 Petri plates with agar to be inoculated. Dip scalpel blade into ethanol and burn the remains off in order to sterilize between plates and strains. Cut a 3 cm X 3 cm square of original mycelium and place upside down onto the agar covered Petri plate to allow the mycelia to have direct contact with the agar. After a Petri dish was inoculated, it was sealed with parafilm and labeled with the correct strain and agar type and placed in a dark controlled environment at 20˚C. Inoculation of Polypropylene Jars Sterilized Hood with Ethanol and UV light. Attached Blender. Gathered full grown controlled strains of Shiitake, Oyster, and Black Poplar mushroom mycelium. Prepare a group of 6 Polypropylene Jars to be inoculated. Add 250 mL of sterile malt-yeast liquid media to blender. Add the mycelium from 1 fully colonized Petri Plate cut into quadrants for easy removal. Blend for 3 quick, 5 high speed bursts to fragment the mycelium. Pipette 10.0 mL of the blended mixture into the grain spawn jars. Obtaining a new sterilized glass pipette between strains. Screwed on lid tight enough so the mycelium doesn’t grown out of the jars and place a zip lock bag over top of the jar. Labeled the jars with the strain. Placed the inoculated jars into a dark controlled room at 20˚C. Measurement The inoculated Petri plate’s and jar’s growth was measured daily and recorded in a table seen below. Augur Augur Augur # PO Type AA Type Le(705) Type Temp. Date 1 41 mm PDA 28 mm PDA 31 mm PDA 68° F 10/10/08 2 41 mm PDA 29 mm PDA 33 mm PDA 3 40 mm PDA 27 mm PDA 32 mm PDA 4 41 mm MYA 33 mm MYA 28 mm MYA 5 41 mm MYA 34 mm MYA 28 mm MYA 6 42 mm MYA 31 mm MYA 24 mm MYA 7 39 mm PDA 26 mm PDA 32 mm PDA 8 40 mm PDA 30 mm PDA 32 mm PDA 9 38 mm PDA 26 mm PDA 31 mm PDA 10 41 mm MYA 36 mm MYA 33 mm MYA 11 41 mm MYA 34 mm MYA 28 mm MYA 12 41 mm MYA 34 mm MYA 29 mm MYA 13 39 mm PDA 31 mm PDA 36 mm PDA 14 41 mm PDA 28 mm PDA 32 mm PDA 15 41 mm PDA 30 mm PDA 35 mm PDA 16 41 mm MYA 37 mm MYA 33 mm MYA 17 41 mm MYA 36 mm MYA 31 mm MYA 18 41 mm MYA 32 mm MYA 27 mm MYA Averag 40.56 31.22 30.83 e mm mm mm Ave. 28.22 32.67 PDA 40 mm mm mm Ave. 41.11 34.11 MYA mm mm 29 mm COX Enzyme Inhibitor Test In Vitro COX-II and COX-I inhibitory activities of mushroom mycelia extracts. Synthetic Test Subjects: Vioxx Celebrex Aspirin Naproxen Ibuprofen Organic Mushroom Test Subjects: Fatty Acid Fractions Palmitic Acid Ergosterol Ergosterol Peroxide Cyclooxygenase (COX) is an inhibitor that can act as a pharmaceutical to treat pain or inflammation. (Wikipedia) COX Enzyme Inhibitor Results Test Subject % Inhibition of COX-I % Inhibition of COX-II Vioxx 23% 67% Celebrex 46% 88% Aspirin 92% 24% Naproxen 63% 61% Ibuprofen 30% 43% Fatty Acid Fraction 80% 88% Palmitic Acid 39% 45% Ergosterol 19% 28% Ergosterol Peroxide 57% 22% Lipid Peroxidation Test The percent inhibition of lipid Peroxidation by ethyl acetate fractions. Synthetic Test Subjects: Butylated Hydrozy-Anisole (BHA) Butylated Hydrozy Toluene (BHT) Tertiary Butylhydroquinone (TBHQ) Organic Mushroom test subjects: Black Poplar Mycelia Oyster Mycelia Shiitake Mycelia Glyceride Fraction Lipid Peroxidation refers to the oxidative degradation of lipids. (Wikipedia) Oxidative damage is known to be one of the major causes to pathological conditions such as cancer, cardiovascular diseases and complications associated with diabetes. Lipid Peroxidation Results Test Subject % Inhibition of Lipid Peroxidation BHA 82% BHT 87% TBHQ 95% Black Poplar 81% Oyster 52% Shiitake 39% Glyceride Fraction 92% Refined COX Enzyme Inhibitor Test In Vitro COX-II and COX-I inhibitory activities of methanolic extracts of mushrooms and glyceride fraction. Synthetic Test Subjects: Aspirin Celebrex Vioxx Organic Mushroom Test Subjects: Black Poplar Mushroom Oyster Mushroom Shiitake Mushroom Glyceride Fraction Refined COX Enzyme Inhibitor Results Test Subject % Inhibition of COX-I % Inhibition of COX-II Aspirin 65% 25% Celebrex 40% 67% Vioxx 0% 95% Black Poplar 39% 87% Oyster 54% 75% Shiitake 34% 92% Glyceride Fraction 43% 92% Mushroom Bioactive Compounds Mushroom Linoleic Ergosterol Ergosterol Sphingolipid Glyceride Acid Peroxide Fraction Black Poplar 0.125 mg/g 0.110 mg/g 0.126 mg/g - - Shiitake 0.218 mg/g 0.056 mg/g 0.062 mg/g 0.030 mg/g - Oyster 0.175 mg/g - 0.140 mg/g 0.060 mg/g 0.041 mg/g Discussion The test results indicate that fatty acids are the most dynamic components of mushroom mycelium which demonstrate both COX-I and COX-II enzyme inhibitions. Palmitic acid exhibited lower COX enzyme inhibitory activity than the fatty acid fraction (FAF),the COX-II and COX-I inhibitory ratio was similar to that of the fatty acid fraction. Ergosterol showed higher inhibitory activity for COX- II than COX-I. Ergosterol Peroxide inhibited the COX-I enzyme in a much higher amount than COX-II enzyme. Discussion continued… The antioxidant properties of linoleic acid and ergosterol have been reported in earlier studies (Zang et al., 2003). The antioxidant activity observed in all three mushrooms in this study is attributed to the presence of linoleic acid and ergosterol. Linoleic acid, an omega 6 fatty acid, is an important dietary component that prevents cardiovascular diseases (Lanzmann-Petithory, 2001). Ergosterol was visible in Black Poplar and Shiitake but not in Oyster mushroom. Studies have shown that ergosterol peroxide has a number of physiological effects such as antimicrobial (Lu et al., 2000) and anti- inflammatory activities (Yasukaqa et al., 1994). Anti-inflammatory properties of ergosterol and ergosterol peroxide have also been reported earlier from our laboratory (Zang et al., 2003). Discussion continued… Recent studies have demonstrated that some of the dietary sphingolipids suppress colon carcinogenesis (Schmelz et al., 2000; Symolon et al., 2004). Similar studies from our laboratory have also shown the anticancer and anti-inflammatory activities of sphingolipids (Diyabalange et al, 2008). Conclusion A number of mushrooms are commonly used in diets and implicated as a source of natural antioxidants (Cheung and Cheung, 2005; Mau et al., 2002). The reports suggest that consumption of mushrooms provide health benefits such as reducing the possibility of cardiovascular diseases, preventing the occurrences of cancer and alleviating inflammatory conditions (Sloan, 2007; Francia et al., 1999). Conclusion continued… In conclusion, this study demonstrates the comparative LPO and COX enzyme inhibitory activities and determined the active components in Black Poplar, Shiitake and Oyster mushrooms. The researcher confirmed previous studies and the abilities to determine the specific levels of compounds and enzymes which are useful for health products. With the assistance of Dr. Mills and Dr. Nair, the results suggest that consumption of Black Poplar, Shiitake and Oyster mushrooms could be very beneficial to health.