Cultivated Edible Mushrooms as a Safe Source for

							                            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.