Continental J. Biological Sciences Volume 3 (2010)

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Biological Sciences 3: 1 - 7, 2010                                                          ISSN: 2141 - 4122

© Wilolud Journals, 2010                                                                                 http://www.wiloludjournal.com



ALCOHOL FUEL FROM BIOMASS: CHALLENGES OF IMPLEMENTATION IN NIGERIA



             Oparaku N.F

National Centre for Energy Research and Development, University of Nigeria, Nsukka, Nigeria. E-mail:greatgodnf @yahoo.com



ABSTRACTS

Nigeria is endowed with reasonable high energy resources; around 48 million hectares of land nearly 60% are lying idle because the land is neither used of food nor for raw materials for alcohol production. There are feed stocks for alcohol productions available in reasonable quantity. These are sugar cane, cassava, sorghum, corn, sugar beets, molasses and varieties of fruits, trees and grasses, all these can be converted directly into burnable fuels, termed as “befouls,” to assist in meeting transportation fuel demands. The two most widely used types of alcohol fuel are methanol and ethanol. Production of ethanol is by hydrolysis of the starch molecules by enzymes into fermentable sugar. The process is the same for starch, sugar molecules once the fermentable sugar is formed. Methanol and ethanol are both clean fuel that can be produced from locally available, renewable resources, using equipment that a local workshop can make and maintain. This can make biofuels an economical option to fossil fuels and new innovation technologies for the conversion of wood and other biomass resources that are not edible into synthesis gas suitable for production of methanol will make alcohol fuel economic viable. In Nigeria, the development of alcohol fuel will help to increase energy for electricity generation and industrial development and to boost agricultural production of crops for food as well as raw materials for commercial production of alcohol fuel. In this paper efforts were made to compare the properties of alcohol fuel with gasoline, application of alcohol fuels in machines, The challenges of implementation and the constraint towards the much talked about alcohol fuel program in Nigeria were analyzed. The challenges before the Nigerian government are enormous, funding research and information dissemination and encouraging production of food through revamping agricultural production. . This should be done by giving incentives and this will encourage farmers to invest in agriculture and agro-bases industries. These and other factors and the constraint were issued discussed in this paper.



KEYWORDS: Alcohol fuel, biomass conversion, methanol, ethanol, challenges, implementation



INTRODUCTION

The energy profile of Nigeria is characterized by acute shortages of petroleum products and associated with high costs, by unreliable and erratic electric power supply, resulting in power outages, load shedding and power rationing. To provide power for domestic, commercial and industrial purposes, many electric generating plants have been installed as stand-by power sources and operated at high cost to the users. These plants pollute the air, and release carbon monoxide which has resulted in several deaths, (Energy Commission of Nigeria, 2009).



<p style="text-align: justify;"> Nigerian Energy biomass are shown in Fig.1

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<p style="text-align: center;">Oparaku N.F: Continental J. Biological Sciences 3: 1 - 7, 2010

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<p style="text-align: justify; text-indent: 36pt;">Table 1: Nigeria’s Energy resources <p style="text-align: justify; text-indent: 36pt;">Energy Commission of Nigeria (2009)

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<p style="text-align: justify;">Advanced bioelectricity production from refined solid biomass (agro-pellets), bioethanol, biomethanol and bio-hydrogen is a viable option for energy production. The refined solid biomass can be used for heating, cooling, co-firing with coal. All these types of biofuels (derived from biomass resources) could be produced

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<p style="text-align: justify;">Commercially but processing cost still limits the investments. The aim and objectives of this paper are; 1. To compare the properties of alcohol fuel with gasoline. <p style="text-align: justify;">
 * 1) Application of alcohol fuels in machines, and finally
 * 2) The constraint and challenges of implementation in Nigeria

<p style="text-align: justify;"> Pre-Treatment of Feed Stock in Biomass Conversion

<p style="text-align: justify;">Feedstock preparation, fermentation and distillation are three processes involved in the ethanol production process. Ethanol can be produces from three main types of biomass feedstock (2) sugars, starches and cellulose. In the case of sugar cane, the cane is first washed, crushes soar as to extract fluid form it and then filtered to separate the bagasse. The resulting liquid would be treated by steaming so as to sterilize and concentrate before fermentation (Charles and Essel, 2002)

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<p style="text-align: justify;">Charles and Essel (2002)

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<p style="text-align: center;">Oparaku N.F: Continental J. Biological Sciences 3: 1 - 7, 2010

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<p style="text-align: justify;">Bioethanol is produced through the conversion (fermentation) of sugars to ethanol. The biomass that exists as complex sugars (polysaccharides) are however first broken down into fermentable sugars through a process of chemical reaction called hydrolysis see Figure 1.

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<p style="text-align: justify;">The simplified fermentation reaction equation for the simple 6-carbon sugar, glucose is:

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<p style="text-align: justify;">Biomass crops used for alcohol and their fermentable sugars are shown in the Table 3 and 4.

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<p style="text-align: justify;">C6H12O6+ Zymaseundefined2CH3CH2OH    +         2CO2

<p style="text-align: justify;">(Glucose)                                         (Ethanol)            (Carbon Dioxide)

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<p style="text-align: justify;">Uses of Alcohol as Fuel

<p style="margin-left: 54pt; text-align: justify; text-indent: -18pt;">·        It is used as fuel and gasoline additive (eg Gasohol)

<p style="margin-left: 54pt; text-align: justify; text-indent: -18pt;">·        It is used in the manufacture of alcohol drinks

<p style="margin-left: 54pt; text-align: justify; text-indent: -18pt;">·        It is used in preservation of specimen for laboratory works

<p style="margin-left: 54pt; text-align: justify; text-indent: -18pt;">·        As ant freezing agent

<p style="margin-left: 54pt; text-align: justify; text-indent: -18pt;">·        Used in the manufacture of acetaldehyde, ethanol and acetic acids

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<p style="text-align: justify;">PERFORMANCE OF ETHANOL IN AUTOMOBILES

<p style="text-align: justify;"> Occurrences of plugged fuel filters are virtually non-existent with alcohol fuel. The “cleansing” nature of ethanol blended fuels can actually keep the fuel system cleaner and lead to improved performance. In the case of dirty fuel systems, contaminants and residues that have been deposited by previous gasoline fill can be loosened. That residue can get caught in the fuel filter. In older cars, especially those

<p style="text-align: justify;">made prior to 1975, replacing the filter solves the problem from that point on—and once car’s fuel system is clean, car’s performance should improve as well.

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<p style="text-align: justify;">The formulation of gasoline has changed dramatically over the past few years without affecting the performance of older cars. Many older cars were designed to run on leaded gasoline, with the lead providing necessary octane performance — and the lead oxides that were formed during combustion provided a cushion that reduced wear on non-case-hardened valve seats. When lead was phased out of gasoline, oil companies added toxic chemicals to raise the octane rating — and other additives to replace the “lubrication” value of lead. Ethanol added to gasoline increases the octane level of the final fuel by three points — and it does so using a natural, renewable additive that works well in older engines.

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<p style="text-align: justify;">PERFORMANCE IN SMALL ENGINES

<p style="text-align: justify;">Ethanol blended fuel is perfectly acceptable in lawn mowers, snow mobiles, and other small engines. Manufacturers of this equipment know that more than 40% of the gasoline sold across the U.S. contains oxygenates, such as ethanol, so they’ve made certain that their engines perform using clean-burning fuels. Ethanol blends may be used anywhere that unleaded gasoline is used—

<p style="text-align: justify;">Chainsaws, from lawn mowers to personal watercraft. Virtually every small engine manufacturer, including Briggs & Stratton, Honda, Toro/Lawnboy, Kohler, and Snapper, approves the use of ethanol-blended fuel in its equipment.

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<p style="text-align: justify;">With the incredible growth in ethanol production,

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<p style="text-align: center;">Oparaku N.F: Continental J. Biological Sciences 3: 1 - 7, 2010

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<p style="text-align: justify;">               Table2: Liquid Fuel from Biomass compared <p style="text-align: justify;">Conclusion: Methanol is Preferred for Mobile Power  (Lynd et.al, 1991).

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<p style="text-align: justify;">Production Of Methanol From Wood Through Gasification

<p style="text-align: justify;">The commission of the European Committee launched a series of pilot biomass gasification projects with a capacity of 12-60 t/day, producing synthesis gas of a quality suitable for methanol production. Production of methanol from synthesis gas is a proven technology. Table.2. Compares the properties of methanol and  ethanol

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<p style="text-align: justify;">To gain experience with a variety of prospective process concept, each project will develop a different technology, though in all cases, the final aim in similar production of a clean synthesis gas, virtually free of hydrocarbons, low in nitrogen; (Lynd et.al, 1991).

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<p style="text-align: justify;">Recent review confirms that the production of methanol will probably be the preferred large scale option relative to methane and ethanol.

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<p style="text-align: justify;">Furthermore, if gasoline production from biomass is ever to become feasible, indirect synthesis via methanol seems to be more promising than the fisher-Tropsch alternative.

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<p style="text-align: justify;">Many studies on the feasibility of methanol from wood have been published, particularly in the US but also in Europe.

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<p style="text-align: justify;">Feasibility and viability will also be influenced by the way the plant can be integrated (for example independent – located in the middle of an easy plantation; or integrated into a power station where part of the synthesis gas is burned directly and another part is used to produce methanol either as the peak shaving fuel or for integration with methanol plant) (Lynd 1989; Lynd  et. al, 1991;Wyman, 1990)

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<p style="text-align: justify;">The last option is relatively attractive because of possible favourable effects related to the mixing of two synthesis gas streams, one with a too high, and the other attractive low H2/CO ratio. Table 3. Compare the properties of the alcohol fuel with gasoline.

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<p style="text-align: justify;">In 2006, U.S. corn farmers produced a near record 10.74 billion bushels of corn. Of that, 1.8 billion bushels went to the production of ethanol and co-products—so there is plenty of room to expand ethanol production without limiting the availability of corn. Average corn yields continue to increase and other nations are growing more corn as well, so the supply continues to grow. At the same time, new raw Materials for ethanol production are being developed including cornstalks, switch grass, vegetable matter, waste from paper/pulp production, and other“cellulosic” sources, see Table 4 and 5

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<p style="text-align: justify;">In Nigeria, the development of fuel will help to boost agricultural production of crops for food as well as raw materials for commercial production of Alcohol fuel.

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1. Low public awareness of sustainable energy methodologies.
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<p style="text-align: center;">Oparaku N.F: Continental J. Biological Sciences 3: 1 - 7, 2010

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<p style="margin-left: 36pt; text-align: justify; text-indent: 36pt;">Table3. Properties of Fuel Alcohols Compared with Gasoline <p style="margin-left: 36pt; text-align: justify; text-indent: 36pt;"> Lynd et. al, (1991)

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<p style="margin-left: 36pt; text-align: justify; text-indent: 36pt;"> Table 4: Some Bioethanol Feedstocks and their Annual  Yield/ha <p style="text-align: justify;">

<p style="margin-left: 36pt; text-align: justify; text-indent: 36pt;">Table5: Average Fermentable Sugar Content of Some Saccharine feedstocks <p style="text-align: justify;">                                Energy Commission of Nigeria( 2009)

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<p style="text-align: center;">Oparaku N.F: Continental J. Biological Sciences 3: 1 - 7, 2010

5. Funding for Research, Development, Demonstration and Diffusion (R,D,D and D) activities is   grossly in-adequate.
<p style="text-align: justify;"> =Implementation Issues= <p style="text-align: justify;">These implementation options for all Energy resources have been proposed by the Energy Commission of Nigeria

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Diffusion programmes in the sector
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<p style="text-align: justify;">The Challenges before the Nigeria Government

<p style="text-align: justify;">Incentives to Farmers

<p style="text-align: justify;">One of the challenges before the Nigeria government is to encourage the production of food through revamping agriculture. This should be done by giving incentives and this will encourage farmers to invest in agriculture and agro-bases industries.

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<p style="text-align: justify;">Increasing Energy Generation to sustain Biofuel Industries

<p style="text-align: justify;"> Nigeria is currently facing a crippling energy crisis. The energy output is put at mere 1400 mega watts (MW). For the country to grow industrially, the energy generated should be in the neighbourhood of 30,000MW.

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<p style="text-align: justify;">Provision of adequate land for crops production

<p style="text-align: justify;">In Nigeria around 48million hectares or nearly 60% the capacity us lying idle because the land is neither used of food nor for raw materials for alcohol production. It therefore makes more economic sense for the country to put the land into crop cultivation for alcohol fuel production.

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<p style="text-align: justify;">Encouraging the Initiatives of Public and Private Agencies to promote Biofuels Production

<p style="text-align: justify;">Nigeria National Petroleum co-operation (NNPC) has created a Renewable Energy Division (RED) to spearhead the development of biofuel industry in the country. Onyekakeyah (2008) RED has launched a biofuel initiative by inviting Expression of interest (EOI) to enlist joint-venture investors into the country.

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<p style="text-align: justify;">Global Biofuels Limited, a private sector company, has also initiated the construction of Nigeria’s first biofuel refinery at Arigidi – Akoka in Ondo State. Other large scale biofuel projects are being initiated in Jigawa and elsewhere.

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<p style="text-align: justify;">Increased funding of Research and Development Centres for Infrastructure Development, Acquisition of Research equipment, Manpower development and training.

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<p style="text-align: justify;">Alcohol fuel holds great prospects for agricultural and industrial development as well as meeting the energy demands of the Nigerian populace.

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<p style="text-align: justify;">At the moment it is still at experimentation stage or pilot plants stage mostly in the research institutions and tertiary institutions.

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<p style="text-align: justify;">CONCLUSION

<p style="text-align: justify;"> Alcohol fuel a clean fuel which when harnessed and appropriate technology for its production developed will help boost energy  production for industries, automobiles and economic development  and this will definitely reduce over dependence in  fossil fuel, with the abundance biomass resources, encouragement from the government is all that is needed  for its full scale take off in Nigeria

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<p style="text-align: justify;">REFERENCES

<p style="text-align: justify;">Charles Y.W. And Essel B.Hagan (2002) Biomass Conversion and Technology. John Wiley and sons. Pages 84-94

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<p style="text-align: justify;">Energy Commission of Nigeria (2009)       Biofuel Training Manual

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<p style="text-align: justify;">Onyekakeyah Luke (2008), “Agriculture and Biofuel Maters Arising, the Guardian, Tuesday May 27, P. 12.

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<p style="text-align: justify;">Lynd, L.R. (1989), “Production of Ethanol from Lignocellulose using Thermophilic Bacteria: Critical Evaluation and Review, Adv in BIOCHEM Eng / BIOTECHNO      38:1.

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<p style="text-align: justify;">Lynd, L.R. (1989),), “Large-Scale Fuel Ethanol from lignocellulose: Potential, Economics, and Research Priorities, Appl. BIOCHEM. BIOTECHNO. 24/25:695.

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<p style="text-align: justify;">Lynd, L.R.; Cushman, J.H.; Nicholas, R.J. and Wyman, C.E. (1991), Fuel Ethanol from   Cellulose biomass, Science 251:1318.

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<p style="text-align: justify;">Wyman, C.E. and Hintman, N.D. (1990), “Ethanol: Fundamentals of Production from    Renewable Feedstocks and Use as Transportation Fuel, Appl. BIOCHEM          BIOTECH Vol. 24/25:735.

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<p style="text-align: justify;">Received for Publication: 20/04/2010

<p style="text-align: justify;">Accepted for Publication: 18/05/2010

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<p style="margin-right: -36pt; text-align: justify;">Continental J. Biological Sciences 3: 8 - 15, 2010                                                       ISSN: 2141 - 4122

<p style="text-align: justify;">© Wilolud Journals, 2010                                                                                 http://www.wiloludjournal.com

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<p style="text-align: center;">Chemical Nature of Ganoderma lucidum (Curtis) Karsten from Woodlands of Edo State, Nigeria.

<p style="text-align: center;">Ihayere, C.A.1, Oghenekaro, A.O 2, Osemwegie, O.O.2 and Okhuoya, J.A.2

<p style="text-align: center;">1Department of Biological Sciences and Biotechnology, WesternDeltaUniversity, P.M.B 10, Oghara, Delta State, Nigeria, 2 Department of Plant Biology and Biotechnology, University of Benin, P.M.B 1154, Benin City, Edo State, Nigeria

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<p style="margin: 0cm 43.2pt 0.0001pt;">ABSTRACT

<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;">Samples of matured and naturally growing sporophores of Ganoderma lucidum (Curtis) Karst., an indigenous medicinal mushroom were collected from three separate local Government areas in Benin City, Edo State, Nigeria and analyzed for chemical substances. Alkaloids, saponins, flavonoids, were present in all the sporophores analyzed irrespective of their location while anthraquinone was absent in all. Sodium (Na) recorded the best values of between 0.924mg/g and 2.137mg/g while lead (Pb) had the least range value (0.017mg/g-0.021mg/g). The fruit bodies of G. lucidum from the University of Benin woodlands recorded the highest values for Na and calcium (Ca) respectively. The best protein, polysaccharide and lipid values recorded were 25.134%, 1.67% and 0.70% (dry weight per gram) of analyzed samples respectively. The spectrum of chemical constituents in the matured sporophores of local G.lucidum picked from the three sampled locations is fundamental to their uses as traditional remedies of diverse ailments.

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<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;">KEYWORDS: Sporophores, Ganoderma lucidum, Chemicals, Alkaloids, traditional.

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INTRODUCTION

<p style="text-align: justify;">Ganoderma lucidum (Curtis) Karst. is a polypore mushroom of the family Polyporaceae belonging to a group of fungi that are relatively benign when compared to their gilled “cousins”, some of which can be very poisonous. Polypore mushrooms have been the ancient “guardians” of the forest and forest peoples since prehistoric times (Wasser, 2002). The fruit body of G. lucidum is identified with a glossy reddish-orange to brownish-black colour. It has a definite stalk which is laterally or eccentrically attached to the cap. Stalkless (sessile) specimens have also been recorded in Nigeria and many parts of the world. G. lucidum is conk-like or kidney shaped with a woody texture, surface lacquered when moist, measuring 5-20cm in diameter and somewhat zoned (Arora, 1991). This mushroom which is one of the white rot fungi known to foresters often causes the root rot of aging and/or diseased trees, making them more easily susceptible to strong wind. The local names of G. lucidum differ dialectically across cultures and tribes. G. lucidum has common socio-cultural uses across Nigeria as traditional herb and/or as an ingredient in herbal preparations, spiritualism and mysticism, a pattern that was also reported in some places around the world (Wasser, 2005). Although, many polypores including G. lucidum are generally too tough to eat, rural people long ago discovered that a boiled tea from the fungus is health strengthening with anti-microbial and stimulatory potencies (Hobbs, 1995).

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<p style="text-align: justify;">Documentary evidence on folk uses of indigenous mushrooms in Nigeria are limited and rudimentary compared to what obtains in China and Japan where mushrooms are exploited for over 4000 years as food and in the treatment of a wide range of human ailments i.e. hepatopathy, chronic hepatitis, nephritis, hypertension, arthritis, neurasthemia, insomnia, bronchitis, asthma and gastric ulcer (Akpaja et al., 2005). Hobbs (1995), Gao, (2002) and Wasser (2002) reported that G. lucidum contains a combination of potent enzymes, polysaccharides (antitumour, immuno-modulating), antioxidants and micronutrients in addition to water, organic and/or volatile soluble compounds such as amino acids, a small amount of protein and inorganic ions, steroids, triterpenes, lipids, alkaloids, glucoside, coumarin glycoside, volatile oil, riboflavin and organic acids including ganoderic acid (Ying et al., 1987). The polysaccharides and triterpenes isolated from G. lucidum are rated major active compounds with outstanding medicinal effects (Eo et al., 2000).

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<p style="margin-right: -36pt; text-align: center;">Ihayere, C.A. et al.,: Continental J. Biological Sciences 3: 8 - 15, 2010

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<p style="text-align: justify;">Information on the chemical nature of naturally occurring Ganoderma species reported in Nigeria i.e. ''Ganoderma. colosum (Fr.) C.F. Baker and Ganoderma. boninense Pat., is inchoate (Ofodile et al''., 2005). Idu and Osemwegie (2007), Okhuoya et al. (2010) have reported the use of this mushroom as immuno-modulator, anti-allergy, antibiotic and anti-hypertensive, and in the treatment of anaemia, obesity and arthritis in Nigeria. Wasser (2005) reported that the global surge in the commerce of medicinal and edible mushrooms amount to $13 billion US dollars. This has challenged us to focus on indigenous wild utility mushrooms in Nigeria especially EdoState as part of ongoing chemical investigation. Literature reports on Ganoderma species concentrated more on the taxonomy, chemical composition, distributions, ethnomycology and cultivation of many American and Asian representatives with little or nothing on African varieties. This study was therefore aimed at evaluating the chemical constituents of G. lucidum, picked from across local Government areas of Edo State, Nigeria, in other to underpin its socio-cultural values and popularity as potent remedies of diverse ailments.

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MATERIALS AND METHODS

Samples and chemicals

<p style="text-align: justify;">Fresh naturally occurring fruit bodies of G.lucidum used for the study were harvested with the use of a cutlass from felled decaying tree logs in randomly selected woodland systems located in Ogbeson village (A), Uselu (B) and University of Benin campus (C). These locations were distributed in Ikpoba-Okha, Egor and Ovia Local Government Areas of Edo State, Nigeria respectively. A total of 6 fruit bodies were randomly collected from 3 distanced (3-5km) woodland systems in a location. The glass and analytical grade chemicals use for this study were supplied by Biochemistry Department of the University of Benin while the study was carried out in the Mushroom Biology Lab of the Department of Plant Biology and Biotechnology, University of Benin, Benin City, Edo State, Nigeria. 

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<p style="text-align: justify;">Chemical Analysis

<p style="text-align: justify;">In the lab, the fresh fruit bodies of G. lucidum from the field were oven dried at 550C for 24 hours, manually cleaned with a small painting brush to remove all extraneous particles, sliced into smaller pieces with the use of a bread knife and further dried at 450 C for 30mins to ensure complete drying after which they were milled into powder using a mechanical blender, bagged and labeled samples A, B and C respectively. Two grams (2g) of the mushroom powder from each of the samples was analysed for metallic elements, alkaloids (anthraquinones, flavonoids, saponins and tannins) and ergastic contents such as protein, lipid and polysaccharides.

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<p style="text-align: justify;">''Metallic element analysis ''

<p style="text-align: justify;">The Fe, Ca, Na, Mg and Pb were analysed using HNO3, H2SO4, HClO4wet-digestion reagents in ratio 10:5:10 method and determined by atomic absorption spectrophotometer (Varian model AA-1475) as outlined by Konuk et al. (2006).

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<p style="text-align: justify;">''Analysis of ergastic substances ''

<p style="text-align: justify;">Protein content was analyzed using the macro Kjeldahl method in which nitrogen contents was first determined and its value multiplied by 6.25 coefficient while the total lipid content was determined using Okalebo et al. (2002) Soxhelet extraction method. Twenty grams (20g) of the G. lucidum powder from each of the sampled locations (Ogbeson village, Uselu and University of Benin campus) were analyzed for polysaccharides using the Sephadex (G125 series) gel extraction and chromatographic methods (Lin et al., 2002)

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<p style="text-align: justify;">Qualitative test of alkaloids

<p style="text-align: justify;">The method of Odebiyi and Sofowora (1978) was used in this test. Two grams (2g) of powdered sample from each of the locations was boiled in 30ml of 95% ethanol for 6 hours in a Soxhelet extractor. The extract was evaporated to dryness using a vacuum evaporator after which the residue was dissolved in 5ml of 1% HCl, shared into two equal parts respectively. Seven drops Mayer’s reagent was added to one part while another seven drops of Dragendoff”s reagent was added to the other. Observed turbidity or precipitation with both reagents indicates the presence of alkaloids.

<p style="margin-right: -36pt; text-align: center;">Ihayere, C.A. et al.,: Continental J. Biological Sciences 3: 8 - 15, 2010

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<p style="text-align: justify;">Saponins and flavonoids were analyzed using the qualitative method of Hertog et al. (1992) respectively while tannins was tested using drops of 10% FeCl3on the filtrate derived from a 500mg/10ml mixture of powdered sample and distilled water (Desphande et al., 1986). Anthraquinones was analysed by shaking 10ml benzene with one grams of the powdered samples respectively after which the filtrate from the mixture was treated with ten mililitre (10ml) of 10% NH4OH fractionated using a separating funnel and observed for colour indicator in the ammonia layer (Odebiyi and Sofowora, 1978).

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<p style="text-align: justify;">Statistical analysis

<p style="text-align: justify;">Each sample was analysed in triplicate and the values were then averaged. Data were assessed by the analysis of variance (ANOVA) as described by Snedecor and Cochran

<p style="text-align: justify;">(1987) and by Duncan-multiple range test with a probability P ≤ 0.05.

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RESULTS AND DISCUSSION

<p style="text-align: justify;">The results of the study showed that alkaloids and their chemical allies i.e. saponins, flavonoids and tannins were present in G. lucidum from the different locations while anthraquinone was absent (Table 1). These compounds in addition to both trace and major elements have been previously investigated in many socio-culturally valuable and edible mushrooms some of which include Chlorophyllum molybditis, Lentinus subnudus, Pleurotus tuberregium, Psathyrella antroumbonata and Schizophyllum commune by Alector (1995) and Alofe et al. (1996). Literature are however scarce on the chemical composition of many Ganoderma species in Nigeria compared to works done on other edible mushrooms (Ofodile et al., 2005; Osemwegie et al., 2006; Jonathan et al., 2008). The presence of alkaloids, flavonoids, saponins and tannins may be one of the reasons for the soaring popularity of Ganoderma in local healthcare provisions in many parts of the world especially in Nigeria (Osemwegie et al., 2006). The effect due to the absence of anthraquinone on the medicinal potency and immune-enhancing capability of this macrofungus is not fully understood despite reports of its presence in novelty plant material employed in phytomedicine (Odebiyi and Sofowora, 1978).

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<p style="text-align: justify;">Table 1: Qualitative parameters of secondary metabolites and alkaloids

<p style="text-align: justify;"> <p style="text-align: justify; text-indent: 36pt;">+ = present - = absent

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<p style="text-align: justify;">G. lucidum from various locations in EdoState had the same range of protein (% dry weight) as those recorded in separate studies elsewhere (Mattila, 2001). G. lucidum collected from forests in Uselu, Egor LGA recorded the highest protein value (25.134%) while those from Ogbeson forests and University of Benin campus recorded 22.75% and 21.21% protein respectively (Table 2). The difference in protein content of the mushroom from the three (3) locations was not significant (P ≤ 0.05). Fasidi and Kadiri (1992) also showed that C. molybditis and P. tuberregium in the South-Western part of Nigeria had protein content of 22.73 and 20.30% (dry weight) respectively. The marginal variation observed in protein contents of mushrooms is not fully understood but may be connected to differences in resource utilization/selectivity, the chemical dynamics and integrity of their macrohabitat. Although, the amino acids content of these samples was not analyzed, the value of the protein content is recognized according to Wasser (2005) to be an acceptable reflection of the amino acid value. The results from the study therefore confirmed that G. lucidum is fit for consumption but Osemwegie et al. (2006) remarked that its rejection was because of its glossy outlook and corky texture.

<p style="margin-right: -36pt; text-align: center;">Ihayere, C.A. et al.,: Continental J. Biological Sciences 3: 8 - 15, 2010

<p style="text-indent: 36pt;">Table 2: Protein values (% dry weight) of G. lucidum from the different sampled locations <p style="text-align: justify;">

<p style="text-align: justify;">Lipid values recorded during the study range from 0.6%-0.7% per gram of each of the sample analyzed respectively (Table 3). The difference in lipid values of the G. lucidum recorded from the three (3) locations was not significant (P ≤ 0.05). This agreed with the work of Fasidi and Ekuere (1993) who in a separate study recorded values ranging between 0.20% - 1.02% for sclerotia of P.  tuberregium cultivated on various waste materials.

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<p style="margin-left: 36pt; text-align: justify; text-indent: 36pt;">Table 3: Lipid composition of G. lucidum samples (% dry weight).

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<p style="text-align: justify;">Polysaccharide values of 1.5 and 1.22% (dry weight) were recorded for fruit bodies of G. lucidum picked from forests in Ogbeson and University of Benin campus respectively while 1.67% (dry weight) was recorded for those picked in Uselu, Egor LGA of Edo State (Table 4). The difference in Polysaccharide content was also observed to be insignificant (P ≤ 0.05). This agreed with Chang (1996) and Wang et al. (1996) who reported polysaccharide values of 1.38% for G. lucidum obtained from China and The Philipines respectively. The polysaccharides found in G. lucidum belongs to either the β-glucans groups which according to Chan et al. (2009) is responsible for the stimulation of many kinds of immune response and/or cells health in many animals and humans. Lentinan and Krestin were some of the β-glucan group that was reported by Wasser (2002) as proceeding through clinical trials in the treatment of cancers and other diseases.

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<p style="margin-left: 36pt; text-indent: 36pt;">Table 4: Polysaccharide values of G. lucidum samples (% dry weight)

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<p style="text-align: justify;">The study also showed that non-toxic elements such as Na recorded the highest value of 2.10 mg/g dry weight followed by Ca (0.90mg/g) and Fe (0.25mg/g) respectively while Mg and Pb recorded the lowest values of 0.05mg/g and 0.02mg/g respectively. Fruit bodies of the sample collected from the University of Benin recorded the highest value for Na and Ca (Fig. 1). The difference in Na and Ca content was observed to be significant (P ≤ 0.05) while that of Mg, Pb and Fe was not significant (P ≤ 0.05). Analysis of fruit bodies from other locations recorded values for Ca which varied from 0.502-1.162mg/g (dry weight) as compared with that from University of Benin with 1.162mg/g dry weight value. This concurred with quantitative record of Konuk et al. (2006) and Olumuyiwa et al. (2008) on mineral composition of edible mushrooms from Turkey and Nigeria respectively but marginal differences were observed which may be as a result of variation in environmental and vegetation status, and level of anthropogenic activities. The values recorded for Ca during this study however concord with the work of Kadiri and Fasidi (1992) which reported 0.642mg/g and 1.25mg/g in L. subnudus and P. tuberregium respectively (Okhuoya and Ajerio, 1988). However, vast scientific literature supported interspecific and intraspecific variations in the elemental and chemical compositions of many utility mushrooms with philosophical rather than data-supported scientific conjectures provided as reasons (Isiloglu et al., 2001; Sanme et al., 2003). The

<p style="margin-right: -36pt; text-align: center;">Ihayere, C.A. et al.,: Continental J. Biological Sciences 3: 8 - 15, 2010

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<p style="text-align: justify;">employment of fungal biomass, molecular and radioactive labeling techniques may provide broader insights to the influence of environmental variables and synecological characteristic on the mechanism of resource utilization, selectivity and assimilation by fungi differing in substrate preference or fungus from similar and different ecozones.

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Mineral elements vs concentration (mg/g dry wgt)

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<p style="text-align: center;">Fig 1: The mineral elements of G. lucidum from different sampled locations (A=1; B=2; C=3)

<p style="margin-left: 47.25pt; text-indent: -18pt;">(A)   Ogbeson village (B) Uselu (C) University of Benin campus

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<p style="text-align: justify;">Sodium (Na) recorded a value of 1.200mg/g for G. lucidum collected from Ogbeson forests, 0.924mg/g and 2.137mg/g for those from the forests of Uselu and University of Benin respectively, fell within the range reported by Kadiri and Fasidi (1992) for Termitomyces robustus (3.926mg/g), L. subnudus (2.298mg/g), Kadiri and Fasidi (1992) for P. tuberregium (2.298mg/g), and Mattila (2001) for Lentinus edodes (1.14mg/g). Vetter (2003) however remarked that the relatively consistent Na level of most edible mushrooms (0.10mg/g - 9.5mg/g) studied is of fundamental nutritional and medicinal benefit to the consumer, especially people with high blood pressure.  

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<p style="text-align: justify;">A lower range of values (0.021 and 0.034) was recorded for Magnesium (Mg) in G. lucidum samples collected from the three locations (A, B and C). This range is in consonance with the value of 0.037mg/g reported in an earlier work done by Wang et al., (1996) but varied from values recorded by Kadiri and Fasidi (1992) for C. molybditis (1.868mg/g) and P. tuberregium (1.484mg/g), and Mattila (2001) for Agaricus bisporus (0.10mg/g) and L. edodes (0.13mg/g). The low level of Mg recorded for G. lucidum may not be unconnected to the chemical nature of the substrates which were mostly wood-based. This according to Okhuoya and Ajerio, (1988) is due to the relatively low level of Mg in soils than calcium and the decrease in uptake of magnesium due to the high concentration of calcium in living tree plants. The level of Iron (Fe) also recorded for this fungus which ranged between 0.156mg/g and 0.176mg/g also concurred with the findings of Wang et al., (1996). Further studies are however necessary for a proper understanding of the origin and dynamics of chemical elemental flux in mushrooms substrates (nutrient base) vis-á-vis their absorption and assimilation.

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<p style="text-align: justify;">The low value observed for Lead (Pb) from the G. lucidum samples from Ogbeson village, Uselu and University of Benin campus woodlands ranged from 0.017mg/g - 0.021mg/g. This was corroborated by Ziegler (2001) and Olumuyiwa et al. (2008) who categorized Pb as one of the trace toxic elements identified in wild edible mushrooms from Nigeria. The presence of Pb and its implication on the edible and medicinal utility of wild mushrooms requires further study even though the knowledge of the source of the

<p style="margin-right: -36pt; text-align: center;">Ihayere, C.A. et al.,: Continental J. Biological Sciences 3: 8 - 15, 2010

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<p style="text-align: justify;">element is common. The relatively higher value of 0.021mg/g recorded for samples of G. lucidum from the University of Benin may be due to increased industrial and vehicular activities and/or emissions within the vicinity of the woodland from which the mushroom was picked (Isilogu et al., 2001).

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<p style="text-align: justify;">It is obvious from this study that, G. lucidum, widely acclaimed for its medicinal properties, contains essential mineral nutrients which are of immense health benefit as antioxidative, vitamins, anti-inflammatory and antimicrobial in human and animals (Ofodile et al., 2005; Ogbe et al., 2008). Wasser (2002) reported that this fungus can boost the human protein level and immune systems through the range of polysaccharides that were anti-tumour (triterpenoids) in nature. Our body structure is made up mainly of bone and cartilage, so we need these minerals especially calcium for our body growth. The shiny/glossy appearance is as a result of the presence of tannin.

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<p style="text-align: justify;">G. lucidum is consumed as tea by cutting whole mushroom into small pieces, sundried or oven dry and simmer in a cup of hot water. It can also be consumed in refined form as tablet. Though the fungus is a fundamental health food, its very woody nature was the reason for its inedibility either in pickled, fried, cooked or raw form. This study has therefore introduced preliminary information on the chemical nature of Ganoderma lucidum indigenous to Nigeria. It has also provided comparative base for quantitative and qualitative chemical data of Nigerian Ganoderma species with widely published varieties from Asia, America and Europe.

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<p style="text-align: justify;">Zhao, J.D. and Zhang, X.Q. (1994) Resources and taxonomy of Lingzhi ( Ganoderma) in China. From program and abstracts of the 1994 International Symposium on Ganoderma Research. (October 24-26, 1994.) Beijing, BeijingMedicalUniversity.

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<p style="text-align: justify;">Ziegler, R.G. (2001). The future of phytochemical databases. American Journal of Clinical Nutrition. 74: 4-5.

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<p style="text-align: justify;">Received for Publication: 05/04/2010

<p style="text-align: justify;">Accepted for Publication: 30/05/2010

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<p style="text-align: justify;">Corresponding author;

<p style="text-align: justify;">Osemwegie, O.O.

Department of Plant Biology and Biotechnology, University of Benin, P.M.B 1154, Benin City, Edo State, Nigeria

<p style="text-align: justify;">[mailto:Email-omorefo@yahoo.com Email-omorefo@yahoo.com],

<p style="margin-right: -36pt; text-align: justify;">Continental J. Biological Sciences 3: 16 - 32, 2010                                                     ISSN: 2141 - 4122

<p style="text-align: justify;">© Wilolud Journals, 2010                                                                                 http://www.wiloludjournal.com

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<p style="text-align: center;">ECONOMIC IMPORTANCE OF SOME UNDEREXPLOITED TREE SPECIES IN NIGERIA: URGENT NEED FOR SEPARATE RESEARCH CENTERS

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<p style="text-align: center;">1K. Okunomo and 2E.O. Egho

<p style="text-align: center;">1Department of Forestry and Wildlife and 2Department of Agronomy, Delta State University, Asaba Campus, Asaba, Delta State

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<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;">ABSTRACT

<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;">This study reviews the importance of some endangered tree species in Nigeria. Trees are very important to people. They provide many products, including food for humans and animals, timber, fuel and medicines. In the tropics (Nigeria inclusive) trees are much more important as food crops and cash crops than in temperate zones. Proper management of trees and forests is necessary to make resources sustainable. Trees play an important part in traditional farming system in Nigeria not just as food and cash crops but also as suppliers of fuel wood and fodder. The problem we are faced with however is the high rate of deforestation. Some endangered but extremely beneficial tree species are disappearing. Furthermore, due to the dearth of research centers that will tackle specific trees by providing detailed information about such trees, Nigerians are denied such benefits. The trees considered here are: Irvingia gabonensis Chrysophyllum albidum, Dialium guineense, Dacryodes edulis, Annona ''muricata. Treculia africana, Garcinia kola Parkia bicolor, Tetrapleura tetraptera and Azadirachta indica''. Detailed information about the botanical characteristics and economic importance has been provided. It is recommended that research centers should be established for this species either separately or combined as in the case of some agronomic crops. This hopefully will reduce extinction and create more awareness about the tremendous benefits derivable from these species.

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<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;">KEYWORDS: Economic benefit, forest exploitation, research centers, endangered species

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<p style="text-align: justify;">INTRODUCTION

<p style="text-align: justify;">The country Nigeria which is officially referred to as the Federal Republic of Nigeria is a country in West Africa and the most populous country in the Africa continent. Nigeria shares land borders with the republic of Benin in the West, Chad and Cameroon in the east, Niger in the North, and borders the gulf of Guinea in the south. Since 1991, its capital has been centrally located city of Abuja, previously, the Nigeria government was head quartered in Lagos.

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<p style="text-align: justify;">The people of Nigeria have an extensive history and based on archaeological evidence, human habitation of the area dates back to at least 9000 B.C. The Benue-Cross river area is thought to be the original homeland of the Bantu migrants who spread across most of central and Southern Africa in waves between the 1st millennium BC and the 2nd millennium AD. However, the Nigerian state came into being on October 1, 1960 when Nigeria declared its independence from the British; and at present consists of 36 states and the federal capital territory. Nigeria re-achieved democracy in 1999 after a sixteen year interruption by a series of military dictators. From 1966 until 1999, Nigeria had been ruled except the short-lived second republic, 1979 – 1983) by military dictators who seized power in coups d’etat and counter-coups.

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<p style="text-align: justify;">The people have been planting trees for thousand of years for food, shelter, ceremonial or religious purposes: the Bible records Abraham planting a tamarisk tree to commemorate the treaty of Beersheba (Gen 21:23). Probably the oldest planted tree still living, of which a reasonable historical record exists, is actually in the tropics, in Sri Lanka where the Bodhi tree (Ficus religious) is recorded as being “planted” miraculously at Anradhapura in about 220 B.C.

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<p style="text-align: justify;">In Nigeria presently, there is a considerable amount of emphasis being given to tree planting due mainly to the climate change being experienced all over the world currently. However, this study’s purview goes beyond these. It is seeking for a comprehensive strategy to develop research centers that will concentrate on providing in-depth information on tree species with a view to alleviating our economic problems.

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<p style="text-align: justify;">TRENDS IN NATIONAL FOREST POLICIES

<p style="text-align: justify;">Nigeria forestry started at the end of the 19th century but was formally launched in 1908 as a department which has the responsibility for development of forest resources and agricultural production i.e forestry started as an all embracing rural land use sector with responsibility for agricultural production, forestry and fisheries production.

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<p style="text-align: justify;">Between 1908 and 1916 there were series of proclamation which aimed at curtailing the effect of cultivation in natural forests, specifically forest crops were singled out for protection. Exploitation firms were compelled to replace trees that were felled by planting a number of seedlings for each felled tree. Some areas were also identified for reservation and in particular fragile areas among water courses and river basins were rigorously protected. The result was that a large number of areas were reserved in various ecological zones of the country with the objectives in the long run that 25% of the superficial area of the country would be under forest reserve after 1918.

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<p style="text-align: justify;">As from 1914 following the amalgamation of Northern and Southern Nigeria a strong forestry department came into being. It was made to reserve forest areas as quickly as possible. Consequently, a lot of forest reserves were created between 1908 and 1930. However, a great deal of agitation ensued from the chiefs and community leaders who felt that their movement and opportunity for shifting cultivation were greatly limited. Eventually reversals were made to the so-called native authorities which were empowered to manage the forest estate. Thus there was a positive movement in term of development policy objectives for the creation of forest reserves between 1908 and 1930. This was followed by backward movement in term of the mis-management of the forest resources by local or native authorities who did not have the capacity, vision and development perspective for the forest estate. Inevitably, there was a slow down in the number of forest reserves being created after 1930. Also because of the importance that agriculture eventually gained as the base of the national economy, some crops that were originally managed or developed by the forestry department were passed on to the agricultural department e.g. rubber species introduced by the early foresters over the past 70 years have been developed as forest crops but since 1935 it has been managed by the agricultural department. Thus forestry got contracted in terms of legal responsibility and in term of a slow down in land acquisition for the sector.

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<p style="text-align: justify;">In 1946 – 1956 development plan was promulgated for the whole country and forestry featured prominently in that plan. However, during the political developments between 1948 and 1950 when the country eventually emerged as a federation, forestry was considered as a separate subject and was passed on to the concurrent list of topics for which the regional zones has the power to develop without interference from the central government. Consequently, separate regional forestry department and policies emerged in accordance to the wishes of the regional government set up in 1954. The federal government had no power to manage the forest resources since the regional governments owned the forest land. Research was the only topic reserved for the federal government.

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<p style="text-align: justify;">Over the last thirty years there has been a great deal of divergence between the regional/state policies and the ideal national forest policy that ought to be pursued in the general interest of the country. For the first ten years or so there was no collaboration between the various regional governments in terms of forest developments. Each regional government enunciated its own policy as well as project on the basis of legal authority without reference to neighboring region and as if forest resources and forestry matters are easily compartmentalized within political boundaries. However, it became obvious by 1960 that forestry affairs and its effects thereof are not always strictly limited to the political boundaries.

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<p style="text-align: justify;">Consequently, a committee known as forest/technical committee was set up in 1961. It was set up to provide opportunities for exchange of ideas and views between the regions chief conservator of forests. The Forestry Technical Committee was originally headed by the Director, Federal/Department of Forest Research. Subsequently, the committee work and activities grew in a conventional manner both in scope and volume to assume an air of authority and policy pronouncement. That committee was transformed in 1970 to what is now known as the National Forestry Development Committee Abo in 1910 by the Federal Department of Forestry Ministry of Agriculture and Natural Resources. The National Forestry Development Committee (NFDC) since then has become a formidable forestry organ in the country.

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<p style="text-align: justify;">Although the 19 states at that time still had the constitutional power to develop forestry resources potential at their convenience. Nevertheless the fact that NFDC met regularly twice a year under the director (FDF) there has been continuing process of collaboration and correspondence between the various state government on the one hand, and the state government and the federal government on the other hand.

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<p style="text-align: justify;">The members of the (NFDC) consists of the chief conservator of forests (CCF), Director, Forestry Research Institute of Nigeria and the Heads of Department of forestry in Nigeria Universities. Because the meetings were held twice a year, a great deal of topics of interest of various state governments are tackled and discussed with a consensus being arrived at the end. e.g. a number of projects have been developed either with federal government direct finance or through the support of the international agencies. Also the FDF have offices in all states of the federation thus providing, a unique opportunity for the federal government to assist forestry development in the states and for the state to take advantage of the opportunity available from the neighboring states. Many a time the states are helped to develop their forest resources than they would have been able to do on their own.

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<p style="text-align: justify;">Although, there is a federal constitution and the regulatory forestry policy that started in 1954 are still largely invoked, and that state forest policies are still constitutionally dependent on the federal government nevertheless there has been a growing together particularly since 1970 in the formulation of long, and short term programmes between the states.

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<p style="text-align: justify;">The situation as of today, is that there is no forestry service that is financially strong and capable to go it alone. They have to depend on the federal department of forestry and occasionally the neighbouring state forestry service for assistance.

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<p style="text-align: justify;">TREE SPECIES REQUIRING SEPARATE RESEARCH CENTERS

<p style="text-align: justify;">Presently, there is only one forestry research institute of Nigeria headed by a director saddled with the responsibility of coordinating forestry research activities in Nigeria. The problem facing the organization at the moment is under funding. However, the major concern of this study is decentralizing the forestry research institute, as is common with agricultural crops by so doing opportunities for in depth research on a particular tree  crops will be readily available. Research activities on food crop will be greatly enhanced with specific findings that will contribute greatly to the economic development of the country.

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<p style="text-align: justify;">The various economic species referred to are listed below:

<p style="text-align: justify;">C. ''Irvingia gabonensis ''

<p style="text-align: justify;">Irvingia gabonensis  and Irvingia wombolu are the two tree species that produce the most economically viable non-timber forest products from the rain forest zone of West Africa (Ladipo, 2000). These Irvingia species are found growing wild in the humid lowland forests of tropical Africa in Angola, Cameroon, Central African Republic (Harris 1990).

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<p style="text-align: justify;">Irvingia gabonensis prefers well drained sites while Irvingia wombolu thrives in wetter conditions. They are also found in compound farms and it is not uncommon to find Irvingia trees protected in cocoa, cola and coffee plantation (Ladipo, 2000).

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<p style="text-align: justify;">Bush mango is planted and maintained in farms in Central and Western Africa. Presently, most farmers maintain mature bush mango trees that are already grown, on their land and will also transplant seedling onto their farms (Ayuk et al, 1999). New seedlings are also raised from selected seeds from trees that are known to produce high yields of good fruits or from seeds procured from the market. However, the most common source of new stock for cultivation is from the forest (Tchoundjeu et al, 1997).

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<p style="text-align: justify;"> Irvingia gabonensis and Irvingia wombolu are very similar and indeed are often difficult to tell apart from herbarium specimen alone (Harris, 1996). There are not many differences between the two species except the edibility of the fruit mesocarp (fruit sweetness), seed characteristics and some tree morphological differences. Some differences listed by Hariss (1996) include the density of fibres in the cotyledon (drawability), the size of the endosperm, the height at first branching, the disc shape of the tree as diagnostic characteristics. Two varieties have been identified (Okafor, 1975) based on the reproductive phenological observation. He reported differences in flowering and fruiting between these varieties.

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<p style="text-align: justify;">Irvingia gabonensis and Irvingia wombolu, the bush mango is the sources of “ogbono” the Irvingia kernel which is popularly used as soup thickener in most West and Central African countries. The popularity of the kernels in local and international markets has highlighted its potentials as a true commercial crop, and this has resulted in more intensive collection of Irvingia fruits from the forests (Ladipo, 2000). Local people gather the fruits of Irvingia gabonensis and Irvingia wombolu and many other mammals consume the fruits in the forests. The juicy pulp of Irvingia gabonensis is sweet and rich in vitamin C and is widely consumed as a dessert fruits or snack in the region (Ladipo, 1999). The fruit can be used for making jam, jelly, juice and wine (Akubor, 1996). Irvingia gabonensis fruits and seeds constitute a significant part of the diets of forest elephants, gorillas, forest pigs, squirrels and other rodents (Tchamba and Seme, 1993).

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<p style="text-align: justify;">The consideration of Irvingia as a strategic crop and one that has immense value for food security cannot be over-emphasized if we consider its nutrient values. This is very important where resource poor farmers whose diet revolves around starchy foods are involved. The attractiveness of Irvingia is derived from the highly valuable by-product of the fruit – the kernel. The kernels which are classified as oilseeds are ground into a paste or cake called ‘dika bread’ which is used as soups, stews or sauce additive for flavouring and thickening (Leakey et al 2003). Irvingia kernels provide carbohydrates and protein, forming an important part of the diet in West and Central Africa (Onyeke et al, 1995). Margarine, Cooking oil, soap, cosmetics and pharmaceuticals are produced from fat extracted from the kernels (Ejiofor et al; 1987).

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<p style="text-align: justify;">Other uses of this socio-economically important fruit crop include medicinal preparations, environmental conservation (Tchoundjeu et al, 2000), fodder and chewing stick. The roots, leaves and bark of Irvingia species are used medicinally in the treatment of diarrhoea, shortening of breast feeding (Ndoye and Tchoundjeu et al, 1995) as well as for hernia, yellow fever and as An anti – poison. Aside from its role as a thickener, the residual kernel cake can be used as a binder in food or pharmaceutical product (Joseph, 1995).

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<p style="text-align: justify;">Irvingia gabonensis and Irvingia wombulu are ranked first in the list of high value agro forestry tree species by farmers in the priority settings survey conducted by the International Centre for Research in Agroforestry (ICRAF) and partners in four countries; Cameroon, Gabon, Ghana and Nigeria (Tchoundjeu et al., 2002). Many rural dwellers depend on NTFPs for their livelihood and income (Baranty et al; 2001).

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<p style="text-align: justify;">The trees are a valuable source of income for West and Central African farmers through the sales of kernels which fetch a price higher than the fruits (Ayuk et al.,1999). Ladipo (1999), reported that the market for kernel products was worth in the region of US $50 million. This market which extends to local, regional and international levels is growing.

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<p style="text-align: justify;">''Chrysophyllum albidum ''

<p style="text-align: justify;">Chrysophyllum albidum is a lowland rainforest tree that is usually planted in villages extending from Sierra Leone to East Africa, it belongs to the family of Sapotaceae, which is relatively large in Nigeria alone. All

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<p style="text-align: justify;">members of the family are trees or shrubs occurring mostly in the rainforest with very few in forest outliers and riparian forest in the savannah regions.

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<p style="text-align: justify;">Under favourable condition, Chrysophyllum albidum may grow up to 36.5 m tall, though it is often small. The leaves are very important in the preparations of various medicines. The fruit is a large berry containing four to five flattened seeds or sometimes fewer due to seed abortion (Keay, 1989). Its fruit pulp which is sweet and pleasantly slightly acidic is edible. Within the head seed coat is the whitish cotyledon which is used for medicinal purpose including leaves and fruits. The plant has in recent times become a crop of commercial value in Nigeria. The fleshy pulp is eaten especially as snack and relished by both young and old (Cenrad, 1999). Chrysphyllum albidum is reported as an excellent source of vitamins, iron, flavours to diets and raw materials to some manufacturing industries (Umelo, 1997 and Adisa, 2000). In addition, its seeds are a source of oil, which is used for diverse purposes. The seeds are also used for local games (Bada, 1997). Traditionally, the fruits are considered as blessings to humanity especially, its potential to effectively improve the reproductive capacity of men and women. Chrysophyllum is not one of the well known indigenous fruit trees but it is also among the known multipurpose forest tree species as the wood of the matured tree is converted for utilization purposes in houses and huts. In certain cases, the wood is used in household articles and tools handles.

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<p style="text-align: justify;">''Dialium guineense ''

<p style="text-align: justify;">In Nigeria, Dialium guineense flowers from September to October and fruits from October to January while in Ghana, from September to November the tree is covered with small white flowers in panicles. Fruits ripen in March too May but could extend beyond July. Animals, which like to eat pulp in which the seeds are embedded, help disperse the fruit.

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<p style="text-align: justify;">However, the fruit can also be transported by water since it floats; transporting by sea currents may lead to long-distance dispersal.

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<p style="text-align: justify;">The importance of Dialium guineense cannot be over-emphasized although, this crop is not too well known and not much has been done on it either. Dialium guineense is edible and widely consumed by the people of Southern Nigeria the pulp is red with a sweet sour, has a stringent flavour similar to baobab but sweeter. It is peeled and eaten raw; it can be a little constipating. The species belongs to the family Caesalpinaceae.

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<p style="text-align: justify;">BOTANICAL CHARACTERISTICS (Dialium guineense) 

<p style="text-align: justify;">It’s about 30 m in height with a densely leafy crown, but often shrubby. Bole without buttresses, Bark is smooth, grey, slash reddish, yielding a little red gum. Leaves sometimes finely hairy, with a common stalk, 5 – 13cm long, with an odd terminal leaflet and usually two pairs of opposite or alternate leaflet. The flowers are usually whitish, in large panicles up to 30 cm long. The branches spread                                                                                                                                             widely and less horizontally. Fruits are usually abundant, more or less circular and flattened but sometimes almost glabrous, up to 2.5cm in diameter.

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<p style="text-align: justify;">The origin of the generic name is not known J.E. Smith a noted 18th Century English Botanist, sought it and could not discover it, nor have modern Botanist. The specific name means “of guinea” (Krugman et al; 1998) enriching, refreshing fruit pulp can also be soaked in water and drank as a beverage. Leaves are bitter they may be used to cook daomoda, a Ghanaian dish that taste both sweet and bitter. It is a good source of fuel. The tree is good for firewood, charcoal and timber. The sap wood is white with distinct ripple marks; the heartwood is red-brown. Because the heartwood has high silicate content, axes and saw  quickly get blunt when used on it. The wood is hard, durable, heavy, light brown, with a fine texture. It is used for vehicles, houses and flooring. The bark and leaves are used against several diseases such as malaria, diabetes etc.

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<p style="text-align: justify;">Harvesting the tree is difficult because the wood is dense. They often have tall buttresses, which have to be slashed before cutting, as much of the wood would be wasted if the trunk were cut above the buttresses. The logs cannot be transported by river as they could sink.

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<p style="text-align: justify;">Dialium guineense grows in dense savannah forest, shadowing canyons and gallery forest. It is found from Senegal to Sudan along the southern border of the sahel. It is the most common and widespread Dialium guineense  in Nigeria. In Ghana, Dialium guineense is found along transition zones bordering high forest in riperean forest of the savannah woodland, in coastal scrub, and in riparian vegetation of the Volta near Ada.

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<p style="text-align: justify;">''Dacryodes edulis ''

<p style="text-align: justify;">Dacryodes edulis is a medium-sized, evergreen tree attaining a height of 18-40 cm in the forest but not exceeding 12 m in plantation, it is generally branched from top down, with a deep, dense crown. The bole is rather short and slightly fluted, about 50-70 cm in diameter and more or less sinuous. The scented pale, grey rough bark exudes a whitish resin, but gums are absent. Leaves are compound, imparipinnate, with 5-8 pairs of glossy leaflets. Based on long term and extensive field observation in Nigeria, two varieties of Dacryodes edulis were discovered. Dacryodes edulis var edulis and ''Dacryodes edulis var parvicarpa. ''The fruit of the former is large, elongated, cylindrical and usually more than 5 x 2.5cm. The fruit is thick and about 3.5-9 cm. The tree often has whorled branching, the branchlets are stout and in ascending order. The fruit of ''Dacryode edulis var. parvicarpa'' is small, rounded and more or less conical, usually less than 5x2.5 cm in size. The fruit pulp is thin, about 2-3.5 cm. Often the tree has bifurcate branching, with slender dropping branchlets.

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<p style="text-align: justify;">The African pear (Dacryodes edulis) is a member of the family Burseraceae (Yombi et al., 1998). Native pear is typical agroforest species that is widely planted and found mainly in home gardens. It produces fruits that are softened in hot water or hot ash and eaten in conjunction with boiled or roasted maize especially during farming season when most of the staple foods such as yam and cocoyam have been exhausted. The fruit is botanically rich in edible vegetable oil (Bassey, 1982). Kapsou (1996), analyzed the fruit characteristics and fatty acid composition of pulp oil of two types of African pear. They discovered that the African pears were significantly different with regards to fatty acid composition.

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<p style="text-align: justify;">Dacryodes edulis has been a priority edible fruit species for domestication work in humid West Africa. The fruit consist of a large seed surrounded by a thin mesocarp. It is rarely taken raw, eaten with maize; and female plants do not flower at the same time, so pollen storage is necessary to control pollination. This species is one of the most preferred trees species by farmers in the hummed lowland of Cameroon. It is used as supplementary feed for chicks.

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<p style="text-align: justify;">Functional Uses

<p style="text-align: justify;">The principal value of the species is in its fruit. The fruit has a leathery, shelled seed surrounded by a pulpy, butyraceous pericarp about 5cm thick which is the proportion eaten either raw or cooked in form of a soft butter. It has a mild smell of turpentine and is oily. The fruits are boiled in salt water, fried or roasted over charcoal. The fruit pulp yields about 48% edible oil, its rich in vitamins and contains a range of amino acids. The kernels which contains about 3.3% proteins, is commonly fed to domesticated livestock such as sheep and goals. The wood is elastic, grayish, white to pinkish, the sapwood and heartwood is difficult to distinguish. The wood has general use for tool handles, particularly axe shafts, and occasionally for mortars and is suitable for carpentry. The bark is used as aromatic on injury. It yields a resin that is used as pitch on calabashes and for mending earthen ware. It can be burnt as a primitive lamp oil or bush candle. The leaves of Dacryodes edulis contain a dye. The wood contains an oil that when used as petrol ether extraction has been found to be composed of fatty acids and esters. The fresh pulp is rich in lipids (55-65%) with a considerable amount of palmitic and linoliec acid. The tree can produce 7-8l/ha of oil. The resin has been reported to yield a peppery essential oil rich in sabinene.

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<p style="text-align: justify;">This species is a perennial cure for a variety of ailments, ranging from ear infections to fevers and oral problems. In Nigeria, the resin is used for treating parasitic skin diseases and jiggers. Pulped bark is used to cicatrice wounds. In the Democratic Republic of Congo a bark decoction is used for gargle mouth wash and for tonsillitis. It is taken in a powdered form with meleguetta pepper as an anti-dysenteric and for anaemia. With palm oil, it is applied tropically to relieve general pains and stiffness and to treat cutaneous conditions. A decoction of the root bark is taken for leprosy.

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<p style="text-align: justify;">Annona muricata (Soursop)

<p style="text-align: justify;">The origin of most of the species of Annona muricata (Soursop) is South America and the Anfilla, however wild soursop is thought to have originated in Africa. The current distribution of the five species covers almost all continents, with soursop and sugar apple, showing the wildest distribution mainly in tropical regions.

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<p style="text-align: justify;">The number of genera and species in the family Annonaceae is still debated. The family has 40 to 50 genera and more than 500 species, most of which are shrubs and small trees. A limited number of species produce edible fruits including many gathered from the wild, and some that have been domesticated. Most of the species are found in the tropics, with only a few genera present in the temperate zone.

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<p style="text-align: justify;">Botanical Description

<p style="text-align: justify;">The name Annonas derives from the Latin “annual harvest” (Lizana and Reginato, 1990). The genus presents numerous unifying characteristics, especially relating to plant height, root system, bark, stem, floral biology, pollination, fruit set and fruit type. There are important variations among Annonas seedlings in the same species, affecting not only the mature foliage and productivity of the plants, but also the fruit size, form, colour, quality and number of seeds in the fruit. These variations are often pronounced enough to have resulted in several botanical names for the same species.

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<p style="text-align: justify;">In general, the Annonas are shrubs or small trees, whose weight varies from 5 to 11m depending on several factors such as species climate, soil and crop management. They are erect or some what spreading in habit, with grey brown bark, often rough and corrugated. Generally stems are ferruginous to grayish; they are tormentoze when young but later becoming glabrous. With few exceptions, Annonas are deciduous, even tropical species, especially when grown in water in areas with dry or cool seasons and without irrigation.

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<p style="text-align: justify;">Description of Annona muricata

<p style="text-align: justify;">The soursop has an erect growth habit with a high canopy height–to– diameter ratio (Pinto and Silva, 1996), although it tends to be low-branching and bushy with upturned limbs (NAS, 1975).

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<p style="text-align: justify;">It is small, slender, evergreen tree 4 to 8m tall when fully mature. It was domesticated in lowland South American as a garden plant.

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<p style="text-align: justify;">The stems are rounded, rough and not pubescent, with a dark-brown colour. The leaves have short petioles, and are oblong-ovate to cylindrical, 14 to 16 cm in length and 5 to 7 cm in width. The flowers of soursop are much larger than those of the other four species. The flowers start to open in the early morning and complete anthesis takes approximately 6hours, depending on the climate. Soursop produces an ovate, conical or heart shaped fruit that is dark green when ripe. The fruit has 127 to 170 seeds, scattered throughout the pulp. They are toxic. Seed size varies from 1 to 2cm in length and from 0.33 to 0.59g in weight with a black colour soon after harvest, but becoming dark brown later (Pinto and Silva, 1996).

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<p style="text-align: justify;">Pulp Properties

<p style="text-align: justify;">Annona Pulp is useful foods because they contain proteins, fatty acids fibres, carbohydrate, minerals and vitamins.

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<p style="text-align: justify;">Soursop pulp is considered to be aromatic and exotic, and is consumed mostly after processing into cold beverages or sometimes eating fresh. The edible portion constitutes 667.5% of total fruit weight. The characteristics flavour of this fruit is produced by amyl and geramyl caproic acids. The processed pulp is used to prepare juices and ice-creams. In Cuba, the pulp is processed to prepare an alcoholic drink called champola. The most important sugars are fructose (1.8%), glucose (2.3%) and sucrose (6.6%). The most common acid in its pulp is citric acid. Source of fruits contains vitamin A and B. Also it is the only ammona with tannius its pulp (Castro et al, 1984).

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<p style="text-align: justify;">Uses of Annona muricata

<p style="text-align: justify;">a. Food Products

<p style="text-align: justify;">Soursop fruits are occasionally consumed fresh or more commonly made into juices and ice creams (Pinto and Silva, 1994). Most people consider it to be acidic for eating fresh, but it is used for making refreshing drinks, ice creams and similar foods nectar (sweetened pulp) can be prepared and used after dilution with 3 pints of water. In Java, Indonesia fruits of soursop are added to soup.

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<p style="text-align: justify;">b. Mechanical Uses

<p style="text-align: justify;">Various plant parts are also widely used in folk medicine, because of the bioactive compounds (mainly acetogenis, alkaloids and flavonoids) found in the roots, leaves, bark, fruits and seeds. The bark contain alkaloids, the leaves have essential oils with parasiticide, anti-diarhoeae, rheumatological and anti-nueragic properties (Moural, 1988). Boiled water infusions of leaves help treat diabetes and gastric upsets and used as kidney ailments. The cooked flowers and petals are used for healing eye inflammations, the treatment requires 2-3 washes a day. Immature soursop fruits have medicinal properties against dysentery, cancer, skin diseases, rashes, fever, malaria, peptic ulcer, colic and Oedema (Khan et al, 1997).

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<p style="text-align: justify;">Treculia africana (African bread fruit)

<p style="text-align: justify;">Treculia africana as commonly known belongs to the family Moraceae. Other members of the family are Ficus exasperata, Millicia excelsa, Artocarpus spp. There are three species in the genus Treculia, Treculia africana (decne), T. obvoidea and T. acuminata (Keay et al., 1964). The first two species are used for furniture making and as fuel wood, but the third one is a slender forest shrub used for construction work.

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<p style="text-align: justify;">Treculia africana is generally found along streams and water courses. Its distributions extend from latitude 150N of Angola and down to SaotomeIsland. It is widely distributed in the following town of Nigeria mostly in Oyo, Ogun, Anambra, Cross River, Delta and Imo State.

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<p style="text-align: justify;">Botanical Characteristics

<p style="text-align: justify;">Treculia africana has a bole usually fluted at the base without buttress. The bark is smooth, light grey to green in colour. It has a copious milky latex when the tree is cut that comes out of it. It grows up to 37 m in length and 3 m girth. Branches and young foliage are glabrous and softy hairy. Leaves are 4 – 8 inch wide. Fruit is greenish, spongy in texture and it is 15 cm long, more in diameter containing numerous individual fruit-like orange – pips. The fruit germinates around February/March every year (Keay et al 1989). The flower is brownish yellow. The flowery period of the tree is between October and February (Keay et al 1989). The seeds are round, sometimes elongated about 1.3 m long, Mesocarp is over 5-inch thick. The leaves are 4-8inches long and 3-4inches broad.

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<p style="text-align: justify;">Economic Importance

<p style="text-align: justify;">Treculia africana and other forest food plants like Pentachlethra macrophylla (oil bean seed), Dacryodes edulis are of high nutritional value which needs to be introduced or added to our food materials consumed. The importance attached to them is the fact that many of them are usually available at strategic period of the year when annual crops that are difficult to store are unavailable or scarce (Okafor, 1978).

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<p style="text-align: justify;">The wood of Treculia africana is used in many furniture manufacturing companies and also for paper production. The fruit pulp and leaves are fed to livestock. The pulp is prepared into a paste like pounded yam, it’s sometimes used as a substitute for pounded yam.

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<p style="text-align: justify;">Proximate composition of Treculia africana

<p style="text-align: justify;">Energy K/Cal                                                                                                                       43.3 - 489

<p style="text-align: justify;">Protein (g)                                                                                                                             15.0 – 20.5

<p style="text-align: justify;">Fat (g)                                                                                                                                   12.2 – 18.6

<p style="text-align: justify;">Total carbohydrate                                                                                                            60.0 – 65.8

<p style="text-align: justify;">Ash                                                                                                                                        2.0 – 2.4

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<p style="text-align: justify;">''Garcinia kola ''

<p style="text-align: justify;">The Garcinia kola commonly known as “bitter kola” which has its name Garcinia named after a French botanist called Laurence Garcin in 18 century (Rai, 2003). The species occurs mostly in West Africa countries such as Cameroon, Central African Republic, Gabon, Zaire, Benin, Cote D’ivoire, Liberia, Nigeria and Sierra Leone. It is a widespread tree of evergreen forests.

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<p style="text-align: justify;">It is an indigenous medicinal tree belonging to the family Gutiferae. It is well branched, evergreen and grown as a medium size trees reaching 12m high in 12 years, and found in moist forests throughout West and Central Africa. The seeds of Garcinia kola have pharmacological uses in treating coughs, throat infections, bronchitis, hepatitis, (inflammation of the liver), liver disorders (Farombi et al., 2005).

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<p style="text-align: justify;">According to Adegoke et al. (1998), seeds of Garcinia kola have inhibitory effects or lipid peroxidation in rat liver homogenate. The seeds, which serve as, a bitter stimulant also serve as snake repellant when they are placed round the compound (Nair, 1990). Other medicinal uses include: purgative, antiparasitic, and antimicrobial. The seeds are used to prevent and relive colic, cure headache or cold. The seeds constituents include; biflaronoids, xanthones and benzophenones and flavonones. The plant has also shown some bronchodilator effect also anti-inflamatory, anti-microbial, anti-bacterial and antiviral properties. In laboratory tests, Garcinia kola was found to halt the deadly disease caused by Ebola virus in its track. Compounds from the plant have also proved effective against some strains of flu, a contagious respiratory disease also commonly known as influenza (Iwu, 1993). Its by-products are also useful: the wood makes excellent fuel-wood, its dense rounded crown makes it an ideal tree for shade around homestead, the branches are used as chewing stick because of its bitter taste and anti-bacterial activities of its extracts. (Taiwo et al., 1999).

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<p style="text-align: justify;">Parkia bicolor (A.Cheu)

<p style="text-align: justify;">Parkia bicolor (A.Cheu) belongs to the family mimosaceae and order Parkiaea and of the genus Parkia (Burkill 2004). It is a useful plant of West tropical Africa. Parkia bicolor extends from Guinea to Zaire. The tree is usually found by the river bank and can grow up to about 100m high. This species is readily distinguished from Parkia biglobosa by its forest habit and its much smaller leaflets which are always opposite (Keay, 1989). The thin layer chromatography of the hexane and ethanol extracts of Parkia bicolor and Parkia biglobosa have been found to display similar properties (Aiyeleagbe, et al; 1996). Extracts of P. bicolor were slightly more active than those of ''Parkia biglobosa. ''

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<p style="text-align: justify;">In the Savanna zone of Nigeria., Parkia biglobosa popular known as the African locust bean tree is well known for its ability to provide shade and is usually planted around the home and used for relaxation. The fermented seeds of Parkia bicolor and Parkia biglobosa are used in all parts of Nigeria and indeed the West Coast of Africa for seasoning traditional soups. The oil from the seeds is also extracted and used for various purposes. Seeds of various species of Parkia have also been investigated for their protein and amino acid contents (Aiyeleagbe et al; 1996).

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<p style="text-align: justify;">A phytochemical analysis of P. bicolor have also reveal the presence of some ingredients such as cardiac glycosides, tannins, glucosides, steroids, alkaloids e.t.c.

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<p style="text-align: justify;">The tree is also utilized as fuel wood among others. Odunfa (1981) stated that the fruit pulp and seeds of Parkia are also known to be rich in protein and amino acids with a high concentration of glutamic acid.

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<p style="text-align: justify;">A phytochemical screening seems to justify the use of the leaf of Parkia for cardiac conditions as appreciable amount was present in P. biglobosa, alone and not P. bicolor (Ajaiyeoba, 1996).

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<p style="text-align: justify;">''Tretra pleura tetraptera (Taub) ''

<p style="text-align: justify;">It is commonly known as Aridan, in Nigeria. It belongs to the family Fabacaea which is widely used in African traditional medicine for the management and control of an array of human ailments including, schistosomiasis, asthma, epilepsy, hypertension and for seasoning, flavouring and enhancing the taste of

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<p style="text-align: justify;">foods, beverages and drugs (Dalziel, 1937). In Eastern parts of Nigeria, the fruits are used to prepare soup for nursing mothers form the first day of delivery and also used as a mosquito repellant (Adewunmi et al., 2001).

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<p style="text-align: justify;">Tetrapleura tetraptera is generally found in the lowland rainforest of tropical Africa. Its distribution extends over large parts of tropical Africa, especiallyWest and East Africa (Ojewale and Adewunmi 2004). It is widely distributed in the following towns in Nigeria mostly in Osun, Niger Delta areas of Nigeria, Ondo and Oyo states.

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<p style="text-align: justify;">Tetrapleura tetraptera is an important spice tree and it is distinguished by the dark green leaves and thick, woody bark and spreading branches and have four longitudinal wing-like rather fleshy ridge about 2cm.

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<p style="text-align: justify;">Botanical Characteristics

<p style="text-align: justify;">Tetrapleura tetraptera plant is a single stemmed, robust, perennial tree with dark green leaves and thick, woody base and spreading branches. The tree reaches 20 to 25 m in height and may attain a girth of 1.5 m and has sharp buttress. It is reddish, hard and heavy wood. It is used for firewood, building poles, pestle, tool handles and carvings. It has a gray/brown, smooth/rough bark with glabrous branchlets.

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<p style="text-align: justify;">The fruits are green when tender and dark red brown when fully ripe. The fruit is also 25cm long with four longitudinal wing-like ridges rarely 3cm broad. The flowers are pinkish cream, turning orange and are placed solitarily or in pairs, axillary or terminal inflorescence. The flowering period of the tree is between May and June. The seeds rattle in the pods, are small, black and hard. The fruits and seed are rich in some macro-elements such as potassium, iron, magnesium and phosphorus but sodium content was low. Sucrose, fructose and glucose were detected in the fruits and seeds (Dosunmu, 1997).

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<p style="text-align: justify;">The leaves are sessile and glabrous, minutely hairy with a common stalk 15 – 30cm long, slightly channeled on the upper surface. It is Sipinnate to eight pairs of pinnae, placed opposite to each other.

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<p style="text-align: justify;">Economic Importance of the Species

<p style="text-align: justify;">The fruit shell, fruit pulp and seed contained varying amounts of nutrients such as protein, lipids and minerals which were comparable and some were even higher than popular spices and ginger (Essien et al., 1994). In Eastern Nigeria, fruits are used to prepare soups for mothers from the first day of delivery to prevent postpartum contraction (Achinewu and Obomanu 1995). Spices are products of plants which are mostly used for seasoning, flavorings and thus enhancing the taste of foods, beverages and drugs (Iwu, 1993).

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<p style="text-align: justify;">Okwu (2003), gave the proximate composition of T. tetraptera as follows: crude protein (7.44% - 17.50%), crude lipid (4.98% - 20.36%), crude fibre (17% - 20.24%), carbohydrate (43.18% - 49.06%) and food energy (234.18% - 42,379.48 gl Cal) the species were also sources of minerals e.g, calcium, phosphorus, potassium, zinc and iron, while the phytochemical screening revealed the presence of tannins phenolic compounds, saponins, alkaloids, steroids and flavoniods which could be subsumed to be responsible for its varied biological and pharmacological properties.

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<p style="text-align: justify;">''Azadirachta indica ''

<p style="text-align: justify;">Azadirachta indica is one of oldest trees of immense benefit to mankind. They not only provide food and shelter but also serve humanity by preventing and curing different ailments. Herbs and spices have been helpful to cure disease. In modern animal feeding, they are forgotten because of use of anti microbial growth promoters (AGP). But due to the prohibition of most of AGP, plant extracts however gained interest in animal feed strategies (Charis et al; 2000). The risk of the presence of antibiotic in milk and meat and their harmful effects on human health led to their prohibition for use in animal feed in the European Union (Cardozo et al; 2004). Azadirachta indica also produce secondary metabolites such as phenolic compounds, essential oils and sarasaponins (Kamel, et al; 2001).

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<p style="text-align: justify;">The practice of herbal medicine dates back to the very earliest period of known human history. There is evidence of herbs having been used in the treatment of diseases and for re-vitalizing body system in almost all ancient civilizations. The Egyptian, the Chinese and previous literature shows that use of herbs in animal feed improved the weight gain of animals. These can be used simultaneously for treating parasitic diseases as well as increasing weight gain and act as growth promoters. The mature tree of Azadirachta indica (Neem) plant can produce 350kg of leaves a year, which may be used for feeding cattle during famines. After the oil has been pressed out from the seeds of Neem, the cake is used as fertilizer but it can also be used as feed. Kudke et al. (1999) posited that up to 10% neem cake may be included in concentrates for cattle and up to 5% for poultry. Hayat et al; (2002) studied comparative prophylaectic effects of indigenous preparations of bakin (Melia azadarach) and ilerala (Momordica charntia) in comparison with the salinomycim against coccodiosis in broiler chicks.

<p style="text-align: justify;">'' ''

<p style="text-align: justify;">Azadirachta indica, is widely known as neem, a tree that has proven benefit for both city and farm dwellers throughout the dry tropics and subtropics. Neem has long been recognized as a versatile multipurpose tree for urban re-greening: agro forestry, fuel wood production, and for a variety of other products including biopesticides. Azadirachta indica is a member of the Meliaceae (mahogany) family. It has been referred to in the past by the botanical names, Melia azadarach (chinaberry). The tree is also known as neem, margosa, limba, minba, nimba, kohomba, and India lilac (Parkar et al; 2003).

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<p style="text-align: justify;">Botany

<p style="text-align: justify;">Neem is a small to medium sized tree with a short, straight bole. The stem branches at 2 – 5m forming a broad, dense, round or oval crown. Total height is 15-25cm, occasionally reaching up to 30 cm, with a stem diameter ranging from 30 to 90cm. Neem is characterized by a long, penetrating lateral root system, which can extend up to 15m, with a relatively short tap root. Neem has moderately thick, fissured, grey outer bark, with a reddish brown inner bark. It is evergreen or deciduous depending on the climate. Leafless periods are usually brief, occurring during extended drought. Leaves are at the tip of branches. The tree produces many small, sweet-scented, white or cream colored, bisexual flowers. The fruit is a smooth, ellipsoidal drupe, 1.2-2.0 long, containing usually one seed. The fruit is initially green and turns yellow as it ripens in about 12 weeks after full bloom. Neem trees are prolific fruit producers, starting as early as 3-5 years, and becoming fully productive at 10-12 years (Charis et al., 2000).

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<p style="text-align: justify;">Ecology

<p style="text-align: justify;">Neem adapts to a broad range of climate and soil conditions. It is normally found at elevations between sea level and 700m. Neem can grow at altitudes up to 1500m, as long as temperatures remain moderate, as it does not withstand cold or frost. Neem tolerates extremely high temperature, but its normal range is about 9.50C – 370C. It is also highly drought tolerant, and once established, it can survive 7-8 month dry seasons. It requires as little as 150mm rainfall per year in areas where the root system can access ground water within 9-12m of the surface, however, it performs best in zones receiving 450-1200mm per year. Neem prefers deep, permeable sandy soils, but can be planted in a wide variety of soil types, including difficult sites where most other species do not perform well. It can thrive on rocky, dry, shallow, infertile soils, but is not recommended for rocky, shallow, infertile soils, but is not recommended for silty or micaceous loams, silty clay soils, saline soils, or where sub-surface hard pan or laterite out cropping occur (NRC, 2000). Neem should also not be planted on sites where soils become waterlogged or seasonally inundated. It prefers a soil pH in the 6.2-7.0 range but can grow within a range of 5.0-8.0 pH. Mature neem trees are light demanding but seedlings tolerate moderate shade during their first growing season especially on dry sites (Dash et al., 2004).

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<p style="text-align: justify;">Distribution

<p style="text-align: justify;">Azadirachta indica is indigenous to South Asia, possibly originating in northern Myanmar and the Assan region of India. Neem’s natural habitat is dry, deciduous, mixed forest, occurring in association with Acacia spp and Dalbergia sissoo (Lemments at al., 2005). It is widespread in India, Pakistan, Myanmar, Sri, Lanka, Thailand, Malaysia and Indonesia. Neem has been introduced and established throughout the tropics

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<p style="text-align: justify;">and sub tropics, especially in drier areas in south east Asia, the pacific islands, Australia, South and Central America, the Caribbean, Sub-Saharan Africa and the Middle Fast (Kael, et al; 2001).

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<p style="text-align: justify;">Economic Importance

<p style="text-align: justify;">i. Agro-forestry and urban forestry:

<p style="text-align: justify;">Neem plays an important role in both urban and rural landscapes. Its well formed crown and short deciduous period has made it a popular choice for shade planting around buildings and along road sides. It is also used on forms as a pasture tree to shade livestock and in boundary rows. Neem is not used as hedgerows or alley cropping, but is used in wind breaks and shelter belts to protect crops from wind damage and soils from erosion (Cardozo et al; 2004).

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<p style="text-align: justify;">ii. Wood products:

<p style="text-align: justify;">Neem produces a moderately dense wood, somewhat similar to mahogany. The wood has a specific gravity of 0.52-0.85, averaging 0.68 (N.R.C, 2000). The heart wood portion is reddish brown, while the sap wood has a yellowish gray or grayish white color. The wood is hard, durable, dull to some what lustrous, aromatic and resistant to insects and fungi. The wood dries with only slight shrinkage, seasons well, and is easy to work, but the rough, interlocked grain does not take a high polish. Neem sawn timber is used in light construction and to make beams, door and window frames, boxes, crates, carts, axles, yokes, cabinets, panels, boats, oars, cigar boxes, carvings, toys, drums, and agricultural implement. It is also used for furniture especially wardrobes, book case, and closets, because the wood repels insects. Neem trees are often managed under pollarding or coppicing systems to produce posts and poles. The round wood is also used as fuel wood and makes very good charcoal. At 14% moisture content the wood gives an energy value of 16.92 mega joules/kg (Lemmens et al., 2005).

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<p style="text-align: justify;">iii. Non-timber products

<p style="text-align: justify;">Useful products can be harvested from almost every part of the neem tree. The bark produces tannins, a fiber used to make rope, and a resin used to make glue. Bark is used medicinally as a remedy for fever, and fruit pulp is also used as a tonic; leaves are used as mulch and green manure, and can also be used as fodder. The leaves have a crude protein content of 12-18%, because they have a bitter taste, livestock usually prefer other foods. Neem leaves mixed in with stored grain house traditionally been used in India to repel insects and prevent food and seed losses.

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<p style="text-align: justify;">iv            Azadirachta

<p style="text-align: justify;">The principal active compound in the leaves is azadirachtin, which repels pests, acts as an anti-feedant, and disrupts insects growth and reproduction. Several bio active compounds are found in the leaves and other tissues, however, the neem seed kernel’s are the main source of azadirachtin. Neem seed contains the most concentrated and accessible amounts of other potentially useful compounds as well. Neem based pesticides have already been approved for various applications and are being produced commercially in several countries (Randhawa et al; 2007).

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<p style="text-align: justify;">v. Neem oil and neem seed cake

<p style="text-align: justify;">Neem seeds will yield 40-50% oil when the dry kernels are crushed or pressed through an oil mill. Neem is used as fuel for lumps. An antiseptic for animal wounds, a lubricant for machinery, an insect repellent, to remove tobacco suckers, and in the production of soap, toothpaste.

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<p style="text-align: justify;">Medicinal Uses

<p style="text-align: justify;">The medicinal plants and herbs have been used for many years in the treatment of various diseases in animal’s and human beings. Nowadays, utilization of these medicinal plants is increasing. These are used in animal feed as growth promoters due to prohibition of most of the antimicrobial growth promoters in animal feeds because of their residual effects. Hayat et al (2004).

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<p style="text-align: justify;">Various parts of the neem tree have been used as traditional Ayurvedre medicine. In India neem oil and the bark and leaf extracts have been therapeutically used as folic medicine to control leprosy, helminthiasis,

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<p style="text-align: justify;">respiratory disorders, constipation and also as a general health promoters. Akilandeswari et al; (2003). Neem oil finds use to control various skin infections. Bark, leaf, root, flower and fruit together cure blood morbidity, biliary infections, itching, skin ulcer’s etc (Almas et al;1999).

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<p style="text-align: justify;">The medicinal uses of Azadirachta are as follows:

<p style="text-align: justify;">Immunostimulant activity: The aqueous extract of neem bark and leaf also possesses anticomplement and immuno-stimulant activity. Neem oil has been shown to possess activity by selectively activating the call medicated immune mechanisms to elicit an enhanced response to subsequent nitrogen.

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<p style="text-align: justify;">Anti ulcer effect: Neem leaf and bark aqueous extracts produce highly potent anti acid and anti ulcer activity.

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<p style="text-align: justify;">Anti fertility effect: Intro-vaginal application of neem oil, prior to coitus, can prevent pregnancy. It could be a novel method of contraception.

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<p style="text-align: justify;">Anti malarial activity: Neem seed and leaf extracts are effective against both chloroquine resistant and sensitive strain malarial parasites.

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<p style="text-align: justify;">Anti cancer activity: Neem leaf aqueous extract effectively suppresses oral squamous cell carcinoma, induced by 7, 12-dimethybenz (a) anthracene (DMIZA), as revealed by reduced incidence of neoplasm. Neem may exert its chemo preventive effect in the oral mucosa by modulation of glutathione and its metabolizing enzymes.

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<p style="text-align: justify;">Anti oxidant activity: The anti-oxidant activity of neem seed extract has been demonstrated in vivo during horse grain germination.

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<p style="text-align: justify;">Anti bacterial activity: Oil from the leaves, seed and bark possesses a wide spectrum of anti bacterial action against gram-negative and gram-positive micro organisms, including M. tuberculosis and Streptomycin resistant strains. Anti microbial effects of neem extract have been demonstrated against streptrococcus motions and S. fuecalis.

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<p style="text-align: justify;">Anti fungal activity: Extracts of neem leaf, neem oil seed kernels are effective against certain fungi including Trichophyton, microspor Trichoseporon, Candida, Geotricum and Cosmetics. It has also traditionally been used for a variety of medicinal purposes, but there is evidence that it should not be ingested orally, neem oil may also have potential in the development of pesticide and fungicides, although it does not contain azadirachtin (N.R.C, 2000). Neem seed cake is the residue left after the oil has been extracted from the kernel. Neem cake is used as a fertilizer with insecticides and fungicidal effect.

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<p style="text-align: justify;">CONCLUSION

<p style="text-align: justify;">The economic importance of the species is highlighted in this paper. However, the urgent and most critical point to consider is the lack or inadequate research effort initiated toward harnessing these economic benefits for the larger society. Azadirachta indica in this study has been shown to possess tremendous medicinal values far more than any of the other species considered. Therefore establishing a research institute for this species individually or collectively with one or two species which hopefully will create room for thorough, in depth, comprehensive and analytical study is absolutely necessary. This will go a long way to promoting the benefits of this species and ultimately enrich the local farmers who may have embarked on the cultivation. Secondly, it will stimulate greater interest in the domestication of this species.

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<p style="text-align: justify;">REFERENCES

<p style="margin-left: 54pt; text-align: justify; text-indent: -54pt;">Achinewu, S.C. and G.G. Obomanu (1995): Studies on Spices of Food value in the South Eastern States of

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<p style="margin-left: 54pt; text-align: justify; text-indent: -54pt;">Taiwo, O., Xu, H.K., and S.E Lee (1999): Antibacterial activities of extracts from Nigerian Chewin sticks.

<p style="margin-left: 54pt; text-align: justify; text-indent: -54pt;">Phytoter Res. 13 (8): 675-679.

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<p style="margin-left: 54pt; text-align: justify; text-indent: -54pt;">Tchamba, M.N. and P.M. Seme (1993): Diet and feeding behaviour of the Forest Elephant in the Santchou

<p style="margin-left: 54pt; text-align: justify; text-indent: -54pt;">Reserve, Cameroon. African Journal of Ecology 31 (2): 165 – 171.

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<p style="margin-left: 54pt; text-align: justify; text-indent: -54pt;">Tchoundjeu, Z., Duguma, B., Marie Laure, T. and N. Marie Laure, (1997): The domestication of

<p style="margin-left: 54pt; text-align: justify; text-indent: -54pt;">Indigenous agro-forestry trees of west and central Africa. CARPE: Limbe Non-wood forest products

<p style="margin-left: 54pt; text-align: justify; text-indent: -54pt;">workshop. Web address: http://carpe.geep.rirginia.edu/products/products2.2.asp

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<p style="margin-left: 54pt; text-align: justify; text-indent: -54pt;"> Tchoundjeu, Z., Kengue, J. and R.R.B. Leakey (2002): Domestication of Dacryyodes edulis: state-of-the

<p style="margin-left: 54pt; text-align: justify; text-indent: -54pt;">arts: Forest, trees and livelihoods 12:3-13.

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<p style="margin-left: 54pt; text-align: justify; text-indent: -54pt;">Umelo, R. (1997): Potentials for Utilization of African Star Apple (Gambaya albida) for jam making in

<p style="margin-left: 54pt; text-align: justify; text-indent: -54pt;">Nigeria. In: Proceeding of a National Workshop in Nigeria; (eds). Denton, O.A., Landipo, D.O. Adetoro,

<p style="margin-left: 54pt; text-align: justify; text-indent: -54pt;">M.A. Sarumi M.B. P 103.

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<p style="margin-left: 54pt; text-align: justify; text-indent: -54pt;">Youmbi, E., Cerceau Larrival, M.T. Veribille, A.M Varonnier Jarreau, M.C (1998): Morphology and in

<p style="margin-left: 54pt; text-align: justify; text-indent: -54pt;">vitro pollen germination of Dacryodes edulis (Burseraceae). Parameters for optimal germination. GRANA

<p style="margin-left: 54pt; text-align: justify; text-indent: -54pt;">37(3): 87-92

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<p style="text-align: justify;">Received for Publication: 18/05/2010

<p style="text-align: justify;">Accepted for Publication: 21/06/2010

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<p style="text-align: justify;">Corresponding author;

K. Okunomo

Department of Forestry and Wildlife, Delta State University, Asaba Campus, Asaba, Delta State

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<p style="margin-right: -36pt; text-align: justify;">Continental J. Biological Sciences 3: 33 - 45, 2010                                                     ISSN: 2141 - 4122

<p style="text-align: justify;">© Wilolud Journals, 2010                                                                                 http://www.wiloludjournal.com

<p style="text-align: center;">

<p style="text-align: center;">COMPARATIVE STUDY OF ENZYMATIC TRANSESTERIFICATION OF Jatropha OIL USING LIPASE FROM Jatropha curcas and ''Jatropha gossipyfolia ''

<p style="text-align: center;">'' ''

<p style="text-align: center;">1Gayatri Nahak, 1Debyani Samantray, 2N.K. Mohapatra and 1R.K. Sahu

<p style="text-align: center;">1B.J.B. Autonomous College, Bhubaneswar, Orissa, 2Academy Management and Information Technology, Bhubaneswar, Orissa

<p style="margin: 0cm 43.2pt 0.0001pt;">ABSTRACT

<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;">Biodiesel consists of monoalkyl esters of long chain fatty acids. It is produced from vegetable oils or fats either by chemical transesterification with methanol or ethanol. The cost of lipases and the relatively slower reaction rate remain as the major obstacles for enzymatic production of biodiesel as opposed to the conventional chemical processes. The enzymatic process offers several advantages over the chemical routes. The handicap of increase in process cost because of the enzyme can be overcome by using efficient production process for enzyme and using reusable derivative of enzymes, such as immobilized enzyme. Numerous strategies available in the area of non-aqueous enzymology can be exploited during the enzymatic alcoholysis for the biodiesel production. The paper reviews the starting oils usually employed in biodiesel production, the process for transforming them to biodiesel playing particular emphasis on enzymatic transesterification the sources of production and characterization of vegetable oils and their methyl ester as the substitute of the petroleum fuel.

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<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;">KEYWORDS: Biodiesel, Enzymatic transesterification, Jatropha oil, Lipase, J. curcas, J. gossipyfolia.

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<p style="text-align: justify;">INTRODUCTION

<p style="text-align: justify;">Many researchers have concluded that vegetable oils hold promises as alternative fuels for diesel engines (Goering et al., 1982; Bagby et al; 1987). However, using raw vegetable oils for diesel engines can cause numerous engine related problems (Korus et al., 1982; Vander Walt and Hugo,1982). The increased viscosity and low volatility of vegetable oils lead to severe engine deposits, injector coking and piston ring sticking (Vellguth, 1983; Clark et al., 1984; Pestes and Stanislao,1984; Perkins and Peterson,1991). However these effects can be reduced or eliminated through transesterification of vegetable oil to methyl esters, commonly known as Biodiesel (Zhang et al., 1988;Perkins and Peterson.,1991). Consequently, considerable effort has gone into developing vegetable oil derivatives that approximate that properties and performance of hydrocarbon-based diesel fuels problems encountered in substituting triglycerides for diesel fuels are mostly associated with their high viscosity, low volatility, and polyunsaturated character (Srivastava et al.,2000). As an alternative to diesel fuel, Biodiesel must be technically feasible, economically competitive, environmentally acceptable and readily available. Now-a-days Biodiesel fuel is used in public traffic for performing from engines, lighting and heating of rooms in specific condition (Haas, 2005; Schlautman et al., 1986; Tomasevic and Siler Marinkove,2003). Yamane et al,2001 recently reported that a biodiesel fuel with good ignitability, such as one with a high methyle oleate content, gives lower levels of NO, Hydrocarbons, HCHO, CH3HO and HCOOH. Since Biodiese is an Oxygenated fuel having an O2 mass fraction of10%. In addition, Sheehan et al, 1998 carried out life cycle analysis and found that the benefit of using Biodiesel is proportionate to the level of blending with petroleum diesel. Three main processes have been investigated in attempts to overcome these drawbacks and allow vegetable oil and waste to be utilized as a viable alternative fuel: Pyrolysis, Micro-emulsification, and Transesterification. Transestrification provides a fuel viscosity that is close to that of No.2 diesel fuel.

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<p style="text-align: justify;">Transesterification is also called as alcoholysis, is the displacement of alcohol from an ester by another alcohol in a process similar to hydrolysis, except that an alcohol is employed instead of water (Meher et al., 2006; Srivastava and Prasad. 2000). Suitable alcohols include methanol, ethanol, propanol, butanol, and amyl alcohol. Methanol and ethanol are utilized most frequently, especially methanol because of its low

<p style="margin-right: -36pt; text-align: center;">Gayatri Nahak et al.,: Continental J. Biological Sciences 3: 33 - 45, 2010

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<p style="text-align: justify;">cost and its physical and chemical advantages. This process has been widely used to reduce the viscosity of triglycerides, thereby enhancing the physical properties of renewable fuels to improve engine performance (Clark et al., 1984). Transesterification of triglycerides produces fatty acid alkyl esters and glycerol. The glycerol layer settles down at the bottom of the reaction vessel. Diglycerides and monoglycerides are the intermediates in this process. The mechanism of transesterification is described as follows.

<p style="text-align: justify;">Figure-1(a)

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<p style="text-align: justify;">

<p style="text-align: justify;">The transesterification reaction with alcohol represented by the general equation shown in Fig-1(a). The first step is the conservation of triglycerides to diglycerides which is followed by the conversion of diglycerides to monoglycerides and of monoglycerides to glycerol, yielding one methyle ester molecule from each glycerides at each step (Freedman et al.,1986 and Noureddimi et al.,1997)

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<p style="text-align: justify;">Although chemical transesterification using an alkali-catalyst process gives high conversion levels of triglycerides to their corresponding methyl esters in short reaction times, the reaction has several drawbacks: it is energy intensive, recovery of glycerol is difficult, the acidic or alkaline catalyst has to be removed from the product, alkaline waste –water requires treatment and free fatty acids and water interfere with the reaction. To overcome such problems associated with chemical catalyst for production of Biodiesel, enzymatic transesterification process using lipase have been developed. The enzymatic process offers several advantages over the chemical routes. The handicap of increase in process cost because of the enzyme can be overcome by using efficient production process for enzyme and using reusable derivative of enzymes, such as immobilized enzyme. Numerous strategies available in the area of non-aqueous enzymology can be exploited during the enzymatic alcoholysis for the biodiesel production. The paper reviews the starting oils usually employed in biodiesel production, the process for transforming them to biodiesel playing particular emphasis on enzymatic transesterification the sources of production and characterization of vegetable oils and their methyl ester as the substitute of the petroleum fuel.

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<p style="text-align: justify;">The interest in the application of enzymes to organic synthesis has been growing rapidly in recent years. A lot of attention has been devoted to attempts at utilizing the catalytic properties of lipase in organic synthesis. The catalytic activity and selectivity of enzymes depend on, among other things, the structure of the reacting substances, the process conditions, the kinds of solvents, and the presence of water (Gryglewicz et al.,2000). Lipase (triglycerol acylhydrolase, E.C.3.1.1.3) are enzymes widely distributed among animals, plants, and micro-organisms that catalyze the reversible hydrolysis of glycerol ester bond and therefore, also the synthesis of glycerol ester.

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<p style="text-align: justify;">In nature, lipase used only for hydrolysis. Under certain circumstances, lipases also catalyze a transeterification reaction. Lipase can be used in low-water environment as excellent tool for the transestrification of commercial triglycerides, and/or their derivatives, to synthesize a growing range of products of potential industrial interest (Pirozzi., 2003).the industrial applications of lipases have grown rapidly in recent years are likely to markedly expand further in the coming year. Lipase may be used to produce fatty acids (Linder et al.,1993), biosurfactants (Edmundo et al., 1998), aroma and flavor compounds (Athawale et al.,2003), lubricant and solvent esters (Hills, 2003), amides and thiol esters (Gandhi 1997). There have been a number of studies, which reported lipase catalyzed transesterification with and without organic solvents. For diesel fuel, ethyl ester is preferred because ethanol can be produced from biomass and is less toxic, but conventional alcoholysis with ethanol gives low yield.

<p style="text-align: justify;">The lipase catalyzed reaction can be classified as follows.

<p style="margin-right: -36pt; text-align: center;">Gayatri Nahak et al.,: Continental J. Biological Sciences 3: 33 - 45, 2010

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<p style="text-align: justify;">I Hydrolysis:

<p style="margin-left: 36pt; text-align: justify; text-indent: 36pt;">     R1COOR2+H2OàR1COOH+R2OH

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<p style="text-align: justify;">ΙΙ. Synthesis: reaction under this category can be further divided.

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<p style="text-align: justify;">1. Esterfication

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<p style="margin-left: 36pt; text-align: justify; text-indent: 36pt;">      R1COOH+R2OHà R1COOR2+H2O

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<p style="text-align: justify;">2. Transesterfication

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<p style="text-align: justify;">Alcoholysis

<p style="margin-left: 90pt; text-align: justify;">

<p style="margin-left: 90pt; text-align: justify;">R1COO R2+R3OHà R1COO R3 +R2OH

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<p style="text-align: justify;">Acidolysis

<p style="margin-left: 90pt; text-align: justify;">

<p style="margin-left: 90pt; text-align: justify;">R1COO R2+R3COOHà R3COO R2 + R1COOH

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<p style="text-align: justify;">Most interesting is the utilization of lipase for catalyzing the synthesis of simple ester of vegetable oil or other agricultured lipid feedstock e.g. the lipase catalyzed alcoholysis of sunflower oil under anhydrous conditions (Mittelbach, 1990). Dosssat et al., 1999 reported transesterification of high oleic sunflower oil with butanol by the immobilized lipozyme R in n-haxane.the reaction was carried out in a continuous packed bed reactor. Without an additional organic solvent, Linko et al.,1994 studied lipase catalyzed transestrification of low erucic acid rapeseed oil and 2-ethyl-1-hexanol. The optimal transestrification condition was an oil/alcohol/molar ratio 1:2:8, a minimum of 1.0 %( W/W) added water and with a temperature of 370C-550C. Under the optimal condition, a nearly complete conversion was obtained in one hour with 14.6 %( W/W) lipase, whereas 0.3 %( W/W) lipase required 10hrs.for similar results. However, at 600C lipase was clearly inactivated under the experimental condition.

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<p style="text-align: justify;">Abigor et al., 2000 reported lipase-catalysed production of alkyl ester by transesterification of palm kernel and coconut oil with different alcohols using PS30 (Pseudomonas cepacia) lipase as a catalyst. In the conversion of palm kernel oil to alkyl esters, without any added solvent to the reaction mixture the highest conversion was given by ethanol(72%), followed by tert-butanol(62%), butanol(42%), propanol(42%), and isopropanol(24%), while only 15% methyl esters was observed with methanol. Through 3-step addition of methanol, Du et al., 2003 amined lipase-catalyzed transeterification of Soya oil in continuous batch operation. They found that in non-continuous batch operation, the optimal oil/alcohol ratio and temperature were 1:4 and 400-500C, because either at higher (1:5) or lower (1:3) methanol concentration would decrease the methyl esters yield to some degree. In this condition, methyl esters yield reach up 92% after 6hrs.reaction. However, during the continuous batch operation lipase lost its activity dramatically when the methanol/oil/molar ratio was 2:1.the optimal molar ratio of oil (alcohol and temperature) were 1:1 and 300C (Du et al., 2003). More details about lipase-catalyzed and enzymatic transestrification of Jatropha oil with methyl and ethyl alcohol will be presented in this paper.

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<p style="text-align: justify;">MATERIALS AND METHODS

<p style="text-align: justify;">Plant Materials

<p style="text-align: justify;">The seeds and leaves of Jatropha curcas and Jatropha gossipyfolia were brought from Department of Forestry, OUAT, Bhubaneswar, and Orissa. The seeds were deshelled manually and mechanically pressed and separated from impurity with the help of separating funnel. Then oil was used for physio-chemical characterization, enzymatic transesterification.

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<p style="text-align: justify;">Chemical and Reagents

<p style="text-align: justify;">Petroleum ether (40-600C), 1% phenolphthalein, 95% ethanol, 0.1N potassium hydroxide,0.5N HCl, KOH, Silica gel, CaCl2, Benzene, Phosphate buffer(pH 7.3)50mM, Sodium taurocholate( Bile salt), Acetone, Ammonium sulphate, 50% H2SO4 and double distilled water.

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<p style="text-align: justify;">Instrumentations

<p style="text-align: justify;">Soxhlet apparatus, Rotary shaker, Cooling Centrifuge, and TLC apparatus and mortar and pestle.

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<p style="text-align: justify;">Oil Extraction

<p style="text-align: justify;">For the extraction of Jatropha two main methods have been identified. 1. Mechanical oil extraction and 2. Chemical oil extraction (Aderibigbe et al,1997 and Forson et al, 2004).

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<p style="text-align: justify;">Figure-1(b): (Flow chart of Oil extraction Unit process)

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<p style="text-align: justify;">Physio-chemical Estimation

<p style="text-align: justify;">All the physiochemical tests were done by the method followed by Sadasivam and Manickam., 2008.

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<p style="text-align: justify;">Estimation of oil content

<p style="text-align: justify;">A piece of filter paper was folded in such a way to hold the seed meal and then second filter paper was wrapped which was left open at the top like a thimble. Then a piece of cotton wool was placed at the top for evenly distribution of solvent as it drops on the sample during extraction. Then a sample packet was placed in the extractor of soxhlet apparatus and oil was extracted with petroleum ether for 6h without interruption by gentle heating. Then it was allowed to cool and dismantle the extraction flask. Then ether was evaporated on a steam or water bath until no odour of ether remains and cooled to room temperature.

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<p style="text-align: justify;">Calculation:

<p style="text-align: justify;">                                    Oil in ground sample (%) =

<p style="text-align: justify;">Oil in dry wt. Basis (%) =

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<p style="text-align: justify;">Estimation of acid value

<p style="text-align: justify;">5 gm oil was dissolved in 50ml of neutral solvent in 250ml conical flask and few drops of phenolphthalein were added. Then it was titrated against 0.1N KOH and shaken constantly until a pink color persists for 15 seconds is obtained.

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<p style="margin-right: -36pt; text-align: center;">Gayatri Nahak et al.,: Continental J. Biological Sciences 3: 33 - 45, 2010

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<p style="text-align: justify;">Calculation

<p style="text-align: justify;">Acid value (mgKOH/gm) =

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<p style="text-align: justify;">Estimation of Saponification Value

<p style="text-align: justify;">5gm of oil was taken into flask and 50ml. of an alcoholic KOH was added from a burette by allowing it to drain for a period of time. Simultaneously a blank was conducted by taking only 50ml. alcoholic KOH without oil. Air condenser was connected to flask and boiled gentle for about one hour. After flask and condenser get cooled, inside of the condenser was rinsed down with a little distilled water. Then 1ml. of indicator was added and titrated against 0.5N HCl until pink color just disappeared.

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<p style="text-align: justify;">Calculation

<p style="text-align: justify;">

<p style="text-align: justify;">Saponification value =

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<p style="text-align: justify;">Biodiesel production

<p style="text-align: justify;">This includes extraction and purification of lipase and its transesterification.

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<p style="text-align: justify;">Free enzyme Preparation

<p style="text-align: justify;">The seeds of J. curcas and J. gossipyfolia were taken in Petri dishes and kept for germination. The germinated seeds were homogenized in mortar and pastel with cold petroleum ether. Similarly the fresh leaves of J. curcas and J.gossipyfolia were taken and washed properly with distilled water and were homogenized in mortar pestle with cold petroleum ether. Then it was centrifuged successively with ether mixture and then finally ground with cold acetone to fine powder, air dried and preserved at -40C. The enzyme was extracted from acetone powder by phosphate buffer (pH7.3). Then they were centrifuged at 15000g for 10min at -40C. The supernatant was preserved for further analysis ad the residue was discarded.

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<p style="text-align: justify;">The crude enzyme extract was partially purified by salting out method using 70% ammonium sulfate. The precipitated proteins from all samples were recovered after discarding the supernatant. The precipitate was redissolved in phosphate buffer (50mM) pH 7.3 and dialyzed to make it free from ammonium sulfate. The dialyzed samples were taken as enzyme source.

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<p style="text-align: justify;">Assay of lipase from acetone powder

<p style="text-align: justify;">The prepared acetone powder (2gm.) was slightly ground with mortar and pestle with buffer solution (30% KOH+ 50% KH2PO4+ 20ml ice cold H2O). Then it was centrifuged at 15000 rpm for 15 min. The pellet was discarded and the supernatant was used as enzyme source (Sadasivam and Manickam., 2008).

<p style="text-align: justify;">

<p style="text-align: justify;">To 20ml. of substrate 5ml. of phosphate buffer was added and the contents were stirred slowly by keeping the beaker on top of a magnetic stirrer-hot plate maintaining the temperature at 35oC and pH 7.0. 0.5ml. of enzyme was added and the pH was recorded immediately at zero time with timer on (pH at zero time). At regular intervals (10 min) or as the pH drops by about 0.2 units 0.1N NaOH was added to bring the pH back to the original level. This titration was repeated for 30-60 min and the volume of NaOH consumed was noted to estimate the protein content in the enzyme sample.

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<p style="text-align: justify;">Calculation

<p style="text-align: justify;"> The enzyme activity as the amount of enzyme required to release one milliequivalent of free fatty acid/ min./gm. sample and specific activity as milliequivalent/ min./ mg. protein.

<p style="text-align: justify;">

<p style="margin-right: -36pt; text-align: center;">Gayatri Nahak et al.,: Continental J. Biological Sciences 3: 33 - 45, 2010

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<p style="text-align: justify;">Enzymatic Transesterification

<p style="text-align: justify;">The enzymatic transesterification was done followed by the method Jatropha oil (10gm) and ethanol (2gm) were taken in the ratio of 1:4 (mole mle-1) in a 100 ml conical flask. To this mixture different concentrations of enzymes showing different activity of enzymes in terms of milliequivalent/min/mg protein were taken in solution form were added and stirred. Then heated to 400C with constant shaking at 200 rpm. Then the samples were withdrawn and analyzed for maximum conversion.

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<p style="text-align: justify;">Test for complete esterification

<p style="text-align: justify;">To 100ml. conical flask containing 2gm. of esters, 5gms.mixture of silica-G: anhydrous CaCl2 (W/W) in the ratio 1:1 was added. It was mixed thoroughly with a glass rod. Then 10ml. of benzene was added and was shaken for ½ to 1min centrifugation was done for short period. The residue was washed with benzene and then it was evaporated the solvent mixture to its original volume of ester oil.

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<p style="text-align: justify;">TLC for ethyl ester

<p style="text-align: justify;">Purification of ester mixture was tested by TLC. A chromatographic plate was prepared by using 20×20m.glass plate over layered with silica gel-G slurry of 2mm. thickness and having a pore size of 250m. The set specification was obtained by making the slurry in water in the production 1:2. The slurry was applied by spreader after adjusting the thickness to 2mm. The plates were dried at room temperature and activated before use 120C for 1hr.

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<p style="text-align: justify;">The ester mixtures were applied at different spots with the unesterified oil at one end as control. At the other end commercial diesel was also applied for comparative study. The applied sample was dried in air and put inside the chromatographic tank containing solvent mixture of petroleum ether and acetic acid in the proportion 80:20:1. A chromatographic running is allowed till the solvent front reach few centimeters below the top. The plates were taken out and dried in air and was spread with the developer (50% H2SO4) and was put inside the hot air oven for 1-2 hr. The absence of spots in case of ester mixture against spots developed by the control confirms the complete esterification.

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<p style="text-align: justify;">RESULTS AND DISCUSSION

<p style="text-align: justify;">The physiochemical properties were assessed in the P.G. Department of Biotechnology, Biochemistry laboratory and results are presented in Table-1. In vitro studies of transesterification were conducted to draw out an inference of maximum yield of Biodiesel using two different sources for enzyme lipase from Jatropha curcas and Jatropha gossipyfolia plant parts. Experiments were conducted to study enzymatic transesterification with standardized protocol with respect to reaction environment. The physiological properties of Jatropha oil suit to go for Biodiesel production as the oil content contains highly unsaturated long chain fatty acid as the best substitute to any other source materials for in vitro studies.

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<p style="text-align: justify;">Effect of ethanol and enzyme concentration on Biodiesel yield in J. curcas and ''J. gossipifolia ''

<p style="text-align: justify;">The preliminary studies to determine the optimum quantity of ethanol, catalyst lipase reaction temperature and reaction time required for transesterification of Jatropha oil were conducted dry varying concentration of ethanol from 10 to 20% lipase concentration 2.0, 2.5 and 3.0gm.equivalent, reaction temperature (400C) and reaction time 8 hour. To 100ml conical flask containing 10gm. Jatropha oil, varying concentration of ethanol 1.0, 2.5 and 3.0gm equivalent. Each mixture was stirred and heated to 400Cwith constant shaking at 200rpm for 8hr. samples were withdrawn after reaction period and analyzed for maximum conversion. In this Table-2 it shows that the combined treatment (in case of J. curcas seed extract) at enzyme 3.0 gm equivalent and 0.2 gm. Ethanol shows higher yield of biodiesel i.e. 8.25gm. In case of leaf extract the combined treatment at 3.0 gm. equivalent and 0.2 gm, ethanol shows higher yield of Biodiesel i.e. 7.10 gm. Where as in case of J. gossipifolia it shows little less as comparison to J. curcas. In Table-3 it shows that the enzyme concentration 3.0 gm. Equivalent in presence of ethanol 1:4 molar ratio i.e. 2 gm. /10gm. of oil (in case of J.gossipyfolia) shows preferably higher yield i.e. 7.85 gm similarly in case of leaf extract the combined value of enzyme at 3.0 gm. equivalent and ethanol at 2.0 gm. gives higher yield i.e. 5.85 gm.

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<p style="margin-right: -36pt; text-align: center;">Gayatri Nahak et al.,: Continental J. Biological Sciences 3: 33 - 45, 2010

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<p style="text-align: justify;">Enzymatic transesterification with specific reference to ester yield by lipase from plant parts of J.curcas and J. gossipyfolia such as seeds, stems and leaves were studied. The extraction, partial purification and enzyme assay were done as per standard protocol. The yields of Biodiesel were studied with the use of lipase only in free preparation. Ethanol was used at various concentrations ranging from 10-25% keeping other factors constant for suitable environmental condition for enzymatic catalyst on the basis of finding (Shah, et al, 2003). As we know lipase possesses unique feature of acting at interface between aqueous and organic phase and its activation involves in making the restriction of active site requiring oil water interface. Therefore studies were made taking water at various levels along with enzyme at various concentrations. The reactions were carried out according to reaction set up and optimization conditions described earlier. The results obtained in summarized and put at a glance in Fig-2 and Fig-3.

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<p style="text-align: justify;">Reaction temperature in case of enzyme catalyzed transesterification shows in conformity with other researches and appears to be at optimum 400C when reaction time allowed 8hrs. The yield of Biodiesel is visibly significant for all sources of enzyme catalyzed reactions that the yield of Biodiesel remains in maximum range with depression of glycerol output. The level of biodiesel yield goes up to above at optimal reaction environment which may exceed beyond with further intensive investigation (Du et al., 2003). Although reports available shows once preferential approach to go for chemical catalysis with a base material as catalyst taking into reaction timing and cost of study as point of consideration but at the same time the data and results show the importance of biochemical transesterification can’t be denied. More over when the removal of glycerol as by-product is considered, biochemical transesterification is no doubt preferred to chemical transesterification. In order to shift the reaction to the right an alcohol excess (molar ratio alcohol:oil = 6:1) and a catalyst (NaOH, KOH at 20% by weight on oil basis) are necessary (Chitra et al., 2005).An optimal ester yield of 98% is achieved after 90 min. of reaction at 60°C (Chitra et al., 2005). Crude glycerol is separated and can be used as a raw material for soap production or other cosmetica.

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<p style="text-align: justify;">Enzyme extract obtained from Jatropha seed as compared to that of leaf shows better result, which obvious for the reason that lipase from the leaf is being metabolized before translocated to other parts of the plant, where as lipase in the germinated seeds is in activated form for hydrolysis of triglyceride and other lipids to support the carbon source for gluconeogenesis in the energy supplement.

<p style="text-align: justify;">

<p style="text-align: justify;">When we look into the efficiency of transesterification of both the species of Jatropha plant, J.curcas is in preferential position as compared to J.gossipyfolia for the reason of compatibility of enzyme with the oil from same source as the substrate i.e. J.curcas oil is being taken for study of transestrification.

<p style="text-align: justify;">Accumulation of emulsion when leaf extract is taken as source is more in both the species compared to seed extract most probably due to increased salt concentration in leaf extract. Under circumstances intermittent withdrawal of emulsions with stepwise addition of ethanol would have been improved the situation in increasing the yield.

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<p style="text-align: justify;">The comparative figures (Fig-2 and Fig-3) of the Biodiesel yield obtained from these two species of Jatropha show the use of lipase from Jatropha seed as better performance followed by Jatropha leaves in both the cases. Since other variable such as alcohol concentration is constant in all the treatments, the activity index of lipase at 3gm.equivalent appears to have important consideration compared to total protein/enzyme content in transesterification as given in the Table-2(a) and Table-2(b).

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<p style="text-align: justify;">Interestingly the enzyme catalyzed transesterification shows compatible results (Table-3 and Table-4) in conversion percentage to the extent of nearly 82-85% with the use of same quantity of alcohol for alcholysis of Jatropha oil as is required for chemical process with reduction in temperature requirement and lengthening reaction time. The reports (Vacek et al., 2001) show higher yield to the extent of 98% because commercial enzyme in purified form are being used. The concentration of glycerol and other by products and enzyme catalyzed transesterification plays a vital role as increased level depressed the conversion in the process of catalysis. Transesterification being an equilibrium reaction, shifting towards product formation required use of excess alcohol proportionally. The data and graphical representation

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<p style="margin-right: -36pt; text-align: center;">Gayatri Nahak et al.,: Continental J. Biological Sciences 3: 33 - 45, 2010

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<p style="text-align: justify;">show 20% alcohol concentration with relation to oil/glycerol both in enzyme and base catalyzed transesterification affect yield of Biodiesel.

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<p style="text-align: justify;">Since removal of glycerol from the product pool during the progress of product catalysis hasn’t been done due to technical constraints, it is worthwhile to mention the yield of Biodiesel may likely to increase further in the system (Belafi-Bako et al., 2002). The decline in ester yield beyond use of 20% alcohol may be attributed to non removal of product after the steady state as it is evident from the result that reduction of Biodiesel yield is visibly significant with increase in accumulation of other by-product.

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<p style="text-align: justify;">Fig-6 shows TLC results for different oil samples along with esterified and unestrified Jatropha oil obtained after incubation for 12hrs. Moreover it is interesting to observe that although the ester mixture confirms the BIS standard, the experimental findings of the chromatogram of the ester mixture clearly indicates variability of ester mixture composition in varying chain length. Therefore in absence of GLC quantification and characterization of ester mixture is not possible in the present investigation. However the fact is that exploitation of the different sources of enzymes as in the study being the cheapest, leave enough scope of further research in the transesterification process to standardize reactant concentrations, reaction timing etc. with modified reactor design for encouraging result in future.

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<p style="text-align: justify;">CONCLUSION

<p style="text-align: justify;">Many studies have been done all over the globe after releasing the importance of Biodiesel. Since then workers are in continuous effort to develop a suitable protocol for effective, efficient and economic conversion of non edible vegetable oil to Biodiesel to meet the challenge for the forth coming energy crises. The result obtained are quite encourgable for further investigation because the process of esterification is confined to eco-friendly enzymatic transesterification using natural biocatalyst from the same source with that oil. It can’t be concluded with the information available beyond all reasonable doubt that the use of enzyme from Jatropha curcas is the best source of catalyst in this process, but the comparative parameter with respect to yield from literature available with that of commercial lipase derived from microbial source is better alternate in quantity terms. However when we consider the cost of production of Biodiesel taking all factors and consideration lower yield of Biodiesel with natural lipase appears to be more profitable proposition. So in the case with species of J.gossipyfolia using as the source of enzyme in transesterification too.

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<p style="text-align: justify;">Therefore the overall use of different species of Jatropha oil isn’t worthy and desirable in terms of quantity and quality of the product, which is confirmed from the ester yield with thorough investigation to devise a standard protocol to go for commercial production of Biodiesel. More research is necessary to get a good insight in the environmental sustainability of this production system. The land use impact is an absolute must to address those sustainability issues.

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<p style="margin: 0cm -18pt 0.0001pt -9.35pt; text-align: justify;">ACKNOWLEDGMENT

<p style="margin: 0cm -18pt 0.0001pt -9.35pt; text-align: justify;">The authors are thankful to (N.K.M) one of the author for providing proper facilities and constant supervision for research work. We are also thankful to Chandrakanti Mohanty for constant encouragement till the completion of research work. Sabitri Nahak is also acknowledging for her timely help to the author (G.N).

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<p style="text-align: justify;">Vellguth G. (1983). Performance of vegetable oil and their monoesters as fuels for diesel engine. SAE paper No. 83-1358. Warrendale, Pa: SAE.

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<p style="text-align: justify;">Zhang Q., Feldman M., and Peterson C. (1988). Diesel engine durability when fueled with methyl ester of winter rapeseed oil. ASAE paper No. 88-1562. ST. Joseph, Mich.: ASEA.

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<p style="text-align: justify;">           Table-1: Physio-chemical properties of Biodiesel <p style="text-align: justify;">

<p style="text-align: justify; text-indent: 36pt;">Table-2(a): Effect of ethanol and enzyme concentration on Biodiesel yield in J. curcas

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<p style="text-indent: 36pt;">Table-2(b): Effect of ethanol and enzyme concentration on Biodiesel yield in J. gossipyfolia

Figure-2: Effect of ethanol and enzyme                                        Figure-3: Effect of ethanol and enzyme

concentration on biodiesel yield in J. curcus                                 concentration on biodiesel yield in J. curcus

<p style="margin-right: -36pt; text-align: center;">Gayatri Nahak et al.,: Continental J. Biological Sciences 3: 33 - 45, 2010

Figure-4: Average yield in lipase                   Figure-4: Average yield in lipase

Catalyzed transesterification J. curcus          Catalyzed  transesterification J. goosiphfolia

under different treatments                             under different treatments

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<p style="text-align: justify;">Figure-6: TLC observation of different oil samples

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<p style="text-align: justify;">Received for Publication: 09/06/2010

<p style="text-align: justify;">Accepted for Publication: 21/07/2010

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<p style="text-align: justify;">Corresponding author:

R.K. Sahu

B.J.B. Autonomous College, Bhubaneswar, Orissa

Email: sahurajani@yahoo.co.in

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<p style="margin-right: -36pt; text-align: justify;">Continental J. Biological Sciences 3: 46 - 50, 2010                                                     ISSN: 2141 - 4122

<p style="text-align: justify;">© Wilolud Journals, 2010                                                                                 http://www.wiloludjournal.com

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<p style="text-align: center;">PHYTOCHEMICAL SCREENING AND MINERAL ELEMENT ANALYSIS OF THE ROOT BARK OF Parinari macrophylla Sabine (Chrysobalanaceae) AND ITS EFFECT ON MICROORGANISMS.

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<p style="text-align: center;">Halilu, M.E1., Abah, J.O1., Almustapha, N.L2 and Achor.M 3

<p style="text-align: center;">1Department of Pharmacognosy and Ethnomedicine, Usmanu Danfodiyo University, Sokoto-Nigeria.

<p style="text-align: center;">2Department of Pure and Applied Chemistry, Usmanu Danfodiyo University, Sokoto-Nigeria.

<p style="text-align: center;">3Department of Pharmaceutics and Pharmaceutical Microbiology, Usmanu Danfodiyo University, Sokoto-Nigeria.

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<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;">ABSRACT

<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;"> Parinari macrophylla (Sabine) Chrysobalanaceae, has been use extensively in the Northern part of Nigeria in Ethnomedicine. It is used to treat numerous diseases which include: Asthma, Skin infections, treatment of wounds, pulmonary troubles, dysentery, inflammations  and it is also used for the treatment of eye and ear  infections. The result of the Phytochemical analysis revealed the presence saponins, tannins, flavonoids, steroids, alkaloids, cardiac glycosides and anthraquinones. The antimicrobial activity of ethanolic extract (30 mg/ml) obtained from P. macrophylla showed that the extract had good antimicrobial activity against Staphylococcus aureus, Escherichia coli, Aspergillus fumigatus and Aspergillus flavus. The zone of inhibitions produced ranges between 16 mm and 17 mm for the bacterial strains while zone of inhibitions of 30 mm and 35 mm was produced by the fungal strains. The MIC produced, ranges between 0.68 mg/ml and 2.20 mg/ml for the bacterial strains while the MIC of 0.25 mg/ml was produced by the fungal strains. The results of mineral element analysis showed the presence of the following elements at various concentrations: Cu, Cd, Pb, Mn, Mg, Fe, Zn, K, Ca and Na. This present research has validate the ethnomedical uses of the plant in the treatment of skin infections.

<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;">

<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;">KEYWORDS: Parinari macrophylla, Phytochemical analysis, Mineral Element Analysis, Antimicrobial Activity and Microorganisms.

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<p style="text-align: justify;">INTRODUCTION

<p style="text-align: justify;">As the world tends towards researching into medicinal plants, we have discovered Parinari macrophylla (Sabine) Chrysobalanaceae, which has been use extensively in the Northern part of Nigeria in Ethnomedicine. It is used to treat numerous diseases which include: Asthma, Skin infections, treatment of wounds, pulmonary troubles, dysentery, inflammations and it is also used for the treatment of eye and ear  infections(Audu et al.,2005)''. P. macrophylla ''is a tree up to 10m high. The plant is obtained in the coastal strip from Senegal to Liberia and to 300km inland in sandy localities and 700-1000 km inland in mailto Niger and Northern Nigeria. The tree appears to survive annual bush fire in Savanna area and in northern sierra-Leon as the only tree of any stature attaining double the normal 20 m, with a girth of 1.30 m (Keay, 1989). The wood is light brown and firmly hard. P. macrophylla have been used by many rural dwellers but have been evaluated scientifically for its ethnomedical usage, phytochemical constituents and its elemental composition. This present study, seeks to provide these information about the the plant.

MATERIALS AND METHODS

<p style="text-align: justify;">COLLECTION OF THE PLANT MATERIALS.

<p style="text-align: justify;">Parinari macrophylla was collected from Birnin Kebbi, Kebbi State, North-Eastern, Nigeria. The plant was identified and authenticated by Auwal Umaru at the herbarium unit, Department of Biological Sciences, UsmanuDanfodiyoUniversity, Sokoto, Nigeria. The parts of the plant collected were: the leaves, the stem, the root bark, the fruits and the flowers for the purpose of identification.

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<p style="margin-right: -36pt; text-align: center;">Halilu, M.E et al.,: Continental J. Biological Sciences 3: 46 - 50, 2010

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<p style="text-align: justify;">DRYING AND STORAGE OF THE SAMPLE

<p style="text-align: justify;">The root bark of Parinari macrophylla was air dried, and then grounded to powder with aid of pestle and mortar. The root bark was stored in polythene bag until required for use. EXTRACTION OF THE PLANT

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<p style="text-align: justify;">MATERIALS

<p style="text-align: justify;">One hundred gram (100 g) of the powdered root bark was extracted with 300 ml of ethanol, using soxhlet extractor. The resulting extract was evaporated to dryness using a rotator evaporator. The percentage yield of the extract was twenty percent (20%).

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<p style="text-align: justify;">PHYTOCHEMICAL ANALYSIS

<p style="text-align: justify;">The phytochemical screening was carried out on the ethanol extract of the root bark of Parinari macrophylla using the standard methods outlined in Brain and Turner (1975), Trease and Evans (2005) and Harbone(1975).

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<p style="text-align: justify;">MINERALIZATION OF SAMPLES.

<p style="text-align: justify;">For the conversion of solid to liquid, a wet digestion technique was used. 0.5 g of the fine powdered samples were placed in beakers for digestion. The contents of the beakers were treated with a mixture of HNO3 and H2O2 in the ratio of 1:1. The beakers with their contents were placed on hot plates in a fume cupboard and heated electrically to boil until all the brown fumes of NO2 disappeared, leaving behind a colourless liquid. After mineralization, samples were transferred quantitatively to 50 mls volumetric flask and made to mark with de-ionized distil water.

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<p style="text-align: justify;">Elemental Analysis.

<p style="text-align: justify;">The aliquots of the digested samples were analysed for metals of interest using Atomic Absorption Spectrophotometer (AAS). Qualitative analysis of the samples were achieved by interpolating the relevant calibration curves prepared from standard metal solution of the aqueous standards.

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<p style="text-align: justify;">ANTIMICROBIAL SCREENING

<p style="text-align: justify;">Test Organisms

<p style="text-align: justify;">The test organisms were standard strains of Aspergillus fumigatus, Aspergillus flavus, Staphylococcus aureus and Escherichia coli. The organisms were obtained from the Department of Biological Sciences (Mycology Laboratory) and Department of Microbiology, Faculty of Science, UsmanuDanfodiyoUniversity, Sokoto, Nigeria.

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<p style="text-align: justify;">Antimicrobial Activity

<p style="text-align: justify;">Well diffusion method (Oboh et al., 2007) was used. The sterilized media was poured into petri dishes. The solidified plates were flooded with the various dilution of the test microorganisms and drained with sterile Pasteur pipette. Wells measuring 8.0mm in diameter were bored into the inoculated plates using cork borer (No.4). The wells were filled with 30 mg/ml of the ethanolic extract. Distilled was used as positive control. Ampicillin was used as negative control for the bacterial strains while fluconazole was used as negative control for the fungal strains. The plates were then incubated at room temperature (270C-300C) for 18 hfor antibacterial activity. For antifungal activity, the plates were observed on daily basis for any activity for five days. The results were obtained using linear measurement with the aid of a calibrated ruler. The results were measured to the nearest millimeter.

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<p style="text-align: justify;">Minimum Inhibitory Concentration (MIC)

<p style="text-align: justify;">Two fold serial dilution of 2 mls of the ethanol extract was made in nutrient broth. Ten dilutions were made and were inoculated with 0.5mls suspensions of the micoorganisms and incubated for 24 h at 37oC. After incubation, subcultures of the mixtures were made unto nutrient agar plate and incubated for 24 h at 37oC (i.e for bacterial strains) and for five days for fungal strains.The minimal inhibitory concentration (MIC), is defined as the lowest concentration that produced no visible bacterial or fungal growth after the incubation time was recorded.

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<p style="margin-right: -36pt; text-align: center;">Halilu, M.E et al.,: Continental J. Biological Sciences 3: 46 - 50, 2010

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<p style="text-align: justify; text-indent: 36pt;">Table 1: Phytochemical Constituents of the root bark of Parinari macrophylla. <p style="text-align: justify;">

TABLE 2:  Antimicrobial Activities of the Ethanolic Extract of  Parinari macrophylla (Zone of inhibition of growth in mm)

Note : The results are mean of 4 readings

NDa= Not determined since ampicillin has no activity on fungal strains.

NDf=Not determined since fluconazole has no activity on bacterial strains.

<p style="text-align: justify;">Table 3: Minimum Inhibitory Concentration (MIC) of P. macrophylla in mg/ml. <p style="text-align: justify;"> <p style="margin-left: 42.55pt; text-align: justify; text-indent: -42.55pt;">Table 4: The concentrations of the elemental constituents of the root bark of Parinari macrophylla expressed in µg/g. <p style="margin-left: 42.55pt; text-align: justify; text-indent: -42.55pt;">

<p style="text-align: justify;">RESULTS

<p style="text-align: justify;">The results of the Phytochemical analysis revealed the presence of saponins, tannins, flavonoids, steroids, alkaloids, cardiac glycosides and anthraquinones (Table.1). The antimicrobial activity of ethanolic extract (30mg/ml) obtained from P. macrophylla revealed that the extract showed a good antimicrobial activity against Staphylococcus aureus, Escherichia coli, Aspergillus fumigatus and Aspergillus flavus (Table. 2). The zone of inhibitions produced ranges between 16 mm and 17 mm for the bacterial strains while zone of

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<p style="text-align: justify;">inhibition ranges between 30 mm and 35 mm was produced by the fungal strains. (Table. 2). The MIC produced ranges between 0.68 mg/ml and 2.20 mg/ml for the bacterial strains while the MIC of 0.25 mg/ml was produced by the fungal strains (table.3). The results of mineral element analysis showed the presence of the following elements at various concentration with Ca and K having the highest concentrations, then followed by Mg and Fe. Cd has the lowest concentration (Table.4).

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<p style="text-align: justify;">DISCUSSION

<p style="text-align: justify;">In the present study, P. macrophylla was investigated for its antimicobial activity against Staphylococcus aureus, Escherichia coli, Aspergillus fumigatus and Aspergillus flavus, P. macrophylla is a medicinal plant commonly used in traditional medicine in Northern Nigeria to treat Asthma, Skin infections, treatment of wounds, pulmonary troubles, dysentery, inflammations and it is also used for the treatment of eye and ear  infections. The results of the antimicrobial activity showed that the ethanolic extract had some antimicrobial activity both on the bacterial strains and the fungal strains (Table.2). The antimicrobial activity demonstrated by the ethanolic extract may be due to the presence of the secondary metabolites detected in the plant. Phenolic compounds like tannins, flavonoids and anthraquinones have been widely reported to show antimicrobial actions (Sharada et al., 2008). According to Audu (2005), the hexane and ethylacetate fractions of P. macrophylla showed antibacterial activity. The antifungal activity is reported and is shown on (Table .2). The antifungal activity may be due to the presence of saponins, tannins and flavonoid (Tra Bi et al., 2008), (Oboh et al., 2008) As it can be seen from table 2, the extract can be compared favorably with fluconazole. The mineral element composition of P. macrophylla have been reported for the first time. The evaluation was carried out to determine where the plant has accumulated some toxic, element since the plant is used in medications. As it can be seen from table 4, the levels of the toxic elements (Pb and Cd) are very low when compared with the recommended daily intake of 10mg/kg.

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<p style="text-align: justify;">CONCLUSION

<p style="text-align: justify;">Our results offer a scientific basis for the use of P. macrophylla in traditional medicine

<p style="text-align: justify;">in Northern Nigeria for treatment of skin disorders. Also the presence some secondary metabolites which may be responsible for the pharmacological activity have been reported. We have also established that the plant has not accumulated toxic elements.

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<p style="text-align: justify;">REFERENCES

<p style="text-align: justify;">Audu, O.T., Oyewale, O and Amupitan J.O. (2005). The Biological Activities of Secondary Metabolites of Parinari macrophylla-Sabine. Chemclass Journal. 2005, Vol. 2 Pp, 19-21.

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<p style="text-align: justify;">Brain, K.R and Turner T.D. (1975). The practical Evaluation of Phytopharmaceuticals. Wright-scientechnica, Pp 90-121.

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<p style="text-align: justify;">Evans, W.C.(2005). Trease and Evans Pharmacognosy. 15th Edition. Elsevier India. Pp135-150.

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<p style="text-align: justify;">Harbone, J.B. (1973). Phytochemical Methods: A guide to modern techniques of plant analysis. Chapman and Hall Ltd. London. Pp49-188.

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<p style="text-align: justify;">Keay R.W.J. (1989). Trees of Nigeria. Clarendon Press Oxford, London P 181.

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<p style="text-align: justify;">Oboh, I.E., Akerele, J.O and Obasuyi, O.(2007). Antimicrobial Activity of The Ethanol Extract of The Aerial Parts of Sida acuta burm.f. (malvaceae). Tropical Journal of Pharmaceutical Research, 6 (4): 809-813

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<p style="text-align: justify;">Sharada L Deore., Khadabadi S.S., Lalita Bhagure and Ghorpada. (2008). In vitro Antimicrobial and Antioxidant Studies on Enicostemma axillare (Lam) Raynal leaves. Natural Product Radiance, Vol. 7(5), pp, 409- 412.

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<p style="text-align: justify;">TraBi, F.H., Koné M.W and Kouamé, N.F. (2008). Antifungal Activity of Erigeron floribundus (Asteraceae) From Côte d’Ivoire, West Africa. Tropical Journal of Pharmaceutical Research, June 2008; 7 (2): 975-979.

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<p style="text-align: justify;">Received for Publication: 09/06/2010

<p style="text-align: justify;">Accepted for Publication: 21/07/2010

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<p style="text-align: justify;">Corresponding author:

Halilu, M.E

Department of Pharmacognosy and Ethnomedicine, Usmanu Danfodiyo University, Sokoto-Nigeria

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<p style="margin-right: -36pt; text-align: justify;">Continental J. Biological Sciences 3: 51 - 62, 2010                                                     ISSN: 2141 - 4122

<p style="text-align: justify;">© Wilolud Journals, 2010                                                                                 http://www.wiloludjournal.com

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<p style="text-align: center;">EFFETS DU STRESS THERMIQUE SUR LA GERMINATION, LA DÉGRADATION DES RÉSERVES PROTÉIQUES ET MINÉRALES DES GRAINES DU GOMBO (Abelmoschus esculentus L.).

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<p style="text-align: center;">Besma Ben Dkhil et Mounir Denden

<p style="text-align: center;">Adresse : Laboratoire d’Agronomie, Institut Supérieur Agronomique Chott-Marièm 4042, Sousse, Tunisie.

<p style="text-align: center;">E-mail : bbendkhil@yahoo.fr

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<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;">Résumé

<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;">L’effet de la température sur la germination des graines du gombo (Abelmoschus esculentus L.) est étudié en relation avec la quantité d’eau absorbée, la mobilisation et la dégradation des réserves protéiques et l’utilisation des éléments potassium, sodium et calcium. Les résultats obtenus ont montré que le gombo est sensible à une variation de température, la germination est optimale à 25°C et elle est totalement inhibée à 10 et 40°C. Une basse température de 10°C a stimulé une augmentation de la teneur en protéines et de l’activité protéasique au niveau de l’embryon. L’inhibition de la germination à 40°C est la conséquence d’une inhibition de l’activité protéasique au niveau des cotylédons et d’une toxicité de l’embryon par l’élément sodium.

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<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;">Mots clés : germination, température, Abelmoschus esculentus, réserves protéiques, prtéases, réserves minérales.

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<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;">Abstract

<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;">In order to study the physiological and biochemical responses of okra seed germination (Abelmoschus esculentus) during time under three temperature regimes (10, 25 and 40°C), an experiment was carried out on laboratory in a dark condition. Temperature effects on germination are studied in relation with water absorption, the degradation and mobilization of stored protein and mineral reserves. Results indicated that 25°C was the optimal temperature that favorises a good aptitude to germinate, whereas low (10°C) and high temperatures (40°C) inhibited germination. Low temperature (10°C) slowed the rate of imbibition, increased proteins content and protease activity. Thermoinhibition of germination at 40°C is the consequence of a decrease on protease activity and toxicity due to sodium accumulation.

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<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;">Key words: germination, temperature, Abelmoschus esculentus, stored protein, protease, minerals reserves.

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<p style="text-align: justify;">INTRODUCTION

<p style="text-align: justify;">La germination est une phase physiologique pendant laquelle la graine passe de l’état de vie ralentie à l’état de vie active (Caboche et al., 1998). Elle est définie comme la somme des évènements qui conduisent la graine sèche à germer : cela commence par l’étape cruciale d’absorption de l’eau par la graine (Othman, 2005), se termine par l’allongement de l’axe embryonnaire et l’émergence de la radicule à travers les structures qui entourent l’embryon (Shereena et Nabeesa, 2006).

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<p style="text-align: justify;">La germination des graines nécessite la mobilisation des réserves accumulées au cours de la maturation dont leur dégradation apportera l’énergie nécessaire à la croissance de la plantule. Cette mobilisation est la résultante des activités hydrolytiques qui libèrent les nutriments à partir des tissus de réserve, d’une part, et des mécanismes de leur transport vers les tissus embryonnaires, d’autre part (Mihoub et al., 2005). Selon les espèces, ces réserves peuvent être majoritairement de nature glucidique, lipidique et/ou protéique (Khemiri et al., 2004).

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<p style="text-align: justify;">La respiration, l’hydrolyse des réserves et les activités enzymatiques demeurent sous la dépendance de la température. En effet, toute variation de la température d’incubation peut affecter en plus de l’activité de

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<p style="text-align: justify;">certaines enzymes, certains processus indispensables pour le contrôle de la germination comme la perméabilité membranaire et l’extensibilité de la paroi (Bewley and Black, 1992; Gul and Waber, 1999). Hawker et Jenner (1993) suggèrent que les hautes températures inhibent la germination des graines en limitant la disponibilité d’énergie et des hydrolysats. Evènement conséquent d’un retard et d’une inhibition de la synthèse et/ou l’activité des enzymes hydrolytiques. De même, les basses températures entraînent une perturbation et un retard de coordination lors de la mobilisation des réserves (Nykiforuk et Johnson-Flanagan, 1994).

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<p style="text-align: justify;">Le gombo est une plante connue pour ses fruits utilisés comme légumes. En Tunisie, la superficie emblavée en gombo est de 188 ha, la zone de production se situe essentiellement dans les régions de Tunis, Bizerte, Béja et Gafsa, la production nationale a atteint 560 tonnes. Certes, il existe des obstacles qui entravent le développement du gombo et qui limitent son extension dans le centre du pays. En effet, la sensibilité de cette espèce durant le stade germination est largement évoquée, elle est généralement attribuée à une imperméabilité des téguments qui s’oppose à la pénétration de l’eau et aux échanges gazeux (Demir, 2001). En plus de ce facteur biotique, s’ajoutent les facteurs abiotiques comme la température et la salinité. Albregts et Howard (1973) ont suggéré la sensibilité du gombo à la salinité et son exigence en chaleur. Il est admis aussi qu’une exposition des graines du gombo à des températures inférieures ou supérieures à leur température optimale de germination peut entraîner l’entrée en dormance secondaire. Les agricultures pratiquent quotidiennement le semis direct des graines, une telle pratique peut aggraver la situation au point où il serait difficile de contrôler les conditions adéquates pour réussir la germination et la levée des plantules.

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<p style="text-align: justify;">L’objectif de notre travail est d’étudier les réponses physiologique et biochimique du stress thermique au cours de la germination des graines du gombo (variété Marsaouia).

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<p style="text-align: justify;">MATÉRIEL ET MÉTHODES

<p style="text-align: justify;">Matériel végétal

<p style="text-align: justify;">La variété du gombo (Abelmoschus esculentus) utilisée est "Marsaouia". Il s’agit d’une variété locale produite par des agriculteurs de la région de la basse vallée de Majerda (Nord du pays).

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<p style="text-align: justify;">Conditions de germination

<p style="text-align: justify;">Les graines du gombo sont stérilisées par l’alcool éthylique (70°C) pendant une minute, puis trempées dans l’hypochlorite de sodium à 15% (v/v) pendant 20 minutes. La mise en germination des graines est réalisée sous hotte à flux laminaire afin de s’assurer des conditions aseptiques et d’éviter toutes contaminations. Après rinçage à l’eau distillée, les graines stérilisées sont ensemencées dans des boites de Pétri de 90 mm de diamètre tapissées de deux couches de papier filtre imbibées d’eau distillée stérile à raison de 25 graines par boite. La germination est effectuée à l’obscurité dans des germoirs à température réglable (25 et 40°C) et dans une enceinte réfrigérée (10°C). Les boites sont soigneusement fermées et entourées avec du parafilm pour éviter l’évaporation de la solution d’imbibition.

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<p style="text-align: justify;">Le critère de germination retenu correspondant à la sortie de la radicule hors des téguments de 2 mm. La germination est relevée tous les deux jours, afin de déterminer l’énergie germinative et le pouvoir germinatif.

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<p style="text-align: justify;">Test de réversibilité de germination

<p style="text-align: justify;">Il s’agit de transférer les graines qui n’ont pas germé au bout de 24 heures aux températures 10 et 40°C à une température optimale de 25°C pendant 6 jours.

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<p style="text-align: justify;">Test de viabilité des graines

<p style="text-align: justify;">Ce test concerne les graines qui n’ont pas germé à 10 et 40°C, elles sont décortiquées puis elles sont trempées dans une solution de triphényl-tétrazolium (0.1%) à l’obscurité. La viabilité des graines s’observe par la coloration en rouge des parties vivantes après une heure de trempage (Guy, 1993 ; 2000).

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<p style="text-align: justify;">Détermination de la teneur en eau

<p style="text-align: justify;">Le poids frais des graines mises en germination à 10, 25 et 40°C a été mesuré toutes les deux heures pendant 24 heures, le poids sec est déterminé après séchage des échantillons à 80°C pendant 48 heures. La teneur en eau des graines est déterminée par la différence entre la masse de matière fraîche et la masse de matière sèche rapportée à la matière sèche, elle est exprimée en ml H2O.g-1MS.

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<p style="text-align: justify;">Dosage des protéines solubles

<p style="text-align: justify;">Le dosage des protéines en solution est déterminé par la méthode de Bradford (1976), la matière fraîche (100 mg) est homogénéisée dans une solution tampon phosphate 0.1 M (pH 7). L’homogénéisât est centrifugé à 13000 rpm pendant 45 min, à 1ml du surnageant on ajoute 5ml de réactif de Bradford et le mélange est incubé pendant 15min à l’obscurité. La variation d’absorbance est mesurée au spectrophotomètre à 595 nm et convertie en mg protéines/ g de matière fraîche. Une gamme étalon est établie à l’aide d’une solution de sérum albumine de bœuf (BSA).

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<p style="text-align: justify;">Détermination de l’activité protéasique

<p style="text-align: justify;">La mesure de l’activité de l’enzyme protéase a concerné les cotylédons et l’embryon de la graine, elle est déterminée selon la méthode d’Anson (Yang and Huang, 1994). Pour cela, 100 mg du matériel végétal frais ont été broyés à froid dans une solution tampon de phosphate 0.1 M (pH 7). Le broyat est ensuite centrifugé à 13000 rpm pendant 45 min, le surnageant récupéré constitue l’extrait enzymatique qui est utilisé pour mesurer l’activité de l’enzyme protéase. Cette dernière est déterminée à partir de 1 ml d’extrait en présence de 1 ml d’une solution de caséine à 1%. Après 20 min d’incubation à 37°C, la réaction est arrêtée par addition de 3 ml d’une solution d’acide trichloroacétique à 10%. La tyrosine libérée est dosée à 280 nm au spectrophotomètre (Camspec M330 UV/Vis). L’activité spécifique est exprimée en A280/min/g de matière fraîche.

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<p style="text-align: justify;">Dosages des ions Na+, K+ et Ca ++

<p style="text-align: justify;">Les graines sont séchées à l’étuve pendant 48 heures à 80°C, puis elles sont placées dans des piluliers contenant chacun 25 ml d’une solution d’acide nitrique (0.1 N). L’extraction des ions dure 48 heures à la température ambiante du laboratoire. Les cations Na+, K+ et Ca++ sont dosés par spectrophotométrie de flamme en émission (type Eppendorf). Les concentrations sont exprimées en µéq/g de matière sèche.

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<p style="text-align: justify;">Analyse statistique

<p style="text-align: justify;">Le dispositif expérimental adopté est randomisé, le nombre de répétitions est de 8 boites par traitement et les expériences ont été répétées trois fois. L’analyse statistique a été réalisée grâce aux procédures des modèles linéaires générales (GLM) du logiciel SPSS (13.0). Le niveau de la signification a été évalué selon le test de Duncan au seuil de 5%.

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<p style="text-align: justify;">RÉSULTATS

<p style="text-align: justify;">Effet de la température sur la germination

<p style="text-align: justify;">La figure 1 montre que les graines ne germent qu’à 25°C avec un temps de latence d’un jour. Le pouvoir germinatif est de 96% après un temps d’incubation de deux jours. Ce taux augmente jusqu’à 99.5% au bout de six jours. Un taux de germination optimal et maximum de 100% est atteint au huitième jour. Cependant, les graines qui sont incubées pendant 12 jours à 10 et 40°C se montrent inaptes à la germination.

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<p style="text-align: center;">Figure 1 : Effets de différentes températures (10, 25 et 40°C) sur le pouvoir germinatif des graines du gombo.

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<p style="text-align: justify;">Test de réversibilité de la germination

<p style="text-align: justify;">Les résultats obtenus dans la figure1 montrent qu’aussi bien à une haute qu’à une basse température la germination est inhibée. Un test de réversibilité parait nécessaire pour préciser s’il s’agit d’une perte de viabilité ou d’une entrée en dormance de l’embryon. Le transfert ultérieur des graines de 40°C à une température optimale de germination (25°C) a donné un taux nul de germination. Par ailleurs, le transfert des graines de 10°C à 25°C a entraîné une reprise de la germination seulement pour quelques graines avec une capacité germinative de 6% (Figure 2).

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<p style="text-align: justify;">Figure 2 : Effet de test de réversibilité de la germination pour des graines imbibées pendant 24 heures à 10°C et 40°C et mises en germination à 25°C.

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<p style="text-align: justify;">Test de viabilité des graines

<p style="text-align: justify;">Le test de viabilité au tétrazolium appliqué sur les graines qui n’ont pas germé à 10 et 40°C a montré que l’embryon et les cotylédons n’ont pas perdu leur viabilité après une durée d’imbibition de 48 heures.

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<p style="text-align: justify;">Effet de la température sur la teneur en eau des graines

<p style="text-align: justify;">Les graines sèches ont une teneur en eau de 0.1 ml/g MS, l’hydratation des graines du gombo est dépendante de la température comme s’est indiquée dans la figure 3. L’absorption d’eau est relativement lente à 10°C et elle est plus importante à 25 et 40°C. La cinétique de la prise d’eau permet de caractériser deux phases distinctes. Une première phase qui dure 6 heures, elle est marquée par une entrée rapide et passive d’eau quelque soit la température. Une deuxième phase qui dure 18 heures, elle est caractérisée par une absorption lente d’eau. Les graines imbibées à 25°C accomplissent ces deux phases avec une meilleure teneur en eau qui est de 1.15 ml/g MS.

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<p style="text-align: justify;">Figure 3 : Evolution de la teneur en eau des graines soumises à l’effet de différentes températures de germination.

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<p style="text-align: justify;">Effet de la température sur la teneur en protéines solubles

<p style="text-align: justify;">La figure 4 illustre la variation de la teneur en protéines solubles dans les cotylédons et l’embryon des graines au cours de leur germination à 10, 25 et 40°C.

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<p style="text-align: justify;">A 25°C, pendant la phase d’imbibition les cotylédons accumulent 152 mg/g MF des protéines. Pendant la seconde phase, les protéines de réserve commencent à se dégrader allouant une teneur de 70 mg/g MF dans les cotylédons. Au moment de la germination, les cotylédons ne retiennent que 44 mg/g MF de protéines solubles.

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<p style="text-align: justify;">Au niveau de l’embryon, la quantité de protéines accumulée pendant la phase rupture du tégument est la plus importante (160 mg/g MF) dont plus que la moitié sera dégradée pour favoriser la croissance de la percée radiculaire.

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<p style="text-align: justify;">A 40°C, une augmentation de la teneur en protéines a été décelée au cours des premières 24 heurs qui suivent le début d’imbibition des graines que ce soit au sein des cotylédons qu’au sein de l’embryon. Notant bien que pendant la phase d’imbibition la teneur en protéines solubles des graines est élevée par comparaison au témoin (25°C). Après 48 heures d’incubation les protéines accumulées dans les cotylédons et l’embryon ont montré une légère diminution.

<p style="text-align: justify;">

<p style="text-align: justify;">A 10°C, la teneur en protéines tend à augmenter après 24 heures d’imbibition notamment au niveau de l’embryon (141 mg/g MF) et elle baisse au niveau des cotylédons après un temps d’imbibition de 48 heures.

<p style="text-align: justify;">

<p style="text-align: justify;">Effet de la température sur l’activité protéasique

<p style="text-align: justify;">L’activité de l’enzyme protéase est sensible à l’effet de la température (Figure 5 a, b et c), elle est stimulée par les températures défavorables à la germination (10 et 40°C).

<p style="text-align: justify;">

<p style="text-align: justify;">A 25°C, pendant la phase d’imbibition, les cotylédons sont le siège d’une activité enzymatique intense c’est pendant cette phase qu’il y aura dégradation des protéines de réserves (Figure 5 a). La phase rupture du tégument est marquée par une diminution de l’intensité de l’activité protéasique de 5 fois moins par rapport à celle atteinte pendant la phase d’imbibition. Une fois que la radicule a percé le tégument de la graine, les protéases ont repris leur niveau initial d’activité malgré la faible teneur en protéines solubles dans les tissus de réserve.

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<p style="text-align: justify;">Au niveau de l’embryon l’enzyme protéase est plus active pendant la phase rupture du tégument et un peu moins au moment de la germination.

<p style="text-align: justify;">

<p style="text-align: justify;">A 10°C, l’imbibition des graines déclenche tôt le processus de dégradation des protéines dans les cotylédons et avec une intensité moins faible au niveau de l’embryon (Figure 5 b). Après un temps d’imbibition de 24 heures, la teneur en protéines des cotylédons diminue ainsi que l’activité de l’enzyme hydrolytique commence à s’inhiber. Néanmoins, au niveau de l’embryon l’activité de l’enzyme protéase est plus intense afin de dégrader les protéines accumulées dans l’organe de croissance.

<p style="text-align: justify;">

<p style="text-align: justify;">A 40°C, d’après la figure 5 c on note qu’au niveau de l’embryon se déclenche une activité enzymatique plus intense que celle atteinte à 25°C, après 24 heures d’imbibition l’activité protéasique baisse conformément à la quantité des protéines qui diminue aussi. Au niveau des cotylédons, les protéases maintiennent leur niveau d’activité jusqu’à 48 heures d’imbibition.

<p style="text-align: justify;">

<p style="text-align: justify;">La variation de l’activité protéasique est significative à 10, 25 et 40°C au cours du temps et au niveau de l’embryon et des cotylédons.

<p style="text-align: justify;">

<p style="text-align: justify;">Effet de la température sur la mobilisation des réserves minérales

<p style="text-align: justify;">Les teneurs en sodium, calcium et potassium des graines non germées à 10 et 40°C sont énumérées dans le tableau 1, elles sont comparées à celles des graines germées à 25°C.

<p style="text-align: justify;">

<p style="text-align: justify;">Il ressort des résultats obtenus que la température a montré un effet significatif sur l’accumulation d’élément sodium. Le test de comparaison des moyennes effectué au seuil de 5% nous a permis de distinguer deux groupes; un premier groupe représenté par la température 25°C et un deuxième groupe formé par les températures 10 et 40°C.

<p style="text-align: justify;">

<p style="text-align: justify;">En effet, la teneur en sodium est nettement élevée (1329 et 1478 µéq/g MS) à 10 et 40°C, alors que cette teneur est de loin plus faible à 25°C (378 µéq/g MS). La teneur en calcium dans les graines n’est pas statistiquement modifiée avec la température, elle varie entre 708 µéq/g MS (40°C) et 735 µéq/g MS (25°C).

<p style="text-align: justify;">

<p style="text-align: justify;">De même, les différentes températures n’ont pas montré de différences significatives pour la teneur en potassium, cette dernière a pris des valeurs qui s’arrangent entre 413 µéq/g MS (10 et 25°C) et 441 µéq/g MS (40°C).

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<p style="text-align: justify;">Figure 4: Variation de la teneur protéines solubles des cotylédons et des axes embryonnaires en présence de différentes concentrations de NaCl pendant la phase d’imbibition (a), la phase rupture de tégument (b) et la phase de germination (c).

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<p style="text-align: center;">Figure 5: Activité des protéases dans les cotylédons et l’embryon des graines mises en germination à 25°C (a),10°C (b) et 40°C (c).

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<p style="text-align: center;">Besma Ben Dkhil et Mounir Denden: Continental J. Biological Sciences 3: 51 - 62, 2010

Tableau 1: Teneurs en éléments sodium, calcium, potassium et chlorure au niveau des graines à 10, 25 et 40°C.

<p style="text-align: justify;">Les moyennes de la même colonne présentant des lettres différentes en exposant diffèrent significativement au seuil de 5% par le test de Duncan.

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<p style="text-align: justify;">DISCUSSION ET CONCLUSION

<p style="text-align: justify;">Le passage de la vie latente à la vie active de la graine nécessite comme il est connu son imbibition. Au fur et à mesure que la teneur en eau augmente, l’intensité respiratoire croît et par suite les besoins en oxygène. Mais le phénomène respiratoire, l’hydrolyse des réserves et les activités enzymatiques qui font suite demeurent sous la dépendance de la température.

<p style="text-align: justify;">

<p style="text-align: justify;">Le seuil de température pour la germination du gombo est de 16°C, une basse température retarde la germination (Hussain et al., 2006). Chez le tournesol, le seuil de température pour la germination est de 4°C, une température d’au moins 8 à 10°C est nécessaire pour une bonne germination suite à un semis direct au champ (Ebrahimi, 2008). Chez le colza, l’optimum thermique se situe entre 15 et 25°C, la germination est inhibée à 5°C (Puppala et al., 1999). Les basses températures ont entraîné un retard ainsi qu’une diminution du pouvoir germinatif des graines du colza (Acharya et al., 1983; Kondra et al., 1983; King et al., 1986; Barber et al., 1991; Wilson et al., 1992).

<p style="text-align: justify;">

<p style="text-align: justify;">Nos résultats montrent que l’optimum thermique pour la germination des graines du gombo est situé à 25°C. Les graines mises en germination à 40°C subissent une inhibition thermique, ce phénomène a été largement démontré chez des graines de céleri (Biddington, 1981), de laitue (Khan, 1980-1981) et d’épinard (Leskovar et al., 1999). Le test au tétrazolium révèle que les différents tissus de la graine restent viables, ce qui nous permet de distinguer que cette haute température n’a pas endommagé l’embryon et il est possible que la quantité d’oxygène qui arrive à l’embryon sous la forme soluble dans l’eau ne soit pas suffisante pour permettre sa germination. Moore (1985) ont rapporté que l’embryon exige plus d’oxygène quand la température de germination augmente, le manque d’oxygène peut provoquer l’entrée en dormance secondaire de l’embryon (dormance induite). Par exemple chez les graines de tournesol, l’optimum thermique de germination est de 25 à 30°C, un traitement de courte durée (1 à 2 jours) à 40-45°C, inhibe la germination. Egalement, nos résultats révèlent une inhibition de la germination sous une température de 10°C, un transfert de ces graines à 25°C entraîne une reprise de la germination mais avec un faible pouvoir germinatif, les graines n’ont pas perdu leur viabilité et l’embryon a entré en dormance. L’ampleur de l’hydratation des graines a été évaluée par la mesure de la teneur en eau sur la base de la matière sèche. Les résultats obtenus montrent que les graines imbibées à 25°C ont accomplit leur germination, elles ont accédé à la phase de protursion de la radicule avec une meilleure teneur en eau. A 10°C, l’absorption d’eau par les graines est moins rapide, ce résultat confirme celui de Nykiforuk et Flanagan (1999) qui indique que des basses températures de 6 et 10°C entraînent une diminution de la vitesse d’absorption d’eau par les graines du colza pendant la phase d’imbibition.

<p style="text-align: justify;">

<p style="text-align: justify;">Par la suite, on constate qu’après 44 heures d’imbibition la quantité d’eau absorbée par les graines à 25°C est avoisinante de celle absorbée à 10 et 40°C. Par conséquent, l’inaptitude à la germination du gombo à 10 et 40°C ne peut pas être attribuée à des difficultés d’absorption d’eau au cours de la phase d’imbibition.

<p style="text-align: justify;">De même, les protéases jouent un rôle primordial durant la germination, elles sont impliquées dans les processus de dégradation des protéines accumulées dans les tissus de réserves et de leur transport sous forme d’acides aminés vers l’embryon. Ces hydrolysats sont absorbés par l’embryon et sont à l’origine de nouveaux acides aminés et de la synthèse de protéines dans les organes en croissance (Ashraf et al., 1995).

<p style="text-align: justify;">L’imbibition des graines stimule énormément l’activité protéasique au niveau des cotylédons, c’est au cours de la phase rupture de tégument que l’activité de l’enzyme est accélérée au niveau de l’embryon et ce

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<p style="text-align: justify;">

<p style="text-align: justify;">qui se synchronise en fait avec une teneur élevée en protéines. Il semble que la radicule de l’embryon du gombo comme celle de l’embryon de petit pois (Murray et al., 1979) possède des tissus de réserve contenant des corps protéiques, et que les étapes précoces de la croissance de l’axe embryonnaire (dés la rupture de tégument) dépendraient d’abord de ses propres réserves puis, ensuite essentiellement de la mobilisation des réserves cotylédonaires. L’apparition de la percée radiculaire est suivie par une rénovation de l’activité des protéases au niveau des cotylédons, cette phase de croissance s’accompagne d’une synthèse de nouveau d’endopeptidases et de carboxypeptidases au niveau des corps protéiques (Côme et Corbineau, 1998).

<p style="text-align: justify;">

<p style="text-align: justify;">Néanmoins, une température défavorable pour la germination n’a pas diminué l’activité protéasique au contraire celle-ci a atteint des valeurs maximales particulièrement pendant les premières 24 heures qui suivent la mise en imbibition des graines. Sous l’effet d’une basse température (10°C) il y avait un retard dans la dégradation des réserves protéiques au niveau des tissus de réserve ce qui résulte une impulsion sur l’activité de ces enzymes.

<p style="text-align: justify;">

<p style="text-align: justify;">Sous une haute température (40°C) l’activité des protéases est énorme au niveau de l’embryon où la teneur en protéines est maximale.

<p style="text-align: justify;">

<p style="text-align: justify;">Au-delà des premières 24 heures et jusqu’aux 48 heures après la mise en imbibition des graines, l’activité des protéases est inhibée simultanément au niveau des cotylédons à 10°C justifiant une accumulation des hydrolysats et donc leur conservation puisque les graines ont entré en dormance, et au niveau de l’embryon à 40°C cette fois ci les hydrolysats ne sont pas utilisés malgré leur disponibilité pour la croissance des axes.

<p style="text-align: justify;">Il est importe de signaler que pendant la germination, les graines utilisent les réserves minérales pour assurer le développement de l’embryon (Malleshi, 1984; Neerjarani et Hira, 1998). Bien que, dans notre étude on ne remarque pas une utilisation de l’élément potassium pendant la phase germinative, il est possible que cet élément serait exploité pendant les phases ultérieures de croissance. L’élément sodium est le plus affecté par la température, une haute température de 40°C et une basse température de 10°C ne permet que l’utilisation des quantités minimes du sodium en comparaison avec une température de 25°C. Les travaux de Salisbury et Ross (1992), ont mis l’accent sur l’effet d’une haute température lors de la germination des graines et qu’elle incite une accumulation des ions autour de la membrane cellulaire, particulièrement les ions Na+ et Cl- qui manifestent une toxicité provoquant de ce fait une inhibition du processus germinatif.

<p style="text-align: justify;">

<p style="text-align: justify;">Une graine du gombo germe à 25°C, où les conditions sont favorables pour acquérir une bonne hydratation en eau dés les premières heures d’imbibition, pour déclencher l’activité protéasique dés la phase imbibition ce qui implique une dégradation des réserves protéiques accumulées au niveau des cotylédons et une utilisation de l’élément sodium pour assurer la croissance de la percée radiculaire. Une basse température de 10°C a entraîné une entrée en dormance de la graine.

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<p style="text-align: justify;">Received for Publication: 09/06/2010

<p style="text-align: justify;">Accepted for Publication: 21/07/2010

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<p style="margin-right: -36pt; text-align: justify;">Continental J. Biological Sciences 3: 63 - 74, 2010                                                     ISSN: 2141 - 4122

<p style="text-align: justify;">© Wilolud Journals, 2010                                                                                 http://www.wiloludjournal.com

<p style="text-align: center;">FERMENTATION OF SORGHUM USING YEAST (Saccharomyces cerevisiae) AS A STARTER CULTURE FOR BURUKUTU PRODUCTION

<p style="text-align: center;">1Mbajiuka Chinedu S, 2Omeh Yusuf S and 3Ezeja Maxwell.I.

<p style="text-align: center;">1Department of Microbiology, 2Department of Biochemistry,College of Natural and Applied Sciences, and 3Department of Veterinary Physiology, Pharmacology and Biochemistry Micheal Okpara University of Agriculture,Umudike,Abia State,Nigeria.

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ABSTRACT
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<p style="margin: 0cm 43.2pt 0.0001pt; text-align: justify;">The traditional method of burukutu production involves malting, mashing, addition of an adjunct, fermentation of sorghum using an old brew as a starter culture for 48 h pasteurization by boiling and maturation. The use of Saccharomyces cerevisiae as a starter culture in burukutu production was compared with the traditional method of brewing burukutu. The microorganisms associated with the traditional method include Staphylococcus species, Streptococcus species, Enterobacter species, Candida species, Aspergillus species and Saccharomyces species. The titratable acidity was gradually increasing from 0.20 to 0.36M for old brew and 0.21 to 0.38M for yeast only. The pH decrease as fermentation proceeds from 5.86 to 3.67f for old brew and from 5.91 to 3.57 for yeast only at room temperature. The percentage of reducing sugar and specific gravity decrease with increase in fermentation period. The fermentation rate was higher for the case of inoculating with Saccharomyces cerevisiae compared with inoculating with old brew. The percentage alcohols of both samples were gradually increasing as the fermentation proceeds but higher in the sample inoculated with Saccharomyces cerevisiae after 48 h with 0.14%. The effect of nutritional contents, shelf life and other qualities of burukutu beer needs to be investigated.

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KEYWORDS:-Traditional methods, Fermentation, Saccharomyces cerevisiae, starter culture, burukutu.

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<p style="text-align: justify;">INTRODUCTION

<p style="text-align: justify;">Cereals are more widely utilized as food in African countries than in the developed world. In fact cereals account for as much as 77% of total calorie consumption in African countries (Alais and Linden,1999;Norman, et al, 1999). A majority of traditional cereal-based food as shown in table 1 consumed in Africa and mainly processed by natural fermentation. Fermented cereals are important as dietary staples for adults in Africa. Major cereals grown in Africa include sorghum, rice, maize and millet (Norman, et al, 1999).

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<p style="text-align: justify;">Sorghum is one of the cereals cultivated in the tropical region of Africa and is about the largest cultivated crop in the northern Guinea savanna areas of Nigeria (Kolawole, et al, 2007). Sorghum is a large variable genus with many cultivars. It constitutes a major source of energy and it serves as a staple food of many of the world’s poorest and least privileged people (Hamad et al, 1993 and Michodjehoun-Mestres, et al, 2005).

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<p style="text-align: justify;">According to Ahmed et al.,1996,sorghum products have poor nutritional value due to their deficiency in lysine, threonine and tryptophan and presence of anti-nutritional factors such as tannins and phytates that interact with proteins, vitamins and minerals, thus restricting their bio-availability. The above factors contribute to anemia and other nutritional diseases in developing countries where the consumption of sorghum products is high(Hassan and El Tinay,1995). However, various techniques have been investigated to improve the protein digestibility and mineral availability of sorghum by reducing its tannin and phytate content. These include malting, fermentation and cooking (Abd Elmoneom et al.,2005 and Okafor,1981.)  Fermentation is the process of anaerobic oxidation of carbohydrates to produce intermediate substrates (organic acid, ethanol etc) with the release of carbon dioxide (Prescott et al, 2005). The advantages of

<p style="text-align: center;">Mbajiuka Chinedu S et al.,: Continental J. Biological Sciences 3: 63 - 74, 2010

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<p style="text-align: justify;">fermentation can not be over emphasized. It is one of the important techniques employed to extend the shelf life of raw food materials while in the technically advanced countries, it is used more to develop and add flavour to variety of diet (Achi, 2005; Isabel et al, 2005). Fermentation is widely used traditionally for processing sorghum into fermented products. Their low pH confers the advantage of microbiological safety  (Tomkins et al., 1988). Sorghum based foods includes burukutu, pito, bogobe, kisra, injera etc. They are mainly fermented; some are non-alcoholic while others are alcoholic beverages(Booney,2005;Sulma et al.,1994 and Okafor,1981).

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<p style="text-align: justify;">Burukutu is a popular indigenous alcoholic beverage of a vinegar-like flavour, consumed in the Northern Guinea savanna region of Nigeria,Republic of Benin and in Ghana (Kolawole et al, 2007 and Norman et al, 1999). It is mainly produced from the grains of guinea corn (Sorghum vulgare and Sorghum bicolor). The traditional process of preparing burukutu involves steeping sorghum grains in water overnight, malting, mashing, fermentation and maturation as described by Norman et al, 1999 and Achi, (2005). It is a batch process carried out on a small scale. Burukutu as an indigenous beer brewed at the cottage level in some parts of West Africa has basic characteristics that include a sour taste due to the presence of lactic acid, a pH of 3.3 to 3.5 and opaque colour because of suspended solids and yeast. It contains vitamins, iron, manganese, magnesium, phosphorus and calcium and also contains about 26.7g of starch and 5.9g of protein per liter (Jogo et al.,2002;Egemba and Etuk, 2007). However, some of the endogenous sorghum microorganisms are pathogenic or may produce toxic substances, such as mycotoxins. (Isabel et al, 2005). But pasteurization of freshly brewed burukutu sample at 600C for 30 min delayed its spoilage for two weeks (Alais and Linden ., 1999).

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<p style="text-align: justify;">Considering the importance of traditional fermentation of cereal-based beverage and the need of using a single microbe as starter culture, it became necessary to investigate the possibility of using a starter culture (such as yeast Saccharomyces cerevisiae) in burukutu production and comparing it with burukutu produced by the normal traditional method (using old brew as starter culture).

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<p style="text-align: justify;">                                               TABLE 1: FERMENTED CEREALS BASED FOODS <p style="text-align: justify;">               Adapted from Norman et al,1999; Achi, 2005 and Achi, 1990.

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<p style="text-align: center;">Mbajiuka Chinedu S et al.,: Continental J. Biological Sciences 3: 63 - 74, 2010

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<p style="text-align: center;">Sorghum grain

<p style="text-align: center; line-height: 150%;">Soak with water overnight

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<p style="text-align: center; line-height: 150%;">Sun-dry the malt

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<p style="text-align: center; line-height: 150%;">Grind the sun-dried malt

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<p style="text-align: center; line-height: 150%;">Mashing (mix with warm water, and adjunct)

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<p style="text-align: center; line-height: 150%;">Sieving

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<p style="text-align: center; line-height: 150%;">Boil mash for 30 min

<p style="text-align: center;">Inoculate with old brew of burukutu

<p style="text-align: center;">(or pure culture of yeast in case of modified method)

<p style="text-align: center; line-height: 150%;">Ferment for 48 hours (2 days) at room temperature

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<p style="text-align: center; line-height: 150%;">Boil to stop fermentation

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<p style="text-align: center; line-height: 150%;">Allow to mature for 2 days

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<p style="text-align: center; line-height: 150%;">Burukutu

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<p style="margin-left: 99pt; text-indent: -99pt;">Figure 1:      Flow Sheet For The Traditional and Modified Method of Burukutu Production From Sorghum Grain.

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<p style="text-align: center;">Mbajiuka Chinedu S et al.,: Continental J. Biological Sciences 3: 63 - 74, 2010

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<p style="margin-left: 99pt; text-indent: -99pt;">A:Homofermentative lactics

<p style="margin-left: 99pt; text-indent: -99pt;">B:Heterofermentative lactics

<p style="margin-left: 99pt; text-indent: -99pt;">C and D:Propionibacterium

<p style="margin-left: 99pt; text-indent: -99pt;">E:Saccharomyces spp

<p style="margin-left: 99pt; text-indent: -99pt;">F:Acetobacter spp

<p style="margin-left: 99pt; text-indent: -99pt;">G:Acetobacter”overoxidizer”

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<p style="margin-left: 99pt; text-indent: -99pt;">Figure 2: Generalized pathways for the production of some fermentation products from glucose by various organisms represented by letters A to G.

<p style="margin-left: 99pt; text-indent: -99pt;">Source:Alais and Linden,1999.

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MATERIALS AND METHODS

MATERIALS

<p style="text-align: justify;"> SORGHUM

<p style="text-align: justify;">The red variety of sorghum (Sorghum vulgare) used for this work was purchased from Umuahia main market in Abia State, Nigeria.

<p style="text-align: center;">Mbajiuka Chinedu S et al.,: Continental J. Biological Sciences 3: 63 - 74, 2010

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<p style="text-align: justify;">OLD BREW

<p style="text-align: justify;">The old brew of burukutu used for inoculation in line with traditional method of burukutu production in this study was obtained from the open market in Gariki, Okigwe, Imo state, Nigeria.

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<p style="text-align: justify;">YEAST

<p style="text-align: justify;">The yeast strain, Saccharomyces cerevisiae used in this work was obtained from the central laboratory at the National Root Crops Research Institute Umudike in Abia State, Nigeria.

METHODS

MALTING

<p style="text-align: justify;">The dehulled and cleaned sorghum grains were steeped overnight in a plastic container with tap water at room temperature (28-300C) without changing the water. The soaked grains were washed and drained. They were uniformly spread on a wet sack cloth, then covered with banana leaves to reduce dehydration. The grains were kept wet by frequent spraying with tap water every morning, and turning over at intervals. Germination was done at room temperature (28-300C) for four days (Michojehoun-Mestres et al, 2005 and Achi, 2005).

<p style="text-align: justify;">SUN DRYING

<p style="text-align: justify;">The germinated or malted sorghum grains were sun-dried in accordance with traditional practice (Kolawole et al, 2007 and Mohammed et al.,1999).

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<p style="text-align: justify;">MILLING

<p style="text-align: justify;">The sun-dried malted sorghum grain was milled into flour and passed through a 0.5mm sieve, using a community plate disc mill. The flour was stored in a polyethylene bag at an ambient temperature prior to analysis according to Kolawole et al, 2007.

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<p style="text-align: justify;">MASHING

<p style="text-align: justify;">The mashing of the sorghum malt was carried out by a method as described by Egemba and Etuk, (2007) and Achi, (2005). 3.0kg of sorghum malt was weighed with a weighing balance and mixed with adjunct (a farinaceous fermented cassava product) and warm water (450C) in the ratio of one part garri to two part malt and six part water (1:2:6). The mixture was stirred and allowed to settle for 30min. when settled, 2L of the clear enzymatic supernatant was decanted and the remaining mash was gradually brought to boil at 1000C for 30min. The mash was allowed to cool at 600C and the clear enzymatic supernatant was added and then kept for 12 h. The mixture was filtered through a sieve mash and rinsed with 30ml of water (450C) to extract the remaining enzymes from the grist. The part of the wort was further boiled for some minutes and allowed to cool for inoculating with pure culture of yeast.

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<p style="margin-left: 72pt; text-align: justify; text-indent: -72pt;">PREPARATION OF STARTER CULTURE OF YEAST FOR INOCULCATION

<p style="text-align: justify;">One gram of dry Saccharomyces cerevisiae weighed with a weighing balance was added to 100ml of the sorghum wort with three tea spoons of glucose and left for six hours to activate the yeast strain. The activated yeast was inoculated into 400ml of sorghum wort for fermentation (Egemba and Etuk, 2007).

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<p style="text-align: justify;">FERMENTATION OF SORGHUM WORT

<p style="text-align: justify;">The fermentation was carried out in a 2L fermentation bucket constructed with plastic tap for 48 hours at room temperature (Nout,1981and Sukki et al.,1994).

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<p style="text-align: justify;">The natural fermentation was carried out by inoculating 100ml of old brew of burukutu into 400ml of the sorghum wort in line with the traditional method of brewing burukutu (Egemba and Etuk, 2007).

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<p style="text-align: justify;">MEASUREMENT OF PARAMETERS

<p style="text-align: justify;">pH

<p style="text-align: justify;">The pH of the samples was determined using pH meter (Hanna pH 211) at interval of 12 h(Richard et al.,1999).

<p style="text-align: center;">Mbajiuka Chinedu S et al.,: Continental J. Biological Sciences 3: 63 - 74, 2010

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<p style="text-align: justify;">TEMPERATURE

<p style="text-align: justify;">The temperature of the samples was determined using pH meter (Hanna pH 211) with temperature electrode.

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<p style="text-align: justify;">TITRATABLE ACIDITY

<p style="text-align: justify;">The titratable acidity was determined by titrating 2ml of samples with 0.1M NaOH to the phenolphthalein end point. The titratable acidity was expressed as the volume of NaOH solution required to neutralize the free acid contain in the sample. This method was described by Ogbadu et al, 1997.

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<p style="text-align: justify;">FERMENTABLE SUGARS

<p style="text-align: justify;">The changes in fermentable sugars were determined using brix meter (Handheld model 1305) at 12h interval according to Onwuka, 2005.

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<p style="text-align: justify;">SPECIFIC GRAVITY

<p style="text-align: justify;">The specific gravity of the samples was determined using brix meter (Handheld Model 1305) with specific gravity readings chart as also described by Bhrise et al.,(1988).

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<p style="text-align: justify;">DETERMINATION OF ALCOHOLIC CONTENT

<p style="text-align: justify;">To determine the percentage of alcohol in the beverage, the specific gravity of the both samples before fermentation starts were compared with specific gravity after fermentation. Following the equation of converting glucose to alcohol by yeast, each glucose molecule is converted into two molecules of ethyl alcohol and two molecules of carbon dioxide.

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<p style="margin-left: 72pt; text-align: justify; text-indent: 36pt;">C6H12O6® 2(CH3CH2OH) + 2(CO2).

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<p style="text-align: justify;">Checking the molecular weight of the molecules, ethyl alcohol is equal to 46.0688 and carbon dioxide is 44.0089.

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<p style="text-align: justify;">From the equation above, each carbon dioxide molecule that leaves the fermentation vessel, one ethyl alcohol molecule must be formed inside the vessel. From the molecular weight, each 44.0098 grams of CO2 that leaves the vessel, 46.0688 grams of ethyl alcohol are formed. In other hand, for each gram of CO2 that bubbles off, about 1.05 grams of ethyl alcohol are produced. Therefore, comparing the specific gravity of the beverage, the final specific gravity is subtracted from the first to give the molecular weight of CO­2 that left the vessel. Then multiply by 1.05 to get the weight of the alcohol per litre of the container. To determine the percentage of alcohol by mass, divide the mass of the alcohol with the specific gravity of the solution after fermentation. Hence this fomular:

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<p style="text-align: justify;">Mass of alcohol

<p style="text-align: justify;">                                      X 100

<p style="text-align: justify;">Final specific gravity

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<p style="text-align: justify;">However, since the percentage of alcohol by mass is higher than the percentage of alcohol by volume because an equal mass of alcohol occupies more volume than water would. To convert from percent alcohol by mass to percent alcohol by volume, the percent alcohol by mass will be divided by density of alcohol which 0.79.

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<p style="text-align: justify;">MICROBIAL ANALYSIS OF THE NATURAL FERMENTATION SAMPLE

<p style="text-align: justify;">Serial dilution of the natural sample (10-3) was inoculated into a nutrient agar prepared with 0.2ml of antifungal (fluconazole) for bacteria isolation and sabouraud dextrose agar for fungi isolation for 24 h and 48 h respectively. Bacterial identification was carried out using gram staining reaction and Fungi identification was carried out by macroscopic and microscopic examination. The media were prepared according to the manufacture’s instruction.The method is according to Glover et al.,(2005).

<p style="text-align: center;">Mbajiuka Chinedu S et al.,: Continental J. Biological Sciences 3: 63 - 74, 2010

RESULTS

MALTING

<p style="text-align: justify;">The sorghum grains used in this project work were dehulled washed and steeped overnight in tap water. They were spread out on a sac bag and kept for germination. The germination of the sorghum grains commenced after 12 h with about 50% of them germinating and the germination increased to about 99.5% after 24 h. At the end of the malting period which was four (4) days plumules attended to about 1.5cm to 2cm in height as shown in plate 1. Besides, there was a scanty growth of moulds on the malting sorghum grains.

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<p style="text-align: justify;">GROUND SUN-DRIED SORGHUM MALT

<p style="text-align: justify;">After the malting process, the germinated sorghum grains were sun-dried and ground into a fine powder. The ground malt releases a pleasant aroma and tasted sugary.

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<p style="text-align: justify;">FERMENTATION

<p style="text-align: justify;">After grinding of sorghum malt, the powder mixed with an adjunct (garri) was mashed with addition of water to get a possible extract or wort which was subjected for fermentation by inoculating with a starter culture as shown plate 2. After 12 h, there was a presence of bubbles which first started in the sample inoculated with Saccharomyces cerevisiae. As the fermentation progress, an alcoholic flavour

<p style="text-align: justify;">was observed from each of the samples. However, the following parameters were tasted at 12 h interval.

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<p style="text-align: justify;">DETERMINATION OF pH VALUES

<p style="text-align: justify;"> As the fermentation proceed. The pH of both inoculated fermenting samples decreases with time as shown in Figure 3.

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<p style="text-align: justify;">TEMPERATURE DETERMINATION

<p style="text-align: justify;">The temperature of the fermenting medium was fluctuating within room temperature which ranges from 28.70C to 300C with increase in fermentation period as presented in Table 3.

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<p style="text-align: justify;">TITRATABLE ACIDITY

<p style="text-align: justify;"> The titration of 0.1M NaoH in 2ml of the samples at phenolphthalein end point shows a gradual increase in the acid content of the fermenting medium with time. The concentration of the acid as shown in Figure 4 was calculated.

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<p style="text-align: justify;">REDUCING SUGARS

<p style="text-align: justify;">During the fermentation process, the levels of fermentable sugars were tasted using refractometer (Handheld model 1305) which was indicated by the appearance of blue color within the range. The percentage of sugar level was observed to be decreasing as the fermentation period progresses as shown in Figure 5.

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<p style="text-align: justify;">SPECIFIC GRAVITY

<p style="text-align: justify;">Specific gravity is a measure of the density of a liquid relative to water. It was decreasing as the fermentation period increases in both samples as illustrated in Figure 6.

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<p style="text-align: justify;">ALCOHOLIC CONTENT

<p style="text-align: justify;">The alcoholic content determined by comparing the specific gravity of the samples before and after fermentation shows that percentage alcoholic content of the samples was increasing with fermentation time but the sample inoculated with Saccharomyces cerevisiae which is 6.7% by volume was higher than that of sample inoculated with old brew which is 6.57% by volume after fermentation. Therefore the percent alcohol differs by 0.14%. This is illustrated in Figure 7.

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<p style="text-align: justify;">IDENTIFICATION OF ISOLATES

<p style="text-align: justify;">The serial dilutions of 10-3 of the natural sample cultured on nutrient agar and sabouraud dextrose agar to identify bacterial and fungal isolates under aerobic conditions shows the list of microorganisms represented in Table 4 and 5 respectively.

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<p style="margin-left: 72pt; text-align: justify; text-indent: -72pt;">TABLE 3:            CHANGES IN TEMPERATURES OF THE FERMENTING SAMPLES WITH TIME. Note: S.C = Saccharomyces cerevisiae, O.B = Old brew of burukutu

TABLE 4: SUSPECTED BACTERIA ISOLATES

TABLE 5: SUSPECTED FUNGI ISOLATES

DISCUSSION

<p style="text-align: justify;">Sorghum grains are an important fermentable cereal used to produce indigenous alcoholic, non-alcoholic beverages, baked products and animal feeds.

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<p style="text-align: justify;">Burukutu is one of the indigenous alcoholic beverages produced by the fermentation of malted sorghum grains within two (2) days. The process involves malting, sun-drying, grinding, mashing, fermentation; pasteurization and maturation as shown in Figure 1.

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<p style="text-align: justify;">During malting of sorghum grains, starch is hydrolyzed into fermentable sugars mainly by amylolytic organisms capable of hydrolysing starchy constituents (Michodjehoun-Mestres et al, 2005 and Achi, 1990). At the end of malting which is four (4) days, the plumule attends to about 1.5 to 2cm in height as similarly observed by Achi, (1990). The scanty growth of moulds observed on the malting grains may be contaminants from the banana leaf used to cover the sorghum grains to prevent dehydration (Norman et al, 1999).

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<p style="text-align: justify;">The release of pleasant aroma and sugary taste by the sun-dried grinded malt shows the effect of malting to improve flavour and conversion of starch to fermentable sugars.

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<p style="text-align: justify;">In the process of fermentation, the appearance of bubbles and release of an alcoholic flavour after 12 h signifies the commencement of fermentation with release of CO2 and formation of alcohol.

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<p style="text-align: justify;">The isolated bacteria such as Staphylococcus species, Enterobacter species, Streptococcus species and Fungi such as Aspergillus species, Candida species and Saccharomyces species as shown in Table 4 and 5 were suspected as those that directly or indirectly participate in the fermentation with natural inoculum of old brew as starter culture. This was in line with Kolawole et al, (2007). According to Mohammed et al.,(1999), Saccharomyces cerevisiae and other yeasts are responsible for the alcoholic fermentation and also contribute to the flavour and acceptability of the product in combination with Streptococcus lactis, Candida mycoderma and Lactobacillus species.

<p style="text-align: justify;">

<p style="text-align: justify;">However, the increase in titratable acidity leads to decrease in pH as fermentation time increases. But the sample inoculated with commercial yeasts which is Saccharomyces cerevisiae was faster than the natural one which was also confirmed with the work of Egemba and Etuk, (2007). This may suggest that the use of only Saccharomyces cerevisiae in sorghum fermentation to produce burukutu will encourages greater capability of producing acid than those of combined effort of those strains of organisms involved in natural fermentation. In addition, the increase in acidity with decrease in pH as fermentation proceed may eliminate or discourage the growth of most spoilage and pathogenic microorganisms that can not withstand such condition hence making the alcoholic beverage safer for consumption and also helps to increase the shelf life in combination with pasteurization by boiling.

<p style="text-align: justify;">

<p style="text-align: justify;">In case of reducing sugars, the percentage of fermentable sugars decrease sharply with the inoculation of Saccharomyces cerevisiae than the old brew with increase in fermentation period as shown in figure five. This may signify the effect of higher utilization of fermentable sugars by Saccharomyces cerevisiae than other strains of microorganisms isolated from the natural fermentation process. Besides, the decrease in specific gravity of the samples shows that some of the sugars have been converted into alcohol which is less dense than water. A similar observation was made by Egemba and Etuk, (2007).

<p style="text-align: justify;">Furthermore, the increase in percentage of alcohol in both fermentation medium also suggests the continuous activity of the microorganisms in conversion of glucose to alcohol as fermentation proceeds. At the end of fermentation, the percentage of alcohol in the sample inoculated with Saccharomyces cerevisiae was higher with 0.14% than the one inoculated with old brew as shown in figure 7. A similar result was gotten by Kolawole et al, (2007) but difference in final percentage of alcohol in the natural method carried out in the laboratory may be as a result of differences in quantity of sorghum flour used and the variety.

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<p style="text-align: justify;">After maturation, the increase in palatable taste and flavour which makes the burukutu more attractive for consumption shows the activity of microorganisms that dominate the beverage during the maturation as described by Achi, (2005) but more developed in the traditional method. The fermentation of Sorghum using only pure culture of palmwine yeasts (Saccharomyces cerevisiae) produced higher alcohol but not with similar aroma and flavour as in the traditional method. The aroma and flavour that makes burukutu attractive for consumption is present in the traditional method and is as a result of  the activities of many other microorganisms present and this conforms with the work of Achi (1990).The implication of this result is that since we are not only interested in the alcoholic production but also in the characteristic aroma and flavour of burukutu that makes it attractive for consumption, the use of a single pure culture as starter culture in the production of  burukutu using sorghum that could be acceptable is not feasible.

<p style="text-align: justify;">

<p style="text-align: justify;">CONCLUSION

<p style="text-align: justify;">This study reveals that fermentation rate were higher for the case of inoculation with commercial yeast (Saccharomyces cerevisiae), compared to inoculating with old brew of burukutu as a traditional method of brewing burukutu hence suggesting that the use of a starter culture Saccharomyces cerevisiae (Yeast) would ensure better fermentation rate with higher production of alcohol in burukutu.

<p style="text-align: justify;">

<p style="text-align: justify;">Our limitations included lack of funds to further our research and the unavailability of digital modern equipment for analysis which left us with the option of improvision sometimes.These limitations are characteristics of third world countries particularly African countries.

<p style="text-align: justify;">

<p style="text-align: center;">Mbajiuka Chinedu S et al.,: Continental J. Biological Sciences 3: 63 - 74, 2010

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<p style="text-align: justify;">However, since our intentions are to improve and modernize the method of burukutu production using sorghum,the aroma, the nutritional value and other qualities of burukutu beer were taken for granted.Therefore,there is the need to investigate these and as well as their respective shell life.The roles played by many other microorganisms isolated from the back drop be investigated. We also suggest that the use of a combination of pure cultures of Lactic acid bacteria and Saccharomyces cerevisiae as a starter culture in burukutu production using sorghum be investigated to see if it could bring out good aroma.

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<p style="text-align: justify;">Achi, O.K. (1990). Microbiology of “Obiolar”: A Nigerian fermented non-alcoholic beverage. Journal of Applied Bacteriology 69: 321-325.

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<p style="text-align: justify;">Achi, O.K. (2005). The potential for upgrading traditional fermented foods through Biotechnology. African Journal of Biotechnology 4(5): 375-380.

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<p style="text-align: justify;">Ahmed, S. ;Mahgoub, S.  and Babiker, B. (1996): Changes in Tannin and cyanide contents and diastic activity during germination and the effects of traditional processing on cyanide content of Sorghum cultivars. Food Chemistry 56: 159-162.

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<p style="text-align: justify;">Alais, C. and Linden, G. (1999) : Food Biochemistry. Aspen Publishers Inc. Gaithersbury,Maryland.USA. Pages 140-143.

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<p style="text-align: justify;">Booney, L. W. (2000): Properties of Sorghum grain and new development of possible significance to the brewing industry. Technical Quarterly 6:227-232.

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<p style="text-align: justify;">Bhrise,V.; Chavan,J. and Kadam,S.(1988):Effects of malting on proximate composition and in vitro protein and starch digestibility of Sorghum grain. ''Journal of Food Science Technology.25 (6):327-329. ''

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<p style="text-align: justify;">Chavan, J.K. and Kadam, S.S. (1989): Nutritional Improvement of Cereals by fermentation.In: Critical Reviews in Food Science and Nutrition 28:349-400.'' ''

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<p style="text-align: justify;">Egemba, K.C. and Etuk, V.E. (2007). A Knetic study of Burukutu Fermentation. Journal of Engineering and Applied Science 2(7): 1193-1198.

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<p style="text-align: justify;">Glover, R.L.K. ;Abaidoo, R.C. ;Jakobsen, M. and Jespersen, L. (2005) : Biodiversity of  Saccharomyces cerevisiae isolated from a surver of pito production sites in various parts of Ghana. Systematic and Applied Microbiology.28 (8):755-761.

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<p style="text-align: justify;">Hamad S.:Bocker,G.:Vogel,R.F. and Hammes, W.P.(1992) : Microbiological and Chemical analysis of fermented Sorghum dough for Kisra production. Applied Microbiology and Biotechnology.37 (6):370-376.

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<p style="text-align: justify;">Hassan,I.A.G. and El Tinay,A.H. (1995):Effect of fermentation on tannin content and in vitro protein and starch digestibilities of two sorghum cultivars. Food Chemistry 53(2):149-151

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<p style="text-align: justify;">Isabel, C.A.; Alexandra, N; Lola, F.D; Antonio, B. and Ivonne, D. (2005). Sorghum Fermentation followed by spectroscopic techniques. Food Chemistry 90:853-859.

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<p style="text-align: justify;">Kolawole, O.M; Kayode, R.M.O, and Akinduyo, B. (2007). Proximate and Microbial Analysis of Burukutu and Pito Produced in Illorin, Nigeria. African Journal of Biotechnology 6(5): 587-590.

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<p style="text-align: justify;">Michodjehoun-Mestres, L; Hounhouigan, D.J; Dossou, J. and Mestres, C. (2005). Physical, Chemical and Microbiological Changes during Natural Fermentation of “gowe”, a sprouted or non sprouted sorghum beverage from West Africa. African Journal of Biotechnology 4(6): 487-496.

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<p style="text-align: justify;">Mohammed, S.I. ;Steenson, L.R. and Kirleis, A.W. (1999) :Isolation and Characterization of microorganisms associated with the traditional Sorghum fermentation for production of Sudanese Kisra. ''Applied and Environmental Microbiology. ''57:2529-2533.

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<p style="text-align: justify;"> Norman, F.H.; Odunfa, S.A; Cherl-Ho, L. and Quintero-Ramirez, R. (1999). Fermented cereals; A global perspective. Food and Agricultural Organization (F.A.O) Bulletin No. 38.

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<p style="text-align: justify;">Nout, M.(1991). Ecology and accelerated natural lactic fermentation of sorghum-base infant food formulas. International Journal of Food Microbiology 12: 217-224.

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<p style="text-align: justify;">Ogbadu, L .J. ; Momo-Jimoh, A. and Ameh, J.B. (1997): Heat Treatment and Chemical preservatives and their effect on the keeping quality of Burukutu beer. ''World Journal of Microbiology and Biotechnology 13:121-125. ''

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<p style="text-align: justify;">Okafor, N. (1981). A scheme for the improvement of fermented foods of Africa, south of the Sahara in global impacts of applied microbiology. Academic press, London. Pp 61-69.

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<p style="text-align: justify;">Okorie, S.U. and Oke, E.N. (2003).Nutritive value of malted sorghum and fermented malted sorghum. Proceedings of the 27th NIFST Conference, Kano. Pp 55-56.

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<p style="text-align: justify;">Onwuka, G.I. (2005). Food Analysis and Instrumentation theory and practice. Naphthah prints Surulere, Lagos. Nigeria Pp 60-101.

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<p style="text-align: justify;">Prescott, L.M; Harley, J.P. and Klein, D.A.(2005). Microbiology (6ih ed) ;.McGraw-Hill.New York.Pages 47-978.

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<p style="text-align: justify;">Richard, K.R; Carl, A.B. and Pradip, D.P.(1999). Encyclopedia of Food Microbiology; 3:2306-2322..

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<p style="text-align: justify;">Sukki, A; Lonner, C; Marklinder, I.; Johansson, M. and Molin, G. (1994). Starter cultures for the production of ogi, a fermented infant food from maize and sorghum. Chemistry, Microbiology Technology Lebensmittel 16(12): 29-33.

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<p style="text-align: justify;">Sulma, I.M; Larry, R.S. and Allen, W.K. (1991). Isolation and characterization of Microorganisms Associated with the traditional Sorghum Fermentation for production of Sudanese Kisra. Applied Journal of Environmental Microbiology 57(9):2529-2533. 

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<p style="text-align: justify;">Togo,  C.A.; Feresu, S. B. and Mutukumira, A.N.(2002):Identification of lactic acid Bacteria from Opaque beer for potential use as a starter culture. Journal of Food Technology 7:93-97.

<p style="text-align: justify;">

<p style="text-align: justify;">Tomkins, A; Alnwick, D. and Haggerly, P. (1988). Fermented Food for Improving Child feeding in eastern and Southern Africa:  A review, household level Food technology. Ottawa International Development Research Centre Pp 136-167.

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<p style="text-align: center;">Mbajiuka Chinedu S et al.,: Continental J. Biological Sciences 3: 63 - 74, 2010

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<p style="text-align: justify;">Received for Publication: 09/06/2010

<p style="text-align: justify;">Accepted for Publication: 21/07/2010

<p style="text-align: justify;">

<p style="text-align: justify;">Corresponding author:

Omeh Yusuf S

Department of Biochemistry

Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria.

E-mail:  [mailto:nduk41@yahoo.com nduk41@yahoo.com]

<p style="margin-right: -36pt; text-align: justify;">Continental J. Biological Sciences 3: 75 - 79, 2010                                                     ISSN: 2141 - 4122

<p style="text-align: justify;">© Wilolud Journals, 2010                                                                                 http://www.wiloludjournal.com

<p style="text-align: justify;">

<p style="text-align: center;">INVESTIGATION OF MICROBES ASSOCIATED WITH THREE VEGETABLES

<p style="text-align: center;">''Talinum triangulares (Gbure), Crassalephalum crepidoides (Ebolo), Ocimum gratissium (Efinrin) ''

<p style="text-align: center;">

<p style="text-align: center;">Yahaya S.A and Ayeni K. E

<p style="text-align: center;">Science Technology Department Federal Polytechnic, P.M.B 420, Offa.

<p style="text-align: center;">

<p style="margin: 0cm 17pt 0.0001pt;">ABSTRACT

<p style="margin: 0cm 17pt 0.0001pt; text-align: justify;">Occurrence of some microbes was investigated in common vegetables such as Talinum trangularea (Gbure);Crassocephalum crepidoides (Ebolo); Ocimum gratissium (Efinrin) The vegetable used were obtained within Offa Community (Owode Market).Two bacterial species were isolated from the vegetables such as Staphylococcus spp, Escherichia coli. Efforts were made to identify the source of the microbes and relevant recommendations were made.

<p style="margin: 0cm 17pt 0.0001pt; text-align: justify;">

<p style="margin: 0cm 17pt 0.0001pt; text-align: justify;">KEYWORDS: Microbes, Staphylococcus, anti oxidants vitamins, autoclave, sterilization.

INTRODUCTION

<p style="text-align: justify;">In many African homes, vegetables form major parts of food. Vegetables are often used in the preparation of soup for human consumption. In certain parts of West Africa, the leaves are often boiled before using it for soup. It was reported by Tindal (1983) that many species probably originated in the Andean region of South America and Mexico and are widely distributed throughout most tropical areas. James (1996) reported that vegetable are excellent source of minerals and vitamins. It was also asserted that vitamins supplied through the consumption of vegetables is very high and of great value, this is because vitamins C, E and A contained in vegetables are believed to defend the body against free radical and therefore, they are termed antioxidants. These vitamins are especially abundant in fruits and vegetables. According to Stone (1995) they are essential for normal functioning of the body and usually obtained from the consumption of vegetables in the year 1665.

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<p style="text-align: justify;">Vegetables, according to Oria and Rafiu (1998) is a branch of horticulture relating to the production of a member of herbaceous plants or plant part.

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<p style="text-align: justify;">METHODS of MEDIA PREPARATION

<p style="text-align: justify;">The types of media used are Nutrient agar and the agars are prepared as follows. 3g of Nutrients agar was weighed on the weighing balance and was dissolved in 300cm3 of distilled water in a conical-flask. The flask was then shaken until the agar dissolved, the flask was then corked with cotton wool and an aluminum foil avoid to contamination. The prepared media were then placed in the autoclave for sterilization at the temperature of 121oC for 15min.

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<p style="text-align: justify;">The Petri-dishes are washed and placed invertly in the oven for sterilization; the Petri-dishes are covered with their lid in order to avoid air contamination and the Petri-dishes are then brought out of the oven, the prepared media was then pored gently into the dishes through one side of Petri-dishes that is gently opened. The dishes containing the media were then allowed to cool at room temperature in order to allow for solidification of the media.

<p style="text-align: justify;">

<p style="text-align: justify;">METHOD OF PREPARATION

<p style="text-align: justify;">The stock samples were prepared by washing the vegetable in the distilled water. This was done by slicing the vegetables into a clean piece of paper and 10g of vegetables was then weighed into a conical flask and 100cm3 of distilled water was added to it.

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<p style="text-align: justify;">METHODS of INOCULATION

<p style="text-align: justify;">The work bench was swambed with acetone for sterility. The inoculating loop was sterilized by placing it in the flame for 2minutes in order to ensure thorough sterilization (burnt red) the loop was then allowed to cool, then used for inoculating the prepared sample into the Petri-dishes containing the solidified nutrient-agar. This nutrient agar allowed the growth of the bacteria.

<p style="text-align: center;">Yahaya S.A and Ayeni K. E: Continental J. Biological Sciences 3: 75 - 79, 2010

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<p style="text-align: justify;">After each inoculating, the loop was sterilized by burning it red before using it for another inoculating and all this were done in the inoculating room also in the presence of flame in order to avoid air contamination of the media. The streaked dishes were then incubated at the temperature of 37oC for 24.

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<p style="text-align: justify;">BACTERIAL CELL IDENTIFICATION TECHNIQUES

<p style="text-align: justify;">There are so many techniques for bacteria identification, but the techniques used in this project are:

<p style="margin-left: 39.75pt; text-align: justify; text-indent: -21.75pt;">(a)      gram staining test

<p style="margin-left: 39.75pt; text-align: justify; text-indent: -21.75pt;">(b)      morphological observation

<p style="text-align: justify;">

<p style="text-align: justify;">METHOD OF STAINING

<p style="text-align: justify;">This was done by making a thin smear of the cell suspension on a clean glass slide and fixed with chemical fixative that is by passing the slice containing the smear over blue flame, then the slide was flooded with crystal violet for 30-60s, then it was flooded with dilute solution of iodine, which decreases the solubility of purple dye. After that it was rinsed off in gentle tap water, then 95% of alcohol was applied which readily removes purple dye-iodine complexes from bacterial, it was flooded with red counter stain that is straining for 30-60s, it was rinsed off in gentle tap water and blotted dry with blotting paper, it was finally examined under oil immersion lens.

<p style="text-align: justify;">

<p style="text-align: justify;">CALATALASE TEST

<p style="text-align: justify;">With the aid of sterile loop, a drop of Hydrogen peroxide was put on the sterile slide and loopful of the culture (organism) was picked and emulsified.

<p style="text-align: justify;">

<p style="text-align: justify;">Effervescence was absent which indicates catalase negative Escherishai coli.

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<p style="text-align: justify;">MOTILITY TEST

<p style="text-align: justify;">An innoculum of the test organism that is Staphyloccus spp was introduced into 2mls of peptone water in the test tube bottle then incubated at temperature of 37oC for 24h then, the test tube was shaken thoroughly for uniform distribution of the test organism in the tube. Also one drop of it was placed on the slide free of grease with the aid of a Pasteur pipette and a cover slide was gently placed on it.

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<p style="text-align: justify;">Finally, it was observed under the microscope at magnification of X40, in which there was no motility observed.

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<p style="text-align: justify;">This indicated that presence of staphylococcus spp because is a non motile gram positive organism.

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<p style="text-align: justify;">RESULTS

<p style="text-align: justify;">The organism listed was isolated from the vegetable under which they were listed.

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<p style="text-align: justify;">''Talinum traiangularea                                     Staphylococci Spp ''

<p style="text-align: justify;">'' ''

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<p style="text-align: justify;">Mg 4000                                                                                                               Cocci in clusters

<p style="text-align: justify;">Fig 1: Staphylococci spp present in Talinum Triangalanes.

<p style="text-align: justify;">

<p style="text-align: justify;">'' ''

<p style="text-align: center;">Yahaya S.A and Ayeni K. E: Continental J. Biological Sciences 3: 75 - 79, 2010

<p style="text-align: justify;">'' ''

<p style="text-align: justify;">'' ''

<p style="text-align: justify;">''Crassocephalum crepidoides                                          Escherichia Coli ''

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<p style="text-align: justify;">Mg x 4000                                                                                  Red Live Bacteria

<p style="text-align: justify;">Fig: 2 Escherichia coli present in crassocephalum crepidoides

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<p style="text-align: justify;">''Ocimum gratissium                                                  Staphylococci Spp ''

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<p style="text-align: justify;">Mg 4,000                                                                                                              Cocci in cluster

<p style="text-align: justify;">Fig 3: Staphylococci spp present in ocimumgratissium

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<p style="text-align: justify;">Table 1: GRAM TEST TABLE <p style="text-align: center;">Yahaya S.A and Ayeni K. E: Continental J. Biological Sciences 3: 75 - 79, 2010

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<p style="text-align: justify;">MOTILITY TEST

<p style="text-align: justify;">Motility test showed that

<p style="text-align: justify;">Specimen A is Staphylococcus spp

<p style="text-align: justify;">Specimen B is Escherichia coli

<p style="text-align: justify;">Specimen C Staphylococcus spp

<p style="text-align: justify;">

<p style="text-align: justify;">CATALASE TEST

<p style="text-align: justify;">From the Catalase test

<p style="text-align: justify;">Specimen A is Catalase Positive

<p style="text-align: justify;">Specimen B is Catalase Negative

<p style="text-align: justify;">Specimen C is Catalase Positive

<p style="text-align: justify;">

<p style="text-align: justify;">DISCUSSION AND RECOMMENDATION

<p style="text-align: justify;">It was discovered that the dungs of the animals that has been infected by these microbes which are used in the planting of these vegetable contribute mainly to the contamination of the vegetables. The dump of the animals which serves as manure for the former serves as sources of the microbes on the vegetables. It was also discovered that most the river water used in irrigating the vegetables beds contain most of these microbes as a result of the human waste that has been deposited in them. Microbes that are also present in the soil also contribute to the contamination of the vegetables.

<p style="text-align: justify;">

<p style="text-align: justify;">It was also found that improper care of the vegetables in the market causes the presence of some of these microbes on them.

<p style="text-align: justify;">

<p style="text-align: justify;">The vegetables were just left on bear ground by the road side which is very unhygienic for human consumption.

<p style="text-align: justify;">Staphylococcus spp is one of the microbes that is causing vegetable contamination. Ketchum (1998) said that most microbial infection is caused by Staphylococcus spp. He also made the following discoveries: <p style="text-align: justify;">
 * That disease cause by Staphylococcus spp are localized pyogenic infection of the skin such as boil and wound infection.
 * It causes systemic infection that can lead to pneumonia.
 * Toxin is produced while the microbes are growing on the vegetable.

<p style="text-align: justify;">James 1996 asserted that Staphylococcus spp are present in the nasal cavity of an infected man as well as on the skin of the animal that feed on contaminated vegetables.

<p style="text-align: justify;">

<p style="text-align: justify;">Watson and Jewar (1993) reported that Diplococcus spp causes variety of infective disease. It is a causative agent of melioodisis disease of man. Bacillus spp is another microbe found on the vegetable.

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<p style="text-align: justify;">Ketchum (1998) reported that it persists for a long time in contaminated field of vegetable and man consume that toxins produced by microbes on the vegetables if not properly sterile.

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<p style="text-align: justify;">RECOMMENDATION

<p style="text-align: justify;">With these advantages; vegetables have gotten many African homes by using them in the preparation of soup for human consumption, I hereby recommend that there should be enough provision for disposal of waste such as public toilet by the government instead of using them as marine for growing vegetables. The government should also make enough provision for the supply of fertilizers to the farmers.

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<p style="text-align: justify;">REFERENCES

<p style="margin-left: 36pt; text-align: justify; text-indent: -36pt;">James M. Jay (1996): Studies in Social and Hygiene, Published by CBS Publishers.

<p style="text-align: justify;">

<p style="text-align: justify;">Oria M. and J. Raffiu (1998) Social in Social Biology, Published by Data Print

<p style="text-align: justify;">

<p style="text-align: justify;">Ketchum P. A (1998): Concept of Applied Biology, Published by Data Print

<p style="text-align: center;">Yahaya S.A and Ayeni K. E: Continental J. Biological Sciences 3: 75 - 79, 2010

<p style="text-align: justify;">

<p style="text-align: justify;">

<p style="text-align: justify;">Stone R.H (1995) New Biology for West African School, University of Ibadan, Published by Spottisoode Ltd

<p style="margin-left: 36pt; text-align: justify; text-indent: -36pt;">

<p style="margin-left: 36pt; text-align: justify; text-indent: -36pt;">Tindal HD (1993): The Scientific Principles of Crop Protection, University of Agriculture, Published by

<p style="margin-left: 36pt; text-align: justify; text-indent: -36pt;">Blanford Press Ltd.

<p style="margin-left: 36pt; text-align: justify; text-indent: -36pt;">

<p style="margin-left: 36pt; text-align: justify; text-indent: -36pt;">Towski Gewar and Watson H.A (1993) Common Vegetables Plantation, Published by CBS Publishers and

<p style="margin-left: 36pt; text-align: justify; text-indent: -36pt;">Distributors.

<p style="text-align: justify;">Received for Publication: 09/06/2010

<p style="text-align: justify;">Accepted for Publication: 21/07/2010

<p style="text-align: justify;">

<p style="text-align: justify;">Corresponding author

Ayeni K. E

Science Technology Department Federal Polytechnic, P.M.B 420, Offa.

E-MAIL:kikeyink@yahoo.com

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