Evaluation of Nutritive Values and Detection of Some Genes Encoding Therapeutic Proteins in Four Mushrooms

Evaluation of Nutritive Values and Detection of Some Genes Encoding Therapeutic Proteins in Four Mushrooms

A mushroom is the soft, asexual reproductive body of a fungus, occurring on its food source or near the surface of the ground (Chang and Miles, 1992). Its nature makes it easy to be perceive with the eyes and hand picked. Toadstool belong to sub-division Basidiomycotina and Ascomycotina (Dickson and Lucas, 1982), which lack chlorophyll, the green colouring matter that many plants use to make their food.

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They grow on lifeless and decomposing biological material. From these decomposing materials, they take in their nutrients through these substrates with the help of very fine thread like structures (mycelium) which penetrate into substratum and generally not visible on the surface (Ibe et al., 2013). The mycelium grows profusely forming the reproductive structures generally known as fruiting body or mushroom which emerge from the substrate (Ibe et al., 2013).

Some mushrooms are edible while some are not. Agaricus, Pleurotus and Volvariella species are eaten all over the world and tropical countries, in particular (Mahendra et al., 2005). Nwokolo (1987), reported that there are about 14,000 mushroom species in existence and that about 50% of these mushroom species are edible. To date, barely approximately 150 varieties are tentatively cultivated, approximately 100 varieties are economically produced; approximately 40 varieties are commercially cultivated while about 10 varieties are produced at industrial scale in some countries (Tewari, 2005).

Nowadays, mushrooms have continued to generate much interest mainly as food (Chang, 1980), and as medicine to cure diseases (Rambelli and Menini, 1983; Oei, 1991; Bushwell and Chang, 1993). The rising concerns in the use of mushrooms as food come from their medicinal and nutritional potential. Literature has made it known that mushrooms found in the tropical region are rich in protein, minerals and vitamins. The nutritional component of mushrooms more especially its amino acid and total protein values are reported coming two times higher than vegetables and four times greater when compared with oranges (Bano, 1993) and considerably above the value of protein in wheat (Aletor, 1990). Chang and Miles (1989) reported that the quantity of crude protein recorded in mushrooms rate only below animal meats, although well above most other edible materials, along with milk, which is an animal product. According to these authors, mushroom protein contains all the essential amino acids required by man. It is not surprising therefore to associate the growing demand for mushrooms with the increasing rise in the unit costs of the conventional sources of animal proteins such as beef, pork, chicken and fish (Aletor 1995; Okwule and Odunze, 2004a).
Mushrooms contain detectable amount of crude fibers. Despite limited facts exists on the Total Dietary Fibre (TDF) content of mushroom. The crude fibre contents values stated by many authors, propose that mushrooms are promising point of supply of dietary fibers (Crisan and Sands, 1978; kurasawa et al., 1982). According to Oso (1977), Okwulehie and Odunze (2004a), mushrooms in most cases hold low fat and oil. On account of the low content of fat and oil in mushroom, they are suggested as good food supplement for persons being treated for heart problems. Studies have also shown that mushrooms are rich in vitamins C, B1, B2, B3 and vitamins D, (Bano and Rajarathnam, 1988).

Mushrooms have been found to be in possession medicinal potential. Bushwell and Chang (1993) reported that mushrooms have anti-cancer and anti-cholesterol effects. The considerable pharmacological activities of mushrooms make them valuable in pharmaceutical industries for development of drugs. Most bioactive compounds which play important function in human and animal physiology have been found abundantly in mushrooms. According to Okwulehie and Odunze (2004b), Russula spices, Pleurotus squarrosulus and Auricularia auricular, all contain appreciable amounts of alkaloids, phenols, saponins and flavonoids which are believed to contribute to the pharmacological activities of mushrooms.
The health embellishing feature of mushrooms also ascribed with existence of other bioactive substances like glycolipids derived from shikimic acid, aromatic phenols, etc (Wasser and Weis, 1999). Good numbers of these compounds gotten from mushrooms and are called biological response modifiers (BRM) which are substances that prompt an individual body react to foreign bodies (Wasser and Weis, 1999). Naturally human body is known to produce these substances in limited amount therefore, externally derived quantity through food or dietary supplements are required (Wasser and Weis, 1999). Mushroom nutriceuticals may finally be the beginning of this external supply on account of these; edible mushrooms have been reported to be safe for human consumption with no side effects (Mizuno, 1999).

This study was aimed at describing the nutritive qualities of four edible mushrooms indigenous to Abakaliki of Ebonyi State with a view to providing useful information for selecting nutrient-rich mushroom species for artificial cultivation programme, because rapid deforestation is about sending these valuable food sources into extinction in the area.

Statement of the Research Problem
The rising interest in the consumption of mushrooms as food stems from their nutritional and therapeutic values. Literatures have reported that mushrooms are rich in protein, minerals and vitamins. The health improving properties of mushrooms have also been ascribed to the presence of some therapeutic proteins. However, almost all the information about the nutritive and medicinal properties of mushrooms from other parts of the world and there are little or no reports on mushrooms from Abakaliki. Moreover, reports on genes encoding medicinal proteins in mushrooms are generally scares and completely unavailable in Nigeria. Hence, this work evaluated the nutritive and assessed the presence of gene encoding some therapeutic proteins in mushrooms from Abakaliki in Ebonyi State.

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Aim and Objectives of the Study
The aim of this study was to evaluate the nutritive and medicinal values of four different mushroom species in Abakaliki area of Ebonyi State while the specific objectives were;
(1) To determine and compare the proximate, mineral and vitamin compositions of four common mushroom species indigenous to Abakaliki.

(2) To determine and compare the amino acid contents of the four mushrooms.

(3) To determine and compare the phytochemical contents of the mushrooms as part of the assessment of its anti nutritional content.

(4) To determine the presence of genes encoding Ostreolysin A (Plo-5), Ling Zhi-8 immunomodulatory protein (LZ-8), Yun-Zhi protein (YZP), Marmorin and Hypsin as an indirect assessment of their medicinal values.

Justification of the Study
This work is an effort to evaluate and compare the nutritive parameters and detect genes encoding therapeutic proteins in four mushrooms in Abakaliki. The work involved determination of the proximate, amino acids, vitamins, minerals, phytochemical parameters and the use of PCR technique to detect some genes encoding therapeutic proteins on the four mushrooms in Abakaliki. The result of this study can form the basis for developing a biotechnology for commercial production of important protein drugs (e.g. anti-cancer and anti-HIV drugs) through isolation of therapeutic protein genes from mushrooms and cloning such genes into fast growing microbial cells like E. coli for mass production of the protein drugs.

Scope and Limitation of the Study
This study covered the common edible mushrooms found in Abakaliki, Ebonyi State. A total of four of the edible mushrooms were characterized and assayed for the presence of genes encoding Plo5 ostreolysin A, Ling Zhi-8 immunomodulatory protein, Yun-Zhi protein, Marmorin and Hypsin as an indirect assessment of their medicinal values. The evaluated nutritional parameters include proximate parameters, amino acid profiles, eight mineral content, vitamins and phytochemical content. The results obtained were compared among the four mushrooms and with those other published works. The study does not involve cultivation of the mushrooms and the use of their extracts to treat any disease condition.

Significance of the Study
Evaluation of nutritive values and detection of some genes encoding therapeutic proteins in four mushrooms in Abakaliki are important because consumption of these edible mushrooms species in the region is on the increase with little or no information on their nutritional profiles and medicinal values. Therefore, the results of this study would be important in filling this information gap with a view to providing information useful for selecting nutrient-rich mushroom species for possible artificial cultivation programme, since rapid deforestation and changing climate are almost sending these important food resources into extinction in the region. Moreover, a successful identification of an important therapeutic protein gene in the mushrooms would pave way to development of a biotechnology for commercial production of the protein drug from microorganisms or even large plants or animals through genetic engineering.

CHAPTER TWO
LITERATURE REVIEW

HISTORICAL BACKGROUND OF MUSHROOM
In some years ago, use of economically grown mushrooms in the United State has been in the increase ranging from 750 million pound range. Even though completely ordinary in this day, mushrooms in the olden days have been more in the rule of illusion and attitude. Egyptian Pharaohs constrained mushrooms for royal families, daunting the plebeian to feed on them plus the Romans elapse order officially making mushrooms as “Food of the Gods”.
Mushroom production can be drawn to about the early 1600s in Europe however it was not just before the 18th century in France that modernized mushroom cultivation approach start to form. Outside of Paris, mushroom farming was carried out on limestone quarries where they produced “champignons de Paris”. Even though cultivation of mushrooms was done on these abandon quarries, modernized mushroom growing approach has come a long way.
Modernized mushroom cultivation started in Kennett Square, Pennsylvania in the year1896. At early stage, mushrooms were cultivated as a side crop to flower production, using the dark spaces underneath potting tables. As the request for edible mushroom raised, special “houses” were made to curb the precise environmental conditions essential for mushroom cultivation.

Even with these approaches, mushrooms cultivation were still not as known available as these present day. Facts has reveal that mushroom growing was still restricted by the temperature and humidity of season, mushrooms were planted in the raining season and gathered in winter and spring. Fresh mushrooms were, therefore, hard to come by in the summer. Present growing method has been able to solve many problems associated with the cultivation process.
Modern mushroom farmer grows their product in modern production facilities. With the opportunity of air conditioning system, that mushroom farmers of the past did not have, presently mushrooms can be cultivated all year round. Although technology has made commercial mushroom production easier, it is still not an easy process as one might think. Mushroom cultivation for commercial purpose begins in an antiseptic condition of a laboratory, where the mushroom spores are created. At this point, the mushroom farmer must take good care of all the environmental conditions.
Modernized mushroom farming currently is among the most demanding agricultural processes presently. With the entire task on mushroom farmers in the cultivation of mushroom, both in the past and present, we are all able to appreciate mushrooms anytime.

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SCIENTIFIC CLASSIFICATION OF MUSHROOM
Scientific Classification of Mushroom
Classification for mushroom:

Kingdom: Fungi

Phylum: Basidiomycota

Class: Agaricomycetes

Order: Agaricales; Auriculariales; Polyporales

Family: Pleurotaceae; Auriculariaceae; Schizophyllaceae; Polyporaceae

Genus: Pleurotus; Auricularia; Schizophyllum; Lentinus

Species: P.ostreatus; A.auricula-judae; S.commune; L.connatus

Binomial name: Pleurotus ostreatus; Auricularia auricula-judae; Schizophyllum commune (Schrot, 1888).

Plate 1 . Photographs of mushroom species used in the study. A is Pleurotus ostreatus, B is

ECONOMIC IMPORTANCE OF MUSHROOM
Nutritional importance:
Mushrooms are the perfect food for everyone due to their nutritional content. They are low in calories, are fat free, cholesterol free, and have very low levels of sugar and salt. They provide a valuable source of dietary fibre, as well as several vitamins and minerals.

Carbohydrates
The carbohydrate content of mushrooms represents the bulk of fruiting bodies accounting for 50 to 65% on dry weight basis (Wani et al., 2010) with monosaccharide resulting to around 10% Wani et al., (2010). Mannitol in mushroom has been reported to contain approximately 81% of the total refined sugars, hence it is effective (Wani et al., 2010). Also Wani et al., 2010 recorded that fresh mushrooms have approximately 0.8% for mannitol, with reducing sugar resulting to 0.3%, glycogen stated for 1.0% and 0.9% for hemicellose.

Proteins
Protein is a valuable component of dry matter of mushrooms (Wani et al., 2010, Bilal et al., 2010). The protein value in mushrooms has been reported according to Lintzel (1941) to be high ranging from 72 to 83%. The nutritional content of mushrooms more especially its protein content relyed ahead of the substratum, extent of pileus, harvesting period and the mushrooms species (Wani et al., 2010). Research on mushrooms has revealed that total protein content found in mushrooms varies from one species to the other. Wani et al., (2010) showed that protein recorded in portobello mushroom varies from 35 to 44% on the dry weight basis. Wani et al., 2010 reported that the quantity of crude protein found in mushrooms rank slightly lower to animal meats but far above most other foods. On a dry weight basis, mushrooms has been stated to account about 19 to 35% proteins as compared to about 6.8% in oryza glaberrima, 11.9% in wheat, 40.1% in soybean and 8.9% in corn ( Wani et al., 2010).
Afiukwa et al., (2015), Verma et al., (1987) and Wani et al., (2010) indicated that toadstool are important for vegetarian because they have some important amino acids which the body need since it can not produce it on its own in large quantity. Bano and Rajarathnam, (1988) showed that the edibility of oyster toadstool proteins is approximately the same when compared with plants (90%) while that of animal protein is 98% (Wani et al.,2010).. Mushrooms has been reported by Hayes and Haddad, (1976) hold all the important amino acids needed by an adult.

Fats
Fat content found in toadstool as recorded by literature is less compared to other proximate parameter. Findings have shown that fats recorded in mushrooms are usually unsaturated fatty acids. Wani et al., (2010) stated that toadstool (mushroom) is mainly good in linolenic acids which are important fatty acid. Orgundana and Fagade (1981) reported about 4.481% fats on dry weight basis on different toadstool species. Wani et al., (2010) investigated on the fibre content noticed on different mushroom species.

Vitamins
Mushrooms have been known to be among the good point of supply of vitamins more mostly B vitamins (Wani et al., 2010). The amount of vitamins recorded in mushrooms has been reported by Wani et al., (2010). Manning (1985) recorded a detailed account of vitamin content on different mushrooms species in addition of some vegetables. Reports according to Wani et al., (2010) stated that mushrooms also contain little amount of vitamin C, A and E.

Mineral constituents
Mushrooms are known to contain appreciable amount of mineral elements. Most mineral constituents recorded in mushrooms are sodium, phosphorus, calcium, potassium, magnesium and elements like copper, manganese, zinc, cadmium result to little constituents (Wani et al., 2010). Potassium, sodium, calcium, magnesium and phosphorus makes up approximately 49 to 65% of the total ash constituents on mushrooms (Wani et al., 2010) while potassium only form about 45% of total ash. Investigation credited to Wani et al., (2010) reported that the sodium and potassium content in portobello mushroom specie resulting to about 300 and 27.3 parts per million respectively. Investigation has revealed mushrooms can be used for bioremediation as literature shows that the can accrue poisonous metals like lead, copper, cadmium, mercury etc (Wani et al., 2010). The amount minerals noticed on wild edible mushrooms was observed to be greater compared to ones grown artificially (Wani et al., 2010).

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Medicinal importance:
Medical roles are another benefit of mushrooms. Their medicinal values have been useful since the ascent days. Mushrooms were used by first century Greek physician Dioscorides in the treatment of tuberculosis as reported by Wani et al., 2010. Even though mushrooms were reported to have important medicinal value in China since 100 A.D. (Wani et al., 2010), scientists were able to find out the main effective ingredient of mushrooms that are health promoting in 1960 (Wani et al., 2010). Scientific have reveal the use of mushrooms in health management for managing uncomplicated and ancient ailment like rashes to current day complicated and universal disease like cancer. Finding has shown that mushrooms contain anti-tumor, anti-cholesterol, and anti-allergic value with liquid extracts from oyster justified well in renal complications (Wani et al., 2010). Wani et al., (2010) recorded that mushrooms can be use in curing some ailments such as liver disease, skin diseases, cardiac ailments, diarrhea, cold, rheumatoid arthritis, anesthesia, diaphoretic, dysentery, gall bladder diseases, etc. Some mushroom species like Ganoderma lucidum has been widely stated in lowering the concentration of serum cholesterol hypertensive patients and reducing blood pressure in such patient (Wani et al., 2010). Lentinus edodus has been used to improve energy and as an anti aging promoter (Wani et al., 2010). Lentinan sulphates gotten from Lentinus species restrict HIV (Gareth, 1990). Drugs produced from mushroom have little or no side effects (Sagakami et al., 1991). Some mushrooms species like puffballs have been reported in use in treating urinary infections (Wani et al., 2010). Extract gotten from maitake not current proved to destroy HIV and improve the function of T-helper cells (Wani et al., 2010). Ganoderma lucidum nutriceuticals have shown encouraging antiviral result like, anti-HIV and anti-hepatitis B (Wani et al., 2010). Mushrooms function as biological response modifiers by improving the certain factors and removing the unfavorable factors from the body and thus they are referred as the fourth active stage of the common cancer management. G. lucidium specie has been proven to function as an antidiabetic and anti-imatory agent (Teow, 1997). Wani et al., (2010) reported that some tribes in India has successively used different mushroom species like, maitake, lion’s mane, splitgill, enoki and reishi for treating cancer, treating joint pains. The medicinal value of mushrooms varies from species. For instance, maitake, shiitake and Ganoderma Lucidum species has been reported for lowering stress. Yamabushitake has reportedly been ideal for improving remembrance; Ganoderma Lucidum used to cause sleep to happen, Cordyceps sinensis specie a traditional Chinese Medicinal mushroom has been touted for physical endurance and sexual performance. Auricularia species has reportedly been used in the distance past for treating piles, stomach pain, ulcers, reflux, and constipation (Wani et al., 2010). Oyster mushroom (Pleurotus tuber-regium and P. ostreatus) has been reportedly a good source for managing high blood pressure, asthma, colds and lowering concentration of serum cholesterol in man (Wani et al., 2010).

Antioxidant activity
Antioxidants are molecule that protects or prevent some types of cells from damage caused by free radicals (Wani et al., 2010). Free radicals are very active chemicals that have the ability to destroy cells. These free radicals has the ability through any means destroy any part of the body, which include proteins, deoxyribonucleic acid, glucose including DNA alteration in addition to malignant growth(Wani et al., 2010). The nascent oxygen (a short-lived free radical of oxygen) is captured through catalyst such as superoxide dismutase, catalase and glutathione peroxidase (Wani et al., 2010). Increased presences of these free radicals always result to oxidative stress. The antioxidants are known for their fight against free radicals in the body, mushrooms are reported better sources of these molecules (Oyetayo et al., 2007; Wani et al., 2010). Sizofiran (a member of the β-(1-3) glucan family) from split gill is active against head and neck tumor (Bilal et al., 2010). Kim and kim (1999) and Wani et al., (2010) stated that extracts gotten from mushroom contains DNA protecting features.

Materials and Methods

Materials
All reagents and chemicals used in this research work were of analytical grade and all equipment used was of standard grade.

Sample collection and preparation:
The samples used for this work were four different species of mushrooms. They were sourced from rural areas of Abakaliki and were identified by a mycologist Dr Amenh of department of Biotechnology, Enugu State University of Science and Technology.
The fresh mushrooms were sun- dried for 3days and then ground into powder using laboratory mortar and piston. The powdered samples were used for the analyses.

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