PHYSICO-CHEMICAL AND TOXICOLOGICAL PROFILES OF FIVE SPECIES OF MUSHROOM IN ANAMBRA STATE NIGERIA AND THEIR POTENTIAL FOR BIOREMEDIATION OF TRACE METAL POLLUTED SOIL

ABSTRACT

Five  species  of  Mushroom  namely  Termitomyces   robustus,  Agaricus  bisporus, Pleurotus tuber-regium, Amanita phalaoides and Amanita verosa were collected from eleven  locations  in  Uke,  Abatete,  Ideani,  Nnobi,  Nnewi  (Okpuno-egbu),  Nnewi (Umudim) and Ozubulu between 2009 and 2012 in Anambra State, Nigeria.   They were  kept  in  clean  collection  bags  and  identified  by a taxonomist.  Some  of the

mushroom samples were later oven dried at 75 0 C for 4 hours and kept for chemical

analysis while some were used for cultivation. During cultivation, seeds from matured mushrooms were scrapped from their veils into already compounded  substrates/soil from their natural habitats and refuse dump soil (Table 3.2).  The seeds were allowed to germinate within 4-5 days, the fruiting bodies/spawns were watered once daily for

14 days. The matured mushrooms were harvested, cleaned and oven dried at 75 0  C

for 4 hours. The dried mushroom  samples (wild and cultivated  respectively)  were homogenized into fine powder using blender with titanium blade and stored in pre- cleaned  bottles  for  chemical  analysis.  These  samples  were  subjected  to  various chemical  analyses  using  standard  methods  by  Association  of  Official  Analytical Chemist  (AOAC).     The  data  were  subjected  to  one  way  analysis  of  variance (ANOVA) at 95 % level using Statistical Package for Social Scientists (SPSS) version

16.0. The moisture content (MC) ranged from 81.79 % to 97.84 %, the highest value was from Amanita phalaoides and the least value was from Agaricus bisporus.  Dry matter (DM) ranged from 2.63 % to 18.36 % showed an indication of high roughages content of the mushrooms. Crude protein (CP) ranged from 8.16 % to 24.67 % which compared favourably with values of seeds and  legumes.  Ash contents ranged from

3.26 % to 14.33 % were indications of high mineral elements present in the studied

mushroom species.  Low values of Lipid (fat/oil) ranged from 1.00 % to 6.68 % gave acceptance of the mushrooms as excellent dietary food for diabetic and coronary heart disease patients. Crude fibre (CF) ranged from 2.62 % to 15.37  %. There were no significant differences at p>0.05 between values of wild and cultivated mushrooms. Carbohydrates  contents  of  32.00-35.40  %,  implied  that  mushrooms  can  function effectively  in  low  fat diet  such as those  required  by  patients  with cardiovascular diseases and diabetes. The vitamin C were detectable at levels ranging from 0.01-0.37 mg/100  g,  values  determined  showed  that  the  studied  mushrooms  were not  good sources of vitamin C, although they could make important contribution to diet. The mean anti-nutritional factors of phytic acid 0.26 mg/100 g, cyanide 0.16 mg/100 g, tannins 0.31 mg/100 g were low when compared with 1.00 mg/100 g found in WHO guideline levels for these toxicants in food. Essential metals concentrations (mg/kg) for wild mushroom samples ranged as follows: Na (152.36 – 777.42), K (166.88 –

933.81), Ca (83.64 -545.00), Mg (476.57–1191.00), Fe (154.68–684.74) while  values

for  cultivated  mushroom  samples  ranged  as  follows:  Na  (332.77  –  1061.12),  K (196.36 – 844.23), Ca (219.69 – 1841.08), Mg (549.66 – 1566.11) and Fe (401.66 –

777.18)  mg/kg.    The  essential  metal  concentrations  were  within  WHO  guideline

values for food. Trace/toxic metal concentrations (mg/kg) for wild mushroom samples ranged as follows: Cd (3.88 – 6.68), Co (0.48 – 1.52), Cr (BDL), Cu (0.12 – 0.72), Mn (8.25 – 24.42), Ni (1.40 – 16.85, Pb (3.60 – 5.03) and Zn (25.00 – 61.17).  Values (mg/kg) for cultivated mushroom samples ranged as follows: Cd (4.40 – 9.88), Co (0.49 – 6.04), Cr (BDL), Cu (0.01 – 0.36), Mn (11.60 – 21.72), Ni (2.19 – 22.05), Pb (3.68 – 6.33) and Zn (9.33 – 68.56).  There were significant differences at (p < 0.05) between mean trace metal concentrations of wild and cultivated mushrooms.  All the soil/substrates samples used for cultivation were polluted with Cd and Cr while 40 % were polluted with Pb.   However, Pb showed elevated values in other metals. There were elevated values of Ni in all the mushroom samples. The rest of the metals were below WHO guideline levels. Bioaccumulation factors ranged as follows: Cd (0.14 –

1.78), Co (0.06 – 3.01), Cr (BDL), Cu (0.01 – 0.35), Fe (1.17 -2.22), Mn  (0.38  –

13.53), Ni (0.08 – 1.95), Pb (0.08 – 1.50) and Zn (0.22 – 10.13).  These values were above acceptable limit in food.

CHAPTER ONE

1.0       INTRODUCTION

1.1      Mushrooms

Mushrooms are a special group of fungi which are saprophytic in nature due to lack of chlorophyll.  They grow in dark, damp places and produce a wide  range of enzymes which progressively breakdown complex substances into simpler inorganic matter1. In many parts  of the world, such as China, United State of America (U.S.A), Canada,  India,  Italy,  Mexico  and  Turkey,  mushrooms  are  highly  priced  and  in massive production for local consumption and export. In U.S.A, the gross domestic products (GDP) for mushroom was seven million tonnes in 2005,   amounting   to   30 million dollars annually1. In Nigeria, mushrooms are grossly under exploited as only a few types are considered edible. There is little evidence of  mushroom cultivation as a commercial  venture  in  Nigeria,  but  this  could  be  used  as  a  means  of  poverty

alleviation   due  to  its  short  cropping   cycle,   cheap   planting   inputs,  less   land requirement,  high  profit  and  quick  returns  on  investment2.  Till  date,  mushrooms collection in Nigeria is mainly from the wild and this practice is fraught with fear of mistaking those regarded as poisonous and non-edible for those regarded as edible2. This  has  been  occasionally  attributed  to  many  deaths  after  mushroom  meals2. Industrialization,  urbanization  and  indiscriminate  refuse  disposal  have  impacted negatively  on  the  environment,  thereby  posing  problems  of  contamination  with pesticides,  petroleum  hydrocarbons,  heavy  metals  and  other  potential  pollutants. Mushrooms have been reported to be good accumulator of trace metals in polluted environment1, 2.

In many countries of the world including Nigeria, edible mushrooms have been priced as delicacies1, 2. Apart from their medicinal values, they constitute an important

food  source  in the  world.  Mushrooms  have  been  reported  to  be  rich  in  protein, glycogen, vitamins, crude fibres and essential mineral compounds3. In fact, Agrahar and Hammam1,  3 reported the rich nutrient contents of mushrooms compared to those of meat and vegetables. Mushrooms such as Flammulina velutipes Lentinus    edodes,

Agaricus bisporus, Pleurotus oestratus, Volvariella volvacea and   Agaricus campestris among others, have been cultivated for food in several countries of the world especially in America, Europe and Asia4.

Health  risk  from  mushroom  consumption  has  been  difficult,  due  to  very

limited knowledge in chemical compositions of the  metals and their bioavailability in man5.  Some  countries  have  established  statutory  limits  for  the  metals  in  edible mushrooms.  It was reported that mushroom breaks down toxic pollutants  into non- toxic substances4, 5. Also reported was the removal of heavy metals and other harmful contaminants   from environment by Shiitake mushroom5, 6. The scavenging of metals from polluted sites by mushrooms was due to remediation and purifying    abilities  of

mushrooms. Mushrooms grow in the presence of heavy metals,  secretes enzymes and detoxify such contaminants7. It was reported that mushroom channels heavy metals from land to fruity bodies for removal from the soil/environment8.  This  is first by denaturing  the  toxins  and  finally  absorbing  such  heavy metals.  Mushrooms  were

reported to be hyper accumulators of heavy metals and radioactive metals that  are toxic  when  consumed   and  thus  has  the  ability  to  eliminate   them   from  the environment9.   Similarly reported was the use of Turkey tail mushroom and Phoenix

oyster mushroom mycelia to eliminate 97 % mercury ion from water10.

The use of mushrooms as food and medicines must have dated from ancient Greek,  Egyptians,  Romans  Chinese,  Mexicans  and  even Africans.  There  are  also evidence of uses in religious and tradomedicinal practices11. Reports show that some

species of mushrooms are poisonous and have claimed the lives of historic  figures, such as Pope Clement VII, King Charles VI of France and Czar Alexis of Russia12. The most celebrated casualty was that of Roman Emperor Claudius Ceasar. There was

however the belief that the mushrooms  that killed him were deliberately  poisoned before  being  introduced  into  his  meal  by  political  enemies13.  In  Nigeria,  daily newspaper  reported  in 1986,  the death of a whole family in  Okpokhumi-Emai  in

Owan East local government area of Edo State after consuming soup prepared with mushrooms. There were many such reports in the literature all over the world14.

1.1.1    Characteristics of Mushrooms

Mushrooms  (Fig.2.2)  grow  in  dark,  damp  places  and  feed  on  decaying matter. Mature mushroom develops spores in the gills located on the under-side of the cap. New mushrooms grow from these spores which are of microscopic sizes15. These are the fungal equivalent of seeds16.When the spores are ripped, millions of them, like

fine powder, are dispersed as they drift away in the wind. If they land in a dark, damp place with adequate decaying matter, they germinate and grow into new mushrooms. They develop a thread-like structure called hyphae. Lots of hyphae grow   together

and form the mycelium. Mushrooms often grow in a ring. The fruiting body starts to grow above the surface like a little button protected by a cap which covers the       veil. The veil splits and falls down around the stalk (stem) and form the annulus. The life cycle of mushroom is between two to three weeks16, 17.

Mushroom is a fungus, which feeds by secreting enzymes and digests food

externally and absorb the nutrients in net like chain called hypha. The net like chain (hypha) is exposed to stimuli in their ecological niche and act as a conscious intellect and respond  to stimuli.  Dense and regular  branching  of hypha endows  fungi with

potentials to pervade any substrate thoroughly18. The higher the mycelium thickness,

the higher the rate of mechanical penetration and breaking down of substrate.  This culminates the higher rate of digestion of substrate through the secretion  of extra- cellular  enzymes.   This  shows  the  potentials   of  bioremediation   capabilities   of

mushroom19. This hypha/mycelium penetrates contaminated soils, thus placing a mat

on them;  it is the process  of breaking  down and adsorption  of toxic  products  or pollutants. Generally, the bonds in hydrocarbon and petroleum products are similar to bonds that hold the plant materials together. The enzymes produced  by mushroom which are lignin peroxidase, manganese peroxidase and laccase penetrate, break and digest or mineralizes these hydrocarbon, petroleum products and pesticides to primary non-solid  products and are liberated  in form of water  and carbon dioxide20. These enzymes act singularly or collectively in aiding mycelium to break down natural or human made resistant  materials21.  Similarly,  it was reported  that the mycelium of Shiitake mushroom exposed to heavy metals of cadmium, copper, lead, mercury and zinc increased the production of enzymes laccase, decolorized them and subsequently

absorbed the heavy metals22.

1.1.2    Economic Importance of Mushrooms

Mushrooms can be of much economic value in tropical rain forest ecology and can play important role in food security programme22, 23. In Australia, mushrooms are their second cash crop after potatoes. In China, United States of America, Russia and  Hungary,  mushrooms  are  highly  priced  and  are  even  exported23.  Globally, countries like United States of America, Mexico, Canada, Italy, India, China, South Africa  and Turkey have engaged  in massive  production  of edible  mushrooms  for export and its  food values24. In Nigeria,  mushrooms  are consumed  mainly in rural

areas, and only a negligible quantity gets to the cities25. Moreover, mushrooms are available only from the wild (that is not cultivated) and collection of wild mushrooms

had been and still is a source of income in rural communities. Nigeria being in the tropics, mushroom production as a commercial venture could be an effective means of poverty alleviation. This is due to its short cropping cycle and low cost of planting

input26. These features of mushrooms can be harnessed in several ways in Nigeria as

is  already  being  done  in  some  African  countries  such  as  Kenya,  Uganda  and Zimbabwe. In the 1990’s, the world production of cultivated edible mushroom was estimated at 7 million tonnes, in the same period the total market value for medicinal and edible mushrooms was estimated to be in excess of USD.30 billion27.

1.1.3    Edible, Medicinal and Poisonous Mushrooms in South-East, Nigeria

The most popular edible mushroom in Nigeria is the Pleurotus tuberregium (Fr.) Singer and it is eaten as food or used as food supplement28. It was reported to be a good substitute for meat in many rural villages especially in south–Eastern States. In the same vein, Chlorophyllum molybditis also featured amongst edible mushrooms in this area and have been analyzed  for its nutrient  contents and considered  safe for consumption29. Amanita and Chlorophyllum species earlier reported as poisonous but are consumed in Nigeria is not totally understood but it is    believed         that        the controversy   may  stem   from   any   one   or   combination   of   factors   relating   to

environment,  genetic  and  physiological  differences  which  were  determinants  of tolerance  level  to  toxins  amongst  racially,  geographically  and  traditional  varied people. The Yoruba and Hausa tribes have been associated  with  mushrooms  more than the other tribal groups. The Yoruba tribes are however reported to have identified and used much greater number of mushrooms for food and  medicine  than  the  Hausa

tribes30.

In Nigeria, a variety of edible and medicinal mushrooms are sourced from the wild  due  to  unorganised  mushroom  farming  culture.  This  practice   (mushroom

scouting/hunting) existed for generations and mostly embarked upon by children and women.   About   twenty-five   edible   mushroom   species   of   good   repute   whose knowledge were handed down generational lines via oral communication have been

identified in Nigeria31. These edible mushrooms, collected from various farmlands,

forests and plantations may be sold or cooked fresh, after treatment with warm salt water, with the addition of essential ingredients like pulped pepper, tomatoes, onions, salt and oil or smoked and/or sun-dried for later use.

Traditionally,  mushrooms are used for nutritional, medicinal and  mythological benefits in Nigeria. The uses of mushroom  genetic resources are  not only of high interest  in  agronomy,  agriculture,  human  food  and  animal  feed  but  also  for  the discovery, production and development of molecules or components with high added

value in industries such as chemical and pharmaceutical industries32. This emphasizes

the relative significance of field study and effective documentations as the bedrock for efficient mushroom exploitation. The nutrients and toxicological profile of edible wild mushrooms in Nigeria have been studied29, 33. There is however dearth of information

on  the  anti-oxidant  property  of  edible  and  medicinal  mushrooms  indigenous  to

Nigeria.    The  level of mushroom  nutriceuticals  on a global scale  confirmed  that mushrooms are good health food and reports abound in Nigeria on their  use   for  the treatment  of malnutrition  in infants,  diabetes,  obesity or  hyperlipidemea, sterility, anaemia, mumps, fever and protein deficiency.  Recently, Ganoderma  species have been successfully tested in poultry farming for the   improvement  of  egg-laying  and disease resistant capacity of birds in Nigeria. At the 2nd  African Conference on edible and medicinal mushrooms in 2009, it was reported   that   beta-glucan   based   dietary

supplements of mushroom origin are effective for the treatment of Buruli ulcer caused

by Mycobacterium ulcerans in Ghana while Ganoderma  lucidum (Leyss.) Karst and

Eimeria tenella can be used for the treatment of infected broiler chickens31, 34.

1.1.4   Statement of Problems

In many parts of the world, such as China, United State of American (USA), Canada,  India,  Italy,  Mexico  and  Turkey,  mushrooms  are  highly  priced  and  in massive production for local consumption and export. In U.S.A, the gross domestic products (GDP) for mushroom were about seven million tonnes in 2005, amounting to

30 million dollars annually48.

In  Nigeria,  mushrooms  are  grossly  underexploited  as  only  a  few  types  are considered  edible.  There  is  insignificant  mushroom  cultivation  as  a  commercial venture in Nigeria, but this could massively be used as a means of poverty alleviation due to its short cropping cycle, cheap planting inputs, less  land requirement,  high profit and quick returns on investment49. Till date, mushrooms collection in Nigeria is mainly from the wild and this practice is fraught with fear of mistaking those regarded as poisonous and non-edible for those regarded as edible. This has been occasionally attributed to deaths after mushroom meals50.

Industrialization, urbanization and indiscriminate refuse disposal have  impacted negatively on the environment, thereby posing problems of contamination  with pesticides,  petroleum  hydrocarbons,  heavy  metals  and  other  potential  pollutants. Mushrooms have been reported to be good  accumulator of trace metals in polluted environment51.   Mushroom are hyper accumulators of heavy metals and radioactive metals that are toxic to consume and are thus eliminated from the environment52.

Edible mushrooms are fungi, which belong to the class Basidiomycetes.  They have been found to provide rich addition to diet in the form of carbohydrate, proteins, fibre, ashes, minerals, valuable salts, vitamins and enzymes. The economic trends in

the  country  has  demanded  looking  inwards  for  solutions  to  the  national  food problems. Protein deficiency in Nigerian infants was highlighted by many authors1, 23,

27.  Nutritionists  have proposed  increasing  the consumption  of vegetable  and  plant

proteins. It has been reported that if the quantity of straw burnt in the field could be utilized to grow mushrooms, millions of tonnes of mushrooms for table use could be produced annually53.

Unsuccessful  attempts  to  grow  mushrooms  have  been  made  at  Obasanjo’s

Farms at Ota, Ogun State, Nigeria. Consumers depend on either imported mushrooms whose  price are not within the reach of many Nigerians or on seasonal flushes which are not dependable,  both  in  quality  and  quantity.Several  factors  contribute  to  the relative neglect of mushroom  growing in developing  countries.  The major limiting factor  in Africa,  Latin America  and  the world  is  lack of the technical  skills and theoretical  knowledge  necessary  for  spawn  production.  Other  important  problems include the identification of local edible species suitable for commercialization. The advantage of local species over imported temperate ones is the possibility without air conditioned  environment.  Information  on  the  biology  and  characteristics  of  local mushroom  species  are  scarce.  There  is  scarcity  of information  on the  studies  of mushroom species in Anambra State, Nigeria.

1.1.5    Aim and Objectives of the Study

The  aim of this study was to  provide  base-line  data on five  mushroom species  and  determine  their  potential  for  remediation  of  metal  polluted  soil  in Anambra State, Nigeria.

The specific objectives were to:

i.     determine  the physicochemical  and toxicological  profiles of  five wild  and cultivated mushrooms in the study area

ii.      determine their nutritional and anti-nutrional properties and iii.      determine their ability to bioremediate metal polluted soil.

1.1.6    Scope of Study

Five   species   of   Mushroom   namely;   Termitomyces   robustus,   Agaricus bisporus,  Pleurotus  tuber-regium,  Amanita  phalaoides  and  Amanita  verosa  were collected  from  eleven  locations  in Uke,  Abatete,  Ideani,  Nnobi,  Nnewi (Okpuno- egbu), Nnewi (Umudim)  and Ozubulu between 2009 and  2012  in Anambra State, Nigeria. Some of the samples were cultivated on soils and substrates, harvested and both  (wild  and  cultivated)  samples  were  subjected  to  chemical  analyses  using standard methods by Association of  Official Analytical Chemists (AOAC). Atomic absorption  spectrophotometer  was  used  in  determining  the  concentrations  of  the heavy  metals   in  the   mushroom   samples   while   some   mineral   elements   were determined using flame photometer. It was expected that the analyses would give an overview  of  the physic-chemical  and  toxicological  profiles  of the  five species  of mushroom  in Anambra State, Nigeria. Also their possible use in biotechnology for bioremediation of trace metal polluted soil was investigated. The mushroom samples were selected using a validated food frequency questionnaire which  was  crried out within Anambra State to ascertain their status and edibility.

1.1.7   Significance of Study

The country has entered economic recession as a result of non-diversifiation of the economy, the fall in oil price has put the country into serious financial crisis. Presently the gross domestic product (GDP) from non-oil sector is at 20 % while in

countries like China, Canada U.S.A and Australia the gross domestic products (GDP) from mushroom alone is about 40 %. Mushrooms as vegetative food will ensure food security  (Fig.2.1),  reduce  some  health  risks  associated  with  consumption  of  high cholesterol and carbohydrate foods. Nigerian unemployment rate is at increase, with mushroom  cultivation,  more  youth  will  be  engaged  and  their  social  life  will  be enhanced.  The  nation  is  confronted  with  various  ecological  and  environmental challenges  such  as  climate  change,  deforestation,  aggressive  soil  erosion,  coastal erosion,  water  pollution,  oil  pollution,  flooding  and  urbanization,  many  of  the indigenous  mushroom  species are disapearing  very fast. This study will provide a platform that may draw the attention of the government to urgently rescure our fast disappearing natural resources including mushrooms. Successes have been recorded in Kenya,  Zimbabwe,  Swizerland,  Malawi,  Tanzania,  Namibia  and others through mushroom farming, it is expected  that in future, Nigeria will have similar  success story. The fear of food poisoning after mushroom meals will be eliminated. The study will provide  a platform  for better  mushroom  management  and  production  in the country.

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