Review of Literature

REVIEW OF LITERATURE

Mushrooms are known for their rich source of biologically active

compounds which offer protection to human body in a natural way.

Mushrooms are rich in dietary fibers, minerals, vitamins, and low in fat. They

are widely consumed as an edible and medicinal resource. Studies have found

that, some species of mushrooms are having therapeutic properties such as

mycochemicals, antioxidants, antimicrobial and hydrolytic enzyme effects.

Since less information with respect of Hypsizygus ulmarius was observed, the

reliable literature survey with reference to Pleurotus and other related

mushroom species were recorded.

2.1 SCREENING OF MUSHROOMS FOR MYCOCHEMICAL PROPERTIES

2.1.1 PHENOLICS

2.1.1.1 Definition and Description

Phenolic compounds are secondary metabolites that constitute one of the

most common and widespread groups of substances (Whiting, 2001; Bennick

2002; Apak et al. 2007).The term "phenolic" or "polyphenol" can be precisely

defined as chemical substance which possesses an aromatic ring bearing one

(phenol) or more (polyphenol) hydroxyl substituents, including functional

derivatives. Natural phenolic compounds accumulate as end-products from the

shikimate and acetate pathways and can range from relatively simple molecules

(phenolic acids, phenylpropanoids,) to highly polymerized compounds (lignins,

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melanins, tannins) (Carey, 2003). More than 8000 polyphenolic compounds

have been identified in various plant species. They have specific health effects,

even though they are non nutritive compounds (Nijveldt et al., 2001).

2.1.1.2 Classification

Phenolic compounds are classified by the number and arrangement of their

carbon atoms in their structure and are commonly found conjugated to sugars

and organic acids (O connel and fox, 2001).

C6 group - which includes simple phenols and Benzoquinones

C6–Cn group - Phenolic acids and hydroxycinnamic acid derivatives

are included in this group.
C6 Cn C6 group - the largest group of phenolic compounds that
includes flavonoids.
(C6 C3)n group - this group includes lignins and lignans with
intermediate and high molecular weight phenolic compounds.
Tannins - are high molecular weight phenols and are classified in to
two main categories as hydrolyzable and condensed tannis.
Hydrolysable tannins are esters of gallic acid, whereas condensed
tannins are polymers of polyhydroxy flavan -3-ol monomers.

2.1.2 FLAVONOIDS

2.1.2.1 Definition and Description

Flavonoids are low molecular weight (Fernandez et al., 2006) bioactive

polyphenols which play a vital role in photosynthesis (Cushnie et al., 2005).

The term "flavonoid" was discovered in 1936, by Hungarian scientist Albert

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Szent from the peels of lemons. Flavonoids are widely distributed among the

plant and fungi kingdom such as vegetables, fruits, seeds, stem, tea, wine and

mushrooms. These are an integral part of diet (Prey et al., 2003; Fernandez

et al., 2006). The average intake of flavonols and flavones was found to be

23 mg/day, among which, flavonol quercetin contributed 16 mg/day (Heim

et al, 2002).

2.1.2.2 Classification

Flavonoids are secondary metabolites characterised by flavan nucleus

and C6-C8-C6 carbon-skeleton (Tsuchiya et al., 2010). The basic structural

feature of flavonoid is 2-phenyl-benzo- -pyrane nucleus, consisting of two

benzene rings linked through a heterocyclic pyran ring (Cushnie et al., 2005).

Flavonoids differ in their arrangement of hydroxyl, methoxy and glycosidic

side groups and in the conjunction between A and B rings. A variation in

C ring provides division of subclasses. According to their molecular structure,

they are divided into eight classes as Flavone, Flavonones, Flavonol,

Isoflavone, Catechin, Dihydroflavonol, Chalcone and Anthocyanidin (Tsuchiya

et al., 2010).

2.1.3 Applications of phenolic and flavonoid compounds

Phenolics and flavonoids have been reported to exert wide range of

biological activities. These includes: antioxidant, antiinflammatory,

antibacterial, antiviral, antiallergic, cytotoxic antitumour, treatment of

neurodegenerative diseases, vasodilatory action. In addition flavonoids are

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known to inhibit lipid-peroxidation, platelet aggregation, capillary permeability

and fragility, cyclo-oxygenase and lipoxygenase enzyme activities. They exert

these effects as antioxidants, free radical scavengers, chelators of divalent

cation. These are also reported to inhibit variety of enzymes like hydrolases,

hyalouronidase, alkaline phosphatase, aryl sulphatase, cAMP

phosphodiesterase, -glucosidase, lipase and kinase (Bimlesh et al., 2011).

Iwalokun (2007) stated that, the qualitative analysis revealed low to

moderate level of tannins, steroid glycosides and carbohydrates in Petroleum

ether and 80% acetone extract of Pleurotus ostreatus. While alkaloids,

flavonoids and anthraquinones were found to be absent. Terpenoids was found

to be high in petroleum ether extract. Quantitative analysis recorded total

phenolics of 325.7 mg Gallic acid equivalent / L and 352.8 mg GAE/ L in PE

and 80% acetone extract. The variations in phenolic content are due to

differences in the mycelial wet and variations in the growth conditions.

Mohammad (2013) revealed that, the quantitatibe analysis phytochemcal

analysis of ethanolic from Pleurotus florida extract showed the presence of

phenolic compound, flavonoid, tannin, glycoside, terpenoid, steroid but not

alkaloid. The amount of total polyphenol content was found to be 0.404 µg

GAE / mg of fresh mushroom. The results showed substantial amount of total

polyphenol.

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Mycochemical screening was carried out by Egwim (2011) by using ten

wild edible mushrooms Cantharrelle cibarius, Laccaria amethysta, Clitocybe

odora, Lepista nuda, Macrolepiotata procera, Lepista saeva, Lactarius

deliciousus, Laccaria laccata, Pleurotus ostreatus and Hericium erinaceus.

The preliminary analysis revealed the presence of alkaloids, saponins, tannins

and flavonoids while steroids and anthraquinones were not detected in any

mushroom species. The valuable alkaloids and flavonoids are responsible for

valuable pharmaceutical products.

Investigation of phytochemical screening of edible mushroom Ramaria

flava in ethanolic extract was carried out by Gezer (2006). The total phenolic

content and Total flavonoid content were found to be 39.83 µg / mg

pyrocatechol equivalent and 8.27 µg / mg quercetin equivalent. Russula delica

ethanolic extract was studied for mycochemicals studies by Aziz (2007). The

results revealed the TPC of 47.01 µg / mg pyrocatechol equivalent and in

contrast TFC was 8.71 µg / mg quercetin equivalent.

Study on mycochemicals of Pleurotus ostreatus PQMZ91109 mycelium

with inorganic and organic nitrogen sources in the culture medium revealed

that, ammonium sulfate resulted in a greater ( 22.77 g mycelium / L)

accumulation of bioactive compounds compared with the organic source (corn,

peptone and yeast extract). The TPC were found to be 83 mg GAE / 100 g,

80 mg GAE / 100 g, 71 mg GAE / 100 g and 66 mg GAE / 100 g in

ammounium sulphate, peptone, corn extract and yeast extract respectively. The

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TFC was 531 mg QE / 100g in ammonium sulphate, followed by corn extract

(477 mg QE / 100g), peptone (450 mg QE / 100g) and yeast extract

(387 mg QE / 100g) (Emanuel, 2012).

Total phenol of Pleurotus ostreatus (Jacq:Fries) cultivated on different

tropical woody substrates showed the total phenolic content ranging from

0.89-2.63 µg / ml. Highest TPC was recorded (2.63 µg / ml) in Pleurotus

ostreatus cultivated with Pycnanthus ongoleubis. The study established the

effect of different tropical woody substrates on high phenolic content (Oyetayo

and Ariyo, 2013).

Pleurotus florida extracts (methanol, ethyl acetate, ethanol and

chloroform) was tested for phytochemicals by Thillaimaharani (2013). The

total phenolic content of four different extract ranged from 0.85 – 6.25 mg

GAE / g dw. Highest and lowest TPC were recorded in ethanolic and

chloroform extract with 6.25 mg GAE / g dw and 0.85 mg GAE / g dw. The

present study reveals that polarity of extraction solvent affect the level of

phenol content.

Pleurotus sajor-caju, Pleurotus florida and Pleurotus aureovillosus

mycelium ethanolic extracts were investigated for mycochemicals that showed

the presence of terpenoids, tannins, steroidal glycosides and carbohydrates.

However cyanogenic glycosides were absent in all species. The quantitative

analysis revealed the amount of phenolics and flavonoids in terms of

pyrocatechol and quercetin equivalents. Highest TPC (7.50 µg mg -1) and TFC

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(1.03 µg mg -1) was observed in Pleurotus florida when compared to Pleurotus

-1
sajor-caju (6 and 0.86 µg mg ) and Pleurotus aureovillosus (6.72 and

0.94 µg mg -1) (Loganathan et al., 2008).

Mondal and co workers (2013) investigated mycochemicals in

methanolic extract of four edible mushrooms. Total phenolic content and total

flavonoid content ranging from 0.06-0.082 and 0.032-0.05 mg /100 mg dry

tissue were observed. Highest phenol content was recorded in Agaricus

bisporus (0.082 mg), while flavonoid content of 0.05mg in Pleurotus sajor-

caju and Pleurotus ostreatus.

The mycochemical screening of Pleurotus pulmonarius LAU 09

revealed the presence of alkaloids, tannins, saponins, steroids, phlobatannins,

flavonoids, cardiac glycosides and anthraquinone. Glycosides were found to be

absent. Significant phenolic content of 42.6% TAE / mg and TFC of 12.17 µg

QE / mg was recorded (Adebayo et al., 2012). Phytochemical analysis of

Pleurotus pulmonarius (Fries) Quel champ recorded the presence of TPC of

28.31 mg GAE / g. The results reveal that the phenolics are responsible for

antioxidant activity (Badole et al., 2008)

The study on Ganoderma lucidium mushroom revealed the presence of

triterpenoids, steroids, glycosides, alkaloids, cardiac glycosides and

carbohydrates. Phenols, tannins, flavonoids and anthracene were found to be

absent (Etim et al., 2014). Phytochemical analysis of Ganoderma lucidum and

Ganoderma philippi extracts (PE, chloroform, methanol and aqueous) recorded

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the presence of carbohydrates, reducing sugars, steroids, terpenoids, alkaloids,

phenolic compounds, flavonoids, saponins and glycosides. Tannins were absent

in both mushrooms. The presence of these primary and secondary metabolites

points to the high nutritional and medicinal values (Ranjeet et al., 2014).

Mycochemical analysis of Agaricus bisporus, Lentinula edodes,

Pleurotus eryngii, Pleurotus ostreatus and Grifola frondosa were investigated

by Dubost, (2007). The total phenolic content in all the mushrooms ranged

from 4.2-10.6 mg GAE / g dw. Among all mushroom tested, Agaricus bisporus

species (white button, crimini and portabella) contained significant amount of

total phenolic content ranging between 8-10.7 mg GAE / g dw.

Mycochemical screening of methanolic extract of wild edible Nigerian

mushrooms was studied by Hamzah (2014). The result revealed the presence of

alkaloids, cardiac glycosides, flavonoids, phenolics, terpenes, steroids and

tannins in various proportions. However, tannins were absent in all mushrooms

except in Termitomyces manniformis and Pleurotus ostreatus while

anthraquinone was present in all except Hericium erinaceus and Pleurotus

ostreatus. The quantitative analysis of mycochemicals in the extract revealed

alkaloid (8.125-135.57 µg / g), tannins (59.27-170.56 mg / g), saponins

(10.17-150.41 mg / g), total phenol (97.16-248.80 mg / g) and total flavonoid

content in the range of 6.41-42.63 mg / g. Among the extracts, Pleurotus

ostreatus and Lactarius deliciosus showed significant high total flavonoids

content of 42.63 mg / g and 34.58 mg / g respectively. Methanolic extract of

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Pleurotus ostreatus showed highest total phenolic content (248.80 mg / g)

while Compsus albarius had significant amount of alkaloids (135.57 mg / g)

and saponins (150.41 mg / g).

Qualitative analysis of mycochemicals from Pleurotus djamor revealed

the presence of flavonoids, saponins, tannin, anthraquinones and terpenoids

while cardiac glycosides and steroids were not detected. Quantitative analysis

recorded 32.55 mg /g GAE of phenols and 1.53 mg /g quercetin of flavonoids

respectively (Sasidhara and Thirunalasundari, 2014). Mycochemicals such as

phenols, flavonoids, saponins and tannins in methanolic extract of Pleurotus

florida was recorded by Prabu (2014).

Ethanolic extracts of Pleurotus pulmonarius, Pleurotus ostreatus,

Pleurotus djamor var. djamor, Pleurotus djamor var. roseus and

Schizophyllum commune was successfully investigated for their mycochemical

properties. The amount of TPC varied from 38.45-51.94 mg TAE / g dw of

extract. However, P. djamor var. djamor had significant TPC of 51.94 mg

TAE / g dw. The total flavonoid content in mushroom extract varied from 1.40-

29.80 mg QE / g dw. The lowest (1.40 mg QE / g dw) and highest (29.80 mg

QE / g dw) values was observed in Schizophyllum commune extract at second

and fourth flushes (Arbaayah et al., 2013). The study signifies the correlation

of TPC and TFC to antioxidant activities.

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The qualitative and quantitative mycocomponents of Pleurotus eous and

Pleurotus platypus reported the presence of tannin, saponins, flavonoids and

anthroquinones. TPC and TFC of Pleurotus platypus cultivated on different

substrates ranged between 725-770 mg / 100g GAE and 446-473 mg / 100gm

TAE). However highest TPC (770 mg/100gm GAE) and TFC (473 mg/100gm

TAE) was recorded in black pod and teak leaves as substrate.

In Pleurotus eous, TPC and TFC ranged between 635-695 mg/100gm GAE and

375-397 mg/100gm TAE. However, highest TPC was recorded when cultivated

on corn straw (695 mg/100gm GAE) and TFC when cultivated on paddy straw

(397 mg/100gm TAE) (Sathyaprabha et al., 2011).

Different extract of Pleurotus squarrosulus was evaluated for total

phenol, total flavonoid by Pal (2010). Highest TPC of 82.5 µg / mg GAE was

observed in hot water extract when compared to cold water (80.93 µg / mg

GAE) and methanol (18.1 µg / mg GAE). Total flavonoid content was found to

be high in hot water extract (5.45 µg / mg QE) when compared to cold water

(3.62 µg / mg QE) and methanol (3.07µg / mg QE).

Total phenolic content, total flavonoid content of Agaricus bisporus and

Agaricus brasiliensis in different extracts (aqueous and 60% ethanol) was

investigated by Gan, (2013). The results revealed that, Agaricus bisporus had

significant higher TPC of 21.47 mg QE / g dw in aqueous extract when

compared to 60% ethanol extract (10.25 mg QE / g dw). Agaricus brasiliensis

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mushroom had higher TFC in 60 % ethanolic extract (5.36 mg GAE / g dw)

when compared to Agaricus bisporus aqueous extract (1.36 mg GAE / g dw).

Termitomyces reticulates and their individual parts (cap and stipe) were

evaluated for phytochemical properties by Loganathan (2010). The result

revealed the presence of alkaloids, saponins, phenols, steroids, carbohydrates,

tannins, cardiac glycosides and anthraquinones. Cyanogenic glycosides were

found to be absent in the mushroom extract. TPC were found to be high in

entire mushroom (3.2 mg / g) followed by cap (2.9mg / g) and stipes

(2.5 mg / g). The amount of TFC recorded high in cap (4.77 µg / mg) followed

by entire mushroom (3.58 µg / mg) and stipes (2.74 µg / mg).

A comparative analysis of mycochemicals present in Volvariella

volvacea using different extracts was studied by Carmel (2014). Methanolic

extract recorded high total phenolics (53.13 mg GAE / g), total flavonoids

(14.35 mg / g) and ascorbic acid levels (1.72 mg / g) when compared to hot

water extract with 36.67 mg GAE / g, 12.54 mg / g and 1.52 mg / g. In the

culture medium of Podaxis pistillaris three effective bioactive compounds

epidi-thiodiketo-piperazines (epicorazine A, epicorazine B and epicorazine C)

were identified by Al-Fatimi in 2006.

Bioactive compounds in methanolic extract of wild edible mushrooms

Lactarius deliciosus, Sarcodon imbricatus and Tricholoma portentosum was

carried out by Barros (2007). The total phenolic and total flavonoid content

was found to be high in Lactarius deliciosus, (17.25 and 8.14 mg / g) when

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compared to Sarcodon imbricatus (3.76 and 2.82 mg / g) and Tricholoma

portentosum (10.8 and 0.40 mg / g).

Mycochemical screening of Pleurotus florida extracts was carried out

by Menaga (2012). Preliminary mycochemical analysis revealed the presence

of carbohydrates, glycosides, phenolic compounds, terpenoids steroids, tannins

fixed oils, saponins and flavonoids in all extracts. Total phenolic content

ranging from 10-62.82 mg catechol equivalent / 100 ml and total flavonoid

content of 6-17.71 mg catechol equivalent / 100 ml was recorded. Highest TPC

of 62.82 mg catechol equivalent / 100 ml and TFC of 17.71 mg catechol

equivalent / 100 ml were recorded. The bioactive components have more

solubility in methanol when compared to other solvents.

Fruiting bodies of Hypsizygus ulmarius was evaluated for nutritional

composition and cellulose degrading ability by Jatav, (2012b). The results

revealed 23.01 % of protein, 52.50% carbohydrate (dry weight basis), 12.20%

crude fiber and 22.05 % ash. The total moisture content was found to be

89.68% (fresh weight basis). The analysis also revealed rich presence of trace

elements such as copper (33.8 ppm), iron (70.55 ppm) and manganese

(30 ppm). Further high cellulose degrading ability was observed.

Methanolic extract of Hypsizygus ulmarius cap and stipe individually

were analyzed for their phytochemical activity (Rajesh and Nageswara, 2013).

The total phenol content was found to be high in cap (26.72 µg / g) when

compared to stipe (22.67 µg / g) while TFC in cap and stipe was found to be

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1.622 and 1.116 µg / g. Hypsizygus ulmarius CO2 and IIHR Hu1 strain was

analyzed for total phenol content by Usha and Suguna (2014). The total phenol

content in H. ulmarius CO2 and IIHR Hu1 strain was found to be in the range

from 8.57 and 6.12 mg/g.

2.2 SCREENING OF MUSHROOMS FOR ANTIOXIDANT PROPERTIES

2.2.1 ANTIOXIDANTS

2.2.1.1 Definition and Description

Antioxidants are chemical compounds which contain polyhydroxy

phenols that protect cells from the damage caused by unstable molecules

known as free radicals. They vary in size, composition and molecular weight.

These antioxidants are capable of preventing the oxidation of molecules. The

efficiency of antioxidants is affected by activation energy of antioxidants,

oxidation or reduction potential, solubility and pH stability. Antioxidants

exhibits its action by breaking chain reaction that results in less reactive radical

or by secondary or preventive antioxidants that function as chelators /deactivate

metals, scavenge singlet oxygen (highly toxic) and removal of ROS

(Noori, 2012).

2.2.1.2 Classification of antioxidants

Antioxidants are classified in to three categories as described by Gutteridge

and Halliwell (Singh et al., 2003).

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1. Primary antioxidants: It is involved in the prevention of oxidant

formation.

2. Secondary antioxidants: Exhibits scavenger of ROS.

3. Tertiary antioxidants: Repairs the oxidized molecules through

sources like dietary or consecutive antioxidants.

Based on solubility antioxidants are classified as

1. Hydrophilic antioxidants: antioxidants that react with oxidants in the

cell cytoplasm and the blood plasma. For example: Ascorbic acid,

Glutathione and Uric acid.

2. Hydrophobic antioxidants: protect cell membranes from lipid

peroxidation. For exampl -tocopherol and ubiquinol.

These compounds may be synthesized in the body or obtained from

the diet.

Antioxidants may be enzymatic or non-enzymatic.

1. Enzymatic antioxidants (catalase, superoxide dismutase, glutathione

peroxidase, glutathione reductase and thioredoxin) directly/indirectly

contribute to defense against the ROS.

2. The non-enzymatic antioxidants are the scavangers of ROS and RNS

which directly reacts with ROS and form disulfides. Non-enzymatic

antioxidants also can be divided into metabolic antioxidants and

nutrient antioxidants. Metabolic antioxidants are the endogenous

antioxidants, which produced by metabolism in the body like lipoid

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acid, glutathione, L-ariginine, coenzyme Q10, melatonin, uric acid,

bilirubin, metal chelating proteins (Droge, 2002; Willcox et al.,

2004). While nutrient antioxidants belonging to exogenous

antioxidants, which cannot be produced in the body but provided

through diet or supplements viz. trace metals (selenium, manganese,

zinc), flavonoids, omega-3 and omega-6 fatty acids (Pham-huy

et al., 2008). Vitamin E and C are the non enzymatic antioxidants

exist within normal cells as well as they can be supplied through diet

(Tiwari et al., 2001).

2.2.1.3 Applications of antioxidant

The antioxidant activity mainly depends on the levels of phenolics and

flavonoids present that play a vital role in the stability of food products, as well

as in the antioxidative defense mechanisms of biological systems (Macheix and

Fleuriet 1990). Recently, natural food derived antioxidants have received

growing attention, because they are known to function as chemopreventive

agents thereby augmenting the body’s natural resistance to oxidative damage

(Kumar et al., 2010). Antioxidants from different source are widely used in the

treatment of neurodegenerative disorders (alzimer’s, parkinson’s), cancer

treatement (prostate, lung and gastric), cardiovascular and renal diseases,

diabetes and autoimmume disorders (Saikat et al., 2010). The overall

effectiveness of a natural phenolic antioxidant depends on the involvement of

the phenolic hydrogen in radical reactions, the stability of the natural

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antioxidant radical formed during radical reactions, and the chemical

substitutions present on the structure (Hall 2001).

Iwalokun (2007) reported that, petroleum ether (PE) and 80% acetone

extract of Pleurotus ostreatus was screened for antioxidant activity that

revealed a disparate vitamin C equivalent antioxidant capacity of 3.6 - 3.8 mM

for Petroleum ether and 4.1-4.4 mM for 80% acetone extract compared to green

tea infusion (6.2-6.4 mM). The observed antioxidant activity in this study was

attributed to organic solvent used, amount of phenolic and terpenoids present,

species and strain variations, difference in their microcidal composition and

concentration, method of extraction and mechanism of actions of active

principles in mushroom.

Ethanolic extract of Pleurotus florida cultivated in Bangladesh was

studied For antioxidant activity by DPPH and reducing power assay. DPPH

scavenging activity of extract showed IC50 value in the range of 0.066 - 0.012

mM GAE while it was 0.154 mM for BHT as standard. The reducing power of

the Pleurotus florida extract was found to be 68.69 µg AAE / mg. The study

reveals that polyphenols are responsible for appreciable antioxidant activity

(Mohammad et al., 2013).

Antioxidant activity of ten wild edible mushrooms Cantharrelle

cibarius, Laccaria amethysta, Clitocybe odora, Lepista nuda, Macrolepiotata

procera, Lepista saeva, Lactarius deliciousus, Laccaria laccata, Pleurotus

ostreatus and Hericium erinaceus was carried out by Egwim, (2011). The

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activity obtained was moderate in the range of 34.46-53.65 nM. Highest

activity was recorded in Laccaria amethysta (53.64 nM) and Lepista nuda,

(3.65 nM) while least activity in Lepista saeva, and Macrolepiotata procera,

(34.46 nM). The results obtained conclude that these mushrooms are good

source of vitamins and flavonoids. Cantharrelle cibarius, Laccaria amethysta,

Clitocybe odora and Lepista nuda species are suggested to act as antioxidant

agents.

Antioxidant activities of edible mushroom Ramaria flava in ethanolic

extract by DPPH method was carried out by Gezer (2006). The activity was

found to be dose dependent with IC50 value of 276 µg / ml. 160 µg of Ramaria

flava ethanolic extract has an equivalent inhibition value (94.7%) of 80 µg

(96.4%) BHA. According to the results obtained high inhibition value is due to

high concentration in phenolic compound.

Study on antioxidant potential of Pleurotus ostreatus PQMZ91109

mycelium with inorganic and organic nitrogen sources in the culture medium

revealed that, the radical scavenging activity of different extract by different

methods exhibited in dose dependent manner. In the DPPH radical scavenging

activity maximum percentage inhibition was obtained at a concentration of

20 mg / ml ranging between 58.76-89.93% with EC50 value of 6.74 mg / ml in

ammonium sulphate extract. In hydroxyl scavenging activity maximum

inhibition of 81.5% was achieved at 20 mg / ml ammonium sulphate extract

with EC50 of 6.54 mg / ml. Superoxide and nitric oxide radical scavenging

activity showed maximum inhibition of 82.4% and 96.31% in ammonium

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sulphate extract at 20 mg / ml with EC50 value of 1.27 and 0.77 mg / ml

(Emanuel, 2012).

Antioxidant activity of Pleurotus ostreatus (Jacq:Fries) cultivated on

different tropical woody substrates was studied by Oyetayo and Ariyo, (2013).

DPPH radical scavenging activity was found to be high (90.17 %) in Pleurotus

ostreatus cultivated with Pycnanthus ongoleubis and least with Pleurotus

ostreatus cultivated with Ceiba pentandra (73.75 %).

Methanol, ethyl acetate, ethanol and chloroform extracts of Pleurotus

florida were tested for antioxidant activity by Thillaimaharani (2013). The total

antioxidant activity was found to be high in ethanolic extract with 230 µg BHT

equivalent / g and least in chloroform extract with 90 µg BHT equivalent / g.

Free radical scavenging activity by DPPH method of four extracts recorded

maximum inhibition with ethanol (79.24%) followed by methanol (71.29%).

Weakest inhibition was observed at ethyl acetate (69.26%) and chloroform

(63.18%) extract. Antioxidant activity of Pleurotus florida extracts was carried

out by Menaga (2013). The mehanolic extract exhibited excellent radical

scavenging activity in all the five different assays performed in a dose

dependent manner with IC50 value 50 µg / ml for DPPH, 110 µg / ml for

reducing power assay, 410 µg / ml for hydroxyl scavenging activity,

510 µg / ml for nitric oxide radical and 542 µg / ml for superoxide radical

scavenging activity.

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Pleurotus sajor-caju, Pleurotus florida and Pleurotus aureovillosus

mycelium ethanolic extracts were investigated for antioxidant activity

(Loganathan et al., 2008). In vitro antioxidant activity by 2,2'-azino-bis

(3-ethylbenzothiazoline- 6-sulphonic acid) (ABTS) method recorded maximum

percentage of inhibition Pleurotus sajor-caju (62.82%), Pleurotus florida

(69.26%) and Pleurotus aureovillosus (42.52%) in all the three mushroom at

250 µg / ml in terms of trolox equivalent.

Mondal and co workers (2013) investigated antioxidant activity in

methanolic extract of four edible mushrooms. Total antioxidant content in

terms of vitamin C content was found to excellent in Pleurotus sajor-caju

(0.041 mg / 100 mg dry tissue) when compared to Pleurotus ostreatus

(0.028 mg / 100 mg dry tissue), Agaricus bisporus (0.037 mg / 100 mg dry

tissue) and Ganoderma lucidum (0.011 mg / 100 mg dry tissue). The study

concludes good amount of antioxidant compounds that is effectively used in

medicine field.

The antioxidant activity of Pleurotus pulmonarius LAU 09 was found to

be in dose dependent manner. Maximum activity of 78.35% and 80.98% was

recorded with 5 mg / ml and 10 mg / ml when compared to ascorbic acid.

Further, antioxidant activity by phosphomolybdenum method was found to be

25.6 and 87.1 µ moles / mg at 1 mg / ml and 5 mg / ml (Adebayo et al., 2012).

The antioxidant activity of Pleurotus pulmonarius (Fries) Quel champ was

found to be in dose dependent manner with IC50 value of 68.84, 72.69 and

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82.76 µg / ml for hydroxyl, nitric oxide and hydrogen peroxide radical

scavenging activity while the percentage inhibition for DPPH was found to be

78.35 % (Badole et al., 2008).

Antioxidant activity of Agaricus bisporus, Lentinula edodes, Pleurotus

eryngii, Pleurotus ostreatus and Grifola frondosa were investigated by Dubost,

(2007). The antioxidant activity of these mushrooms in terms of oxygen radical

absorbance capacity (ORAC total), hydroxyl, peroxynitrite and superoxide

radical averting capacity (HORAC, NORAC, SORAC) revealed that ORAC

total valued ranged between 39-138 µmol TE / g dw. Agaricus bisporus

was found to have highest values among mushrooms tested with a range

between 80-131 µmol TE / g dw. HORAC among mushroom was found to

be 3-13.6 µmol CAE / g dw. There was significant difference found within

Agaricus bisporus mushrooms. However, Portabella contained the highest

value (81.3 µmol CAE / g dw) while maitake mushroom contained the lowest

value (2.67 µmol CAE / g dw). The NORAC among mushrooms tested was in

the range 2-9 µmol TE / g dw. The lowest value was observed in Maitake

(2 µmol TE / g dw) and highest in Portabella mushroom (9 µmol TE / g dw).

However, there was significant difference within Agaricus bisporus

mushroom species. SORAC values of the mushrooms ranged between

0.37-2.6 k units SOD eq / g dw. Portabella contained higest value (2.69 k units

SOD eq / g dw) while, Maitake recorded the least (0.37 k units SOD eq / g dw).

The results declare that higher the total phenolic content, higher the antioxidant

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activity which mainly depends upon growing conditions of mushroom and

amount of secondary metabolites produced.

Antioxidant activity of methanolic extract of wild edible Nigerian

mushrooms by DPPH method was in dose dependent manner. However,

Lactarius deliciosus, had the highest activity of 71.49% at 500 µg / ml. the

reducing power of selected mushroom extract were proportional to the

concentration 1000 mg / ml. Pleurotus ostreatus and Lactarius deliciosus, had

better reductive power than other mushroom extracts irrespective to the

concentrations. The study supports the antioxidant actions due to the presence

of flavonoids and phenolic compounds (Hamzah et al., 2014).

Pleurotus djamor revealed the reduction capability of DPPH in dose

dependent manner. At 100 µg / ml of mushroom extract the percentage

inhibition was found to be 76.44% when compared to ascorbic acid (99.3 %).

The IC50 value of extract and ascorbic acid was 64.72 µg / ml and

29.42 µg / ml. The study suggests that antioxidant activity was due to the

presence of phenols and flavonoids (Sasidhara and Thirunalasundari, 2014).

Ethanolic extracts of Pleurotus pulmonarius, Pleurotus ostreatus,

Pleurotus djamor var. djamor, Pleurotus djamor var. roseus and

Schizophyllum commune was successfully investigated for their antioxidant

potential. The extract showed positive antioxidant activity by DPPH method

with IC50 values varied from 2.75-12 mg / ml for all samples tested. However,

Schizophyllum commune extract showed highest activity in all flushes.

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Reducing power of the extract ranged between 2-10 mg / ml. The greatest

reduction of ferricyanide complex to ferrous form was observed in Pleurotus

djamor var. djamor extract at 10 mg / ml concentration for both first (1.226)

and second (1.230) flushes (Arbaayah et al., 2013). The study signifies the

correlation of TPC and TFC to antioxidant activities.

Pleurotus eous and Pleurotus platypus reported dose dependent

antioxidant activity of 91.4-95.2% in P. platypus and 85.5-87.4% in Pleurotus

eous. Highest activity of 95.2% / 100 g was observed in Pleurotus platypus

with paddy straw as substrate whereas in banana substrate, Pleurotus eous

showed 87.4%/ 100 g (Sathyaprabha et al., 2011). Different extract of

Pleurotus squarrosulus was evaluated for in vitro antioxidant activity by Pal

(2010). The antioxidant activity against hydroxyl, DPPH, nitric oxide, ferrous

chelating ability, reducing power and super oxide was found to be high in hot

water extract with IC50 value of 268, 340, 320, 75, 1140 and 1473 µg / ml when

compared to other extracts. Results showed that the hot water extract has

maximum antioxidant property and could be utilized as a promising source of

therapeutics.

Antioxidant activity of Agaricus bisporus and Agaricus brasiliensis in

different extracts (aqueous and 60% ethanol) was investigated by Gan (2013).

The antioxidant activity by DPPH method was significantly higher in Agaricus

brasiliensis 60% ethanol with EC50 value of 1.67 mg / ml. FRAP was found to

be high in Agaricus brailiensis 60% ethanol (107.24 µ mole Fe2+ equivalent / g

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dw) and Agaricus bisporus in aqueous extract (186.72 µ mole Fe2+ equivalent

/ g dw). The results declare that strong positive correlation exist between TPC

and FRAP assay in both extracts and TFC and FRAP in aqueous extract.

Termitomyces reticulates and their individual parts (cap and stipe) were

evaluated for antioxidant activity by Loganathan (2010). Strong antioxidant

potential was observed in all different the assay performed. In reducing power

assay, ABTS and DPPH models the entire mushroom ethanolic extract showed

maximum EC50 value of 2.58, 1.37 and 4.92 mg / ml when compared to their

individual parts. The results conclude that the sample with highest antioxidant

contents show higher antioxidant activity with low EC50 values.

Antioxidant potential of Ganoderma lucidum ethanolic extract showed

maximum inhibition of reducing power at 200 mg / ml (1.38 OD), DPPH at

250 mg / ml (72.24%), hydroxyl radical scavenging activity at 25 mg / ml

(64.69%), superoxide activity at 300 mg / ml (73.54%) and nitric oxide at 200

mg / ml (65.87%). The results conclude that appreciable amount of secondary

metabolites with free radical scavenging activity that make the mushroom ideal

nutritional supplement with good medicinal properties (Rajasekaran et al.,

2011). The study on Ganoderma lucidium mushroom antioxidant potential was

studied by (Etim et al., 2014). Maximum percentage inhibition of 71.43% was

recorded at 0.125 mg / ml when compared to standards Vitamin C and

Buthylated hydroxyl anisole (BHA) by DPPH method. The IC50 value of the

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extract was found to be 0.23. The results suggest that, the antioxidant capacity

was due to presence of alkaloids and other secondary metabolites.

A comparative analysis of antioxidants present in Volvariella volvacea

using different extracts was studied by Carmel (2014). The antioxidant activity

was found to be in dose dependent manner in both extracts. However

maximum percentage inhibition of 68.78% at 125 mg / ml for ABTS, DPPH at

250 mg / ml (80.32%), superoxide at 300 µg / ml (74.23%) and 75.52% at 200

µg / ml for nitric oxide was recorded in methanolic extract. Hydroxyl

scavenging activity was found to be high in hot water extract at 250 µg / ml

(69.34%). The results declare that the antioxidant activity of edible mushroom

is due to the considerable amount of phenolics, flavonoids and ascorbic acid.

In vitro antioxidant study of Pleurotus eous aqueous extracts (PEAE-1,

PEAE-2, PEAE-3) was studied by Sasikumar (2011). DPPH radical scavenging

activity was found to be maximum at 2.5mg / ml with 83.76% inhibition for

PEAE-1, 80.68% for PEAE-2 and 84.76% for PEAE-3. The EC50 value of 1.4,

1.24 and 1.35 mg / ml was recorded for PEAE-1, PEAE-2 and PEAE-3.

Research work on antioxidant activity of Ganoderma lucidum (Curt: Fr.)

P. Karst. (Aphyllophoromycetideae) from tropical South India was reported by

Soniamol (2009). The chloroform extract showed maximum activity by DPPH

method at 1000 µg / ml with 91.12%. The IC50 value of the extract by hydroxyl

and nitric oxide radical scavenging method was found to be 144.66 and

21.66 µg / ml.

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The antioxidant activity of Hypsizygus ulmarius recombinant laccase by

DPPH method exhibited higher free radical scavenging activity at 500 µg / ml

with 36% of inhibition. The IC50 value was found to be at 563.47 µg / ml.

Further the cytotoxic effect of recombinant laccase of Hypsizygus ulmarius on

MCF-7 (human breast cancer) cells characterized by MTT cell viability assay

showed significant reduction of viable cells with IC50 value of 3.75 µM and

increase in dead cells with increase in enzyme concentration. The results say

that this could be due to antioxidant potential of enzyme (Ravikumar et al.,

2013a).

Methanolic extract of Hypsizygus ulmarius (cap and stipe) were

analyzed for their antioxidant activity (Rajesh and Nageswara, 2013). The IC50

value obtained for cap and stipe extract by DPPH (1.242 and 1.556 mg / ml),

reducing power (4.39 and 4.98 mg / ml), FRAP (9.007 and 20.317 mg / ml) and

hydrogen peroxide (2.920 and 2.995 mg / ml) method exhibited good

antioxidant activity. Hypsizygus ulmarius CO2 and IIHR Hu1 strain was

analyzed for antioxidant potential by reducing power assay by Usha and

Suguna (2014). The reducing power was found to be high in Hypsizygus

ulmarius CO2 strain (0.89 OD) when compared to Hypsizygus ulmarius IIHR

Hu1 strain (0.64 OD) at 2.5 mg / ml. In this study a direct co relation exist

between mushroom antioxidant activity and total phenolic content.

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2.3 SCREENING OF MUSHROOMS FOR ANTIMICROBIAL PROPERTIES

2.3.1 ANTIMICROBIAL ACTIVITY

Antimicrobial agent is a compound produced by bacteria, fungi or of

synthetic in nature that kill microorganisms or inhibit their growth. They are

widely employed to cure bacterial diseases. Antimicrobial agents that

reversibly inhibit growth of bacteria are called bacteriostatic whereas those

with irreversible lethal action on bacteria are known as bactericidal (Rajesh and

Rattan, 2008). Ideally, antimicrobial agents disrupt microbial processes or

structures that differ from those of the host. They may damage pathogens by

hampering cell wall synthesis, inhibiting microbial protein and nucleic acid

synthesis, disrupting microbial membrane structure and function, or blocking

metabolic pathways through inhibition of key enzymes (Willey et al., 2008).

Before an antimicrobial agent is accepted for use in human beings it

should have following properties such as selective toxicity, bactericidal,

effective against a broad range of bacteria, ant allergic, active in plasma and

body fluids, long shelf life, less expensive, stable and preferably water soluble

(Rajesh and Rattan, 2008).

The innovative research for antibiotics has improved mankind’s health

status by confining life threatening infections. Although there are numerous

classes of drugs that are routinely used to treat infections in humans, there are

several reasons why the discovery and development of new antimicrobial

agents are important. The dramatically rising prevalence of multi-drug resistant

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microbial infections has become a serious health care problem. This increased

resistance has limited the selection of antimicrobials that may be used to treat

specific organisms. Consequently, the search for new antimicrobial agents

always remains as an important and challenging task for medicinal chemists.

The antimicrobial study carried out by Iwalokun (2007) in Pleurotus

ostreatus petroleum ether and 80% acetone extract revealed that PE extract

exhibited good antimicrobial activity ranging from 3-7.8 mm for gram positive,

5-8.2 mm for gram negative and 8.1-10.8 mm for fungi when compared to

acetone extract (3-10.5mm). The observed antimicrobial activity in this study

was attributed to amount of phenolic and terpenoids present, species and strain

variations, difference in their microcidal composition and concentration,

method of extraction and mechanism of actions of active principles in

mushroom.

The antimicrobial activity of Pleurotus florida cultivated in Bangladesh

was studied by Mohammad (2013). The Antibiogram of extract tested against

Escherichia coli, Pleurotus aerugnosa, Salmonella typhi, Serratia marcescens,

Staphylococcus aureus and Salmonella paratyphi A showed ZOI in the range of

9.20-17 mm. The MIC of the extract was found to be highest for Salmonella

paratyphii A at 1300 µg / ml while Serratia marcescens and Staphylococcus

aureus had the lowest MIC of 1000 µgm / ml. The results obtained had greater

amount of phenols responsible for maximum antibacterial activity.

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Antimicrobial activity of edible mushroom Ramaria flava ethanolic

extract tested against seven gram negative bacteria, five gram positive bacteria

and one yeast showed best activity ranging from 4-20 mm against Salmonella

enteritidis, Klebsiella pneumoniae, Staphylococcus aureus cowan, Bacillus

subtilis, Bacillus cerus, Staphylococcus aureus, Yersinia enterecolitica,

Micrococcus luteus and Micrococcus flavus. Antibacterial activity did not

appear in Pseudomonas aeruginosa, Escherichia coli, Morganella morganii,

Proteus vulgaris and Candida albicans. The most susceptible bacteria were

found to be Yersinia enterecolitica, Micrococcus luteus and Micrococcus flavus

with 11, 13 and 20 mm ZOI. The study reveals that Ramaria flava were not

effective as commercial drugs, but when used in higher concentrations can be

used as antibiotics (Gezer et al., 2006). Russula delica ethanolic extract

exhibited narrow spectrum of antibacterial activity against array of

microorganisms with ZOI ranging from 4-15.5 mm in Salmonella enteritidis,

Klebsiella pneumoniae, Yersinia enterecolitica, Staphylococcus aureus cowan,

Micrococcus luteus, Micrococcus flavus Bacillus subtilis, Bacillus cerus and

Candida albicans. However, antibacterial activity was not found in Proteus

aeruginosa, Escherichia coli, Morganella morganii, Proteus vulgaris, and

Staphylococcus aureus (Aziz et al., 2007).

Study on antimicrobial potential of Pleurotus ostreatus PQMZ91109

mycelium with inorganic and organic nitrogen sources in the culture medium

revealed that the ammonium sulphate extract did not exhibit MIC for

Escherichia coli. Least MIC was observed in Bacillus cerus, Listeria innocua

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and Pseudomonas aeruginosa at 2.5 mg / ml while for Candida albicans and

candida species were at 1.25 mg / ml. However, Staphylococcus auerus

showed at 12.5 mg / ml. The findings reported that ammonium sulphate as

nitrogen source exhibited increased phenolic and flavonoids content, good

antioxidant and antimicrobial activity (Emanuel, 2012).

Antimicrobial activity of Pleurotus ostreatus (Jacq:Fries) cultivated on

different tropical woody substrates showed that the ethanolic extracts obtained

from cultivated Pleurotus ostreatus exhibited good zone of inhibition (ZOI)

against different micro organisms ranging from 5.33-20.33 mm. The extract of

Pleurotus ostreatus cultivated on Pycnanthus ongoleubis substrate exhibited a

better activity against almost the organisms except Pseudomonas aeruginosa

and Bacillus subtilis. However, best inhibitory effect of 18-20 mm was

observed with Staphylococcus aureus. The MIC of extracts on the tested

organisms ranged from 2.5-20 mg / ml. Highest was recorded for Klebsiella

pneumonia and Pseudomonas aeruginosa (20 mg /ml). The study established

the effect of different tropical woody substrates on antimicrobial activity

enhancing nutraceuticals properties (Oyetayo and Ariyo, 2013).

Pleurotus florida extracts (methanol, ethyl acetate, ethanol and

chloroform) was tested for antimicrobial activity by Thillaimaharani (2013)

against eight human pathogenic bacteria (Escherichia coli, Salmonella typhi,

Klebsiella pneumoniae, Vibrio parahaemolyticus, Klebsiella oxytoca, Proteus

murabilis, Vibrio cholera and Streptococcus species). Among them, ethanolic

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extract showed maximum (23 mm) ZOI against Streptococcus species and

minimum against Vibrio parahaemolyticus (4 mm). However, minimum

antibacterial activity was observed in chloroform extract with 11 mm against

Escherichia coli and no activity found against Vibrio cholera. Further

antifungal activity of extracts tested against Trichophyton rubrum,

Epidermophyton floccosum and Microsporum gypseum was studied. Ethanol

and chloroform extract showed maximum and minimum ZOI. Ethanolic

extract of Pleurotus florida produced MIC at 25 mg / ml against Escherichia

coli and Klebsiella oxytoca followed by Streptococcus species (50 mg / ml) and

Proteus murabilis (75 mg / ml). In case of fungi, ethanolic extract showed MIC

at 50 mg / ml against Epidermophyton floccosum.

Pleurotus sajor-caju, Pleurotus florida and Pleurotus aureovillosus

mycelial ethanolic extracts were investigated for antimicrobial activity

(Loganathan et al., 2008). The antimicrobial effect tested against different

organisms showed inhibition zone of 10-22 mm. Most susceptible bacteria

were found to be Micrococcus flavus (22 mm). ZOI was completely absent in

all the three extract against Klebsiella pneumonia, Proteus vulgaris, and

Pseudomonas aeruginosa. The report suggests that difference in antioxidant

activities was due to strains used, extraction solvent and extraction method.

Mondal and co workers (2013) investigated antimicrobial activity from

methanolic extract of four edible mushrooms that exhibited good ZOI ranging

from 3.7-17 mm for gram positive bacteria and 4.5-40.1 mm for gram negative

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bacteria. Highest ZOI was observed in S. aureus (16.6 mm) and least in

Bacillus species (4.5 mm) by Pleurotus ostreatus methanolic extract. Agaricus

bisporus PE and acetone extract showed maximum ZOI against Vibrio cholera

(40.1 mm) and minimum against Staphylococcus aureus (3.8 mm). Ganoderma

lucidum PE and methanolic extract showed highest and lowest ZOI against

Vibrio cholera (16.6 mm) and Escherichia coli (5.3 mm). Among fungi

species, maximum growth inhibition of 77.78% was observed in Aspergillus

flavus by Pleurotus ostreatus PE extract and minimum 57.78% in Penicillium

patulum by Pleurotus sajor-caju acetone extract. The study concludes

significant antimicrobial activity that is effectively used in medicine field.

Akyuz (2009) investigated antimicrobial activity of Pleurotus eryngii

var. ferulae methanolic extracts grown on various agrowaste. Excellent zone of

inhibition ranging from 7.7-10.3 mm against microorganisms such as Bacillus

megaterium Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae,

Cadida albicans, Cadida glabrata, Trichophyton and Epidermophyton species

were observed when compared to aqueous extract. Excellent antimicrobial

activity against micro organisms were observed with ZOI ranging from 7-30

mm. Maximum ZOI was seen in Staphylococcus aureus with 30 mm and least

in Escherichia coli (7 mm). ZOI was nil in Pseudomonas aeroginosa. The

results exhibit the phenols play an important role in antioxidant potential

(Adebayo et al., 2012).

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The study on Ganoderma lucidium mushroom revealed strong

antibacterial activity against Escherichia coli (12 mm), Klebsiella pneumonia

(12 mm), Proteus mirabilis (13 mm) and Streptococcus species (14 mm) was

observed at 1000 mg / ml (Etim et al., 2014).

Antimicrobial activity of Lactarius deliciosus, Sarcodon imbricatus and

Tricholoma portentosum wild edible mushrooms methanolic extract was

carried out by Barros (2007). The methanol extract showed good antimicrobial

activity. Lactarius deliciosus entire mushroom extract inhibited Bacillus cerus,

Pseudomonas aeruginosa, Bacillus subtilis Candida albicans and

Cryptococcus neoformans in the concentrations tested. Tricholoma

portentosum extract was effective against Bacillus cerus, Bacillus subtilis and

Cryptococcus neoformans while Sarcodon imbricatus exhibited activity against

Bacillus cerus and Cryptococcus neoformans. Escherichia coli were found to

be resistant to all the mushroom extracts. The results showed that the

antibacteral activity of the mushroom is due to the phenolics.

In the culture medium of Podaxis pistillaris three effective bioactive

compounds were identified. These Bioactive components exhibited effective

antibacterial acitivity against Staphylococcus aureus, Micrococcus flavus,

Bacillus subtilis, Proteus mirabilis, Serratia marcescens and Escherichia coli.

However, MIC against Staphylococcus aureus is 25 µg / ml for epicorazine A,

50 µg / ml for epicorazine B and 75 µg / ml for epicorazine C (Al-Fatimi,

2006).

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Antimicrobial activity of Pleurotus florida extracts was carried out by

Menaga (2012). Among the extracts studied for antbiogram, ethanolic and

methanolic extract demonstrated highest activity. Ethanolic extract exhibited

highest activity against Pseudomonas sp., and Camphylobacter sp., while,

methanolic extrat against Escherichia coli, Salmonella typhi, Staphylococcus

aureus, Camphylobacter sp., and Vibrio sp. Aqueous extract showed highest

ZOI (24 ± 1.5 mm) against Vibrio sp. whereas ethyl acetate and hexane extract

showed least and no activity against most of the organisms tested. In this study

Pleurotus florida cultivated along with horse gram as nutrient supplement

resulted in high mushroom yield and phenolic content when compared to

Pleurotus florida cultivated on rice straw as substrate. Significant antimicrobial

activity in methanolic extract is recorded due to the presence of phenolic

compounds and other secondary metabolites.

Biosynthesis of silver nanoparticles and antimicrobial study of edible

mushroom Hypsizygus ulmarius revealed that effective reduction of silver

metal nanoparticles by nitrate dependent reductase and a shuttle quinone

extracellular process at 3 mM with increased productivity and sharp and

intense surface plasmon at 386 nm. The synthesized silver nanoparticle

exhibited bactericidal activity against Staphylococcus aureus and Pseudomonas

aeruginosa. Maximum ZOI for Staphylococcus aureus (0.15 mm) and

Pseudomonas aeruginosa (0.17 mm) was seen in 0.5 mg / ml and 0.4 mg / ml

(Shivshankar et al., 2013).

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2.4 SCREENING OF MUSHROOMS FOR CARBOXYLESTERASE

2.4.1 CARBOXYLESTERASE

Carboxylesterase (EC.3.1.1.1, carboxyl ester hydrolases) are

hydrolyzing enzymes widely distributed in nature that have been identified,

isolated and characterized. They occur in multiple molecular forms and

exhibits unique enzyme characteristics such as substrate specificity,

regiospecificity and chiral specificity. These enzymes are preferably catalyze

the hydrolysis of both endogenous and exogenous short chain fatty acid esters

(Chandrashekharaiah et al., 2011).

The major functions of these enzymes have also been implicated in

carbon source utilization, pathogenicity and detoxification (Ewis et al., 2004).

Particularly, the potential application of these enzymes for the synthesis of

short chain esters has attracted the interest of a broad range of industrial fields

like foods, pharmaceuticals and cosmetics. Among these flavor acetates from

primary alcohols constitute compounds with a great application due to their

characteristic fragrance and flavor (Romero et al., 2005).

The carboxylesterases are also involved in fruit ripening, abscission, cell

expansion, reproduction as well as hydrolysis of ester containing xenobiotic

molecules. Other significant functions of the carboxylesterases include

metabolism and subsequent detoxification of many agrochemicals,

pharmaceuticals (Redinbo and Potter, 2005; Potter and Wadkins, 2006),

metabolism of a number of therapeutics such as cholesterol-lowering drug-

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lovastatin (Tang and Kalow, 1995), the antiinfluenza drug, Oseltamivir

(Tamiflu) (Shi et al., 2006), the narcotic analgesic meperidine (Demerol)

(Zhang et al., 1999), cocaine and heroin (Pindel et al., 1997), resolution of

racemic mixtures and optically pure compounds (Bornscheuer, 2002) and in

soft- and pro-drug design (Bodor and Buchwald, 2000, 2003, 2004).

Xanthophyll esterase was purified from culture supernatant of Pleurotus

sapidus with molecular mass 430 kDa by Holger (2005). The enzyme activity

was optimum at pH 5.8 and 40 0C. This carboxylesterase represents the first

enzyme of Basidiomycetous fungus that has been characterized at the

molecular level.

The production and activity of Agaricus bisporus endoxylanase and

-xylosidase were studied in compost and liquid cultures by James (2000). The

result revealed drastic increase of endoxylanase during fruiting body

development and decreased after harvest, whereas b-xylosidase activity was

observed during fruiting body development only. Using activity staining and

-xylosidase

activity were separated from liquid cultures and compost extract.

Purification and characterization of alkaline esterase from from the

fruiting body of the medicinal and edible mushroom Sparassis crispa was

carried out. The molecular weight of purified enzyme was found to be 60 kDa

and it also demonstrated high substrate specificity towards p-nitrophenyl

acetate. The optimum enzyme activity was observed at pH 8.0 and temperature

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50 0C respectively. The enzyme stability was found even at alkaline pH

conditions and at the temperature ranging from 30-40 0C. The Km and Vmax of

purified enzyme was found to be 0.2 mM and 0.5 U / mg proteins respectively

(Gayathri et al., 2011).

Characterization of esterase from edible mushroom species, Amanita

vaginata var. vaginata and Tricholoma terreum was reported by Ertunga

(2009). The enzyme from both sources had the highest activity in the presence

of p- nitrophenyl butyrate (pNPB) as a substrate. The enzyme activity in

Amanita vaginata var. vaginata was found to be optimum at pH 8.0 and

temperature 30 0C. The Km and Vmax values for the enzyme were 71 µM

and 14.4 U / I. The enzyme found in Tricholoma terreum exhibited enzyme

stability in the pH range 3-10 and temperature range 10-40 0C. The Km and

Vmax value for Tricholoma terreum was found to be 9.6µM and 34.6 U / I.

The esterase from both the sources showed and increased enzyme activity in

the presence of dithiothreitol (DTT) and metal ions such as K+, Al3+, Ni2+ and

Li+.

Nora (1994) reported the presence of acetylxylan esterase from

Schizophyllum commune and the molecular mass of the enzyme was found to

be 31 kDa. Acetylxylan esterase was stable at pH range 6.2-8.5 upon incubation

at 25 0C for 7 h. The pH and temperature optimum of the enzyme activity was

7.7 and 30-45 0C respectively. Acetylxylan esterase activity was inhibited by

metal ions like Ca2+ and Co2+.

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Extracellular carboxylesterase from Fusarium graminearum by

Donaghy and McKay (1992) showed optimum esterase activity at 37 0C

temperature and pH 9.0. Increased activity was recorded with whey as

substrate, supplemented with Tween 40. Good activity was observed when

fungus was grown on a semi-solid bran culture medium.

Dry mycelium of Aspergillus oryzae efficiently catalyzed the

esterification between free acetic acid and primary alcohols (geraniol and

ethanol). Highest activity was observed in medium containing Tween 80 as

carbon source. Maximum production was obtained using 12.4 g of geranyl

acetate at 80 0C and 4.1 g of ethyl acetate at 50 0C after 24 h (Molinaria et al.,

2000).

Feruloyl esterases designated as FAE-1 of 50 kDa and FAE-2 of 55 kDa

from Aspergillus niger (CFR 1105) grown on wheat bran showed maximum

specific activity in solid state fermentation on day 2 when compared to

submerged fermentation on day 3. The temperature optima for both enzymes

were found to be 50 0C and pH optima of 9 and 6 respectively (Hedge and

Muralikrishna, 2009).

Three ferulic acid esterases from the filamentous fungus Chrysosporium

lucknowense C1 was purified and characterized by Kuhnel (2012). The

enzymes were more active at pH 7.0 and temperature of 45 0C. All the enzymes

released ferulic acid and p-coumaric acid from a soluble corn fiber fraction.

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Ferulic acid esterases FaeA1 and FaeA2 also released dehydrodiferulic and

dehydrotriferulic acids.

Hypsizygus ulmarius recorded maximum production of laccase on

6th day incubation with 3% inoculum, xylose and KNO3 at pH 5 and temperature

30 0C. Fold purification was found to be 5.11 with 53.33 % recovery at

optimum pH 6.0 and temperature 40 0C respectively. The enzyme activity was

enhanced by Mn2+and Cu2+ ions and reduced by Fe2+, Na2+ and Co2+ ions

(Ravikumar et al., 2012a).

2.5 SCREENING OF MUSHROOMS FOR ALKALINE PROTEASE

2.5.1 ALKALINE PROTEASE

Proteases are the most important hydrolytic enzyme and have been

studied extensively since the advent of enzymology (Gupta et al., 2002a).

Proteases are a complex group of enzymes collectively known as

peptidylpeptide hydrolases and are responsible for the hydrolysis of peptide

bonds in a protein molecule. They are a single class of enzymes which occupy

a pivotal position with respect to their applications in both physiological and

commercial fields. Proteolytic enzymes are ubiquitous in occurrence, being

found in all living organisms (bacteria, fungi, plants and animals). Proteases

execute a large variety of functions, extending from the cellular level to the

organ and organism level, to produce cascade systems such as homoeostasis

and inflammation. These and are essential for the complex processes involved

in the normal physiology of the cell (cell growth and differentiation.) as well as
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in abnormal pathophysiological conditions. In general, extracellular proteases

catalyze the hydrolysis of large proteins to smaller molecules for subsequent

absorption by the cell whereas; intracellular proteases play a critical role in the

regulation of metabolism (Rao et al., 1998).

The extracellular proteases are of commercial value and find multiple

applications in various industrial sectors. Today, proteases account for

approximately 40% of the total enzyme sales in various industrial market

sectors, such as detergent, food, pharmaceutical, leather, diagnostics, waste

management and silver recovery. Proteases constitute a group of enzymes

which differs in properties such as substrate specificity, active site and catalytic

mechanism, pH and temperature for activity and stability profiles. Proteases are

currently classified into six broad groups: Serine-Threonine-Cysteine-

Aspartate-Glutamic acid-Metalloproteases. Based on optimal pH, they are

classified as acidic, neutral and alkaline protease.

Alkaline proteases (EC.3.4.21, 22, 24) are hydrolytic enzymes that hold

a huge share in the world enzyme market accounting for about 60% of total

enzyme sale. These enzymes have either a serine amino acid or a metal ion at

the catalytic centre and have been reported to operate under extreme

physiological conditions of temperature of (20 to 70 0C), pH (up to 12) and in

presence of organic solvents and detergents. Alkaline proteases are among the

most commercially exploited group of enzymes with applications in various

industries (Gupta et al., 2002b).

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Although alkaline proteases are primarily used as detergent additives,

they have wide range of applications in the fields of research and industries. In

food industry, they have been routinely used for various purposes such as

cheese making, baking, preparation of soya hydrolysates, and meat

tenderization (Ferrero et al., 1996). Proteases are invariably used in tonics,

especially for indigestion. Tannery is another major niche where these enzymes

are used for dehairing and refining of hides and skins to get quality leather,

Extraction of silver from used X-ray films and degumming of silk to improve

its luster (Kanehisa, 2000; Puri, 2001) are other areas of appliance. Alkaline

proteases are also used for waste treatment, delignification of hemp (Dorado

et al., 2001), pest control (Kim et al., 1999) and synthesis of peptides (Isono

and Nakajima 2000).

Pleureryn an aspartic protease with molecular weight 11.5 kDa was

isolated from fruiting bodies of the edible mushroom Pleurotus eryngii. The

maximum enzyme activity was found at pH 5.0 and temperature 45 0C. The

activity of the protease was adversely affected by pepstatin A. The isolated

protease exhibited inhibitory activity against HIV-1 reverse transcriptase their

by effecting translation in a rabbit reticulocyte lysate system (Wang and Ng,

2001).

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Proteolytic activity from sporophores of Agaricus bisporus mushroom

was found to be high in early stage of sporophore development when compared

to mycelium. Gill tissues of mushroom had a higher activity than the stipe or

pileus (Burton et al., 1994).

Protease from fresh fruiting bodies of the edible mushroom Pleurotus

citrinopileatus with molecular mass of 28 kDa was purified by Cui (2007).

Maximum activity was obtained at pH 10 and temperature 50 0C. Enzyme was

found to be activated by metal ions such as K+ and Li+, but found to be

inhibited by Al3+, Cu2+ and Hg2+ ions. The Km and Vmax values were found to

be 3.44 mg / ml and 0.139 mg ml 1 min 1 respectively. The enzyme was devoid

of ribonuclease and antifungal activities.

Pleurotus sajor-caju was found to produce maximum protease enzyme

on fourth day of incubation in a media containing 3% corn and 0.8%

ammonium nitrate under optimum growth temperature and pH conditions.

During purification, 2.72 fold purification was achieved with a recovery of

53% enzyme activity. Maximum enzyme activity was observed at 60 0C

temperature and pH 8.0. The molecular weight of purified protein was 48 kDa.

Metal ions such as Ca2+ and Na2+ were found to decrease the enzyme activity

(Ravikumar et al., 2012b).

Cordyceps militaris protein (CMP) was purified from the dried fruiting

bodies of C. militaris with molecular mass of 12 kDa and pI of 5.1. Protease

activity was found to be maximum at temperature 37 0C and pH range 7-9.

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 Review of Literature

PMSF strongly inhibited the enzyme activity. Strong antifungal effect and

cytotoxicity was observed against Fusarium oxysporum and human breast and

bladder cancer cells (Park et al., 2009).

Proteases from Grifola frondosa (ProGF) was purified with optimum

temperature 55 0C and pH 7.0. About 80 % of maximal enzyme activity was

recorded with pH range of 4.5–8.5 and temperature range of 50–75 0C. The

protease was substrate-specific mainly cleaving at Ala14-Leu15, Tyr16-Leu17 and

Pro28-Lys29 bonds. The peptide concentration increased as the average peptide

chain length decreased indicating the presence of both endopeptidases and

aminopeptidase activities (Nishiwaki et al., 2009).

Antithrombotic activity of a protease purified from a medicinal

mushroom Ganoderma lucidum exhibited concentration dependent inhibitory

effects on platelet aggregation induced by adenosine diphosphate (ADP).

Protection of mice against thrombotic death, paralysis induced by collagen and

epinephrine in a dose dependent manner was observed. Fibrinolytic activity

was also observed along with coagulation (Kumaran et al., 2011).

Fibrinolytic protease of molecular mass 21.32 kDa in monomeric form

was purified from Schizophyllum commune. The result revealed the increase of

specific activity by 9.29-fold in culture broth. The protease activity was

inhibited by EDTA and enhanced by magnesium. The maximum protease

activity was observed at pH 5.0 and at temperature 45 0C (Chung et al., 2010).

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A protease with a molecular mass 30 kDa was isolated from fruiting

bodies of the wild edible mushroom Termitomyces albuminosus with optimal

pH 10.6 and temperature 60 0C. The enzyme was stable in the presence of 2 %

Tween 80 and 4 M urea. More than 80 % of the enzyme activity was retained

in 2 % Triton X 100, 54 % in 10 mM EDTA and 31 % in 2 % SDS. The

enzyme was strongly inhibited by PMSF suggesting that it is serine protease.

The protease was inhibited by Hg2+, Cu2+ and Fe3+ metal ions. The Km and
1
Vmax values were 8.26 mg · ml and 0.668 mg · ml 1· min 1
respectively

(Zheng et al., 2011).

Two fibrinolytic proteases designated as FFP1 and FFP2 with molecular

mass 32 kDa and 42 kDa was purified from the culture supernatant of

Fomitella fraxinea mycelia. Both enzymes had the same optimal temperature

(40 0C), but different pH optima of 10 and 5 respectively. FFP1 activity was

completely inhibited by PMSF and aprotinin indicating as serine protease. The

activity of FFP2 was enhanced by the addition of Co2+ and Zn2+, whereas

inhibited by Cu2+, Ni2+, and Hg2+. Furthermore, FFP2 activity was strongly

inhibited by EDTA & 1,10-phenanthroline indicating that the enzyme is a

metalloprotease. Both enzymes readily hydrolyzed fibrinogen. Km and Vmax

values of FFP1 were found to be 0.213 mM and 39.68 units / ml respectively

(Lee et al., 2006).

A 20.9 kDa metalloprotease was isolated from dried fruiting bodies of

wild mushroom Lepista nuda that functioned maximum at pH 7.0 and

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 Review of Literature

temperature 50 0C respectively. The Km and Vmax values were found to be

-1
6.36 mg / ml and 9.11 min-1. The activity was adversely inhibited by

EDTA-2Na and enhanced by Fe2+ ion. Inhibitory activity against HIV-1

reverse transcriptase, hepatoma Hep G2 and leukemia L1210 cells were also

observed (Wang et al., 2011).

A novel protease with a molecular mass of 15 kDa was purified from

fresh fruiting bodies of the wild mushroom Amanita farinose with optimal

pH 8.0 and temperature 65 0C. The protease inhibited proliferation of human

hepatoma HepG2 cells (Sun et al., 2011).

The effect of different temperature and growth stages of blue oyster

mushroom on the activity of celluloytic and pectinolytic enzyme in terms of

loss in viscosity was reported by Jatav (2012a). The cellulase and

polygalacturonase transeliminase activity was high after inoculation,

polygalacturonase (PG) was maximum at primordia initiation stage, while

polymethyl galacturonase and pectin transeliminase (PTE) were maximum at

young stage after inoculation. All the enzymes investigated were more active at

25 0C.

Serine protease of molecular mass 28 kDa designated as hmsp was

isolated from fresh fruiting bodies of edible mushroom Hypsizygus marmoreus

by Zhang, (2010). The enzyme was found to be thermo liable, exhibiting

temperature and pH optima of 50 0C and 7.5. Protease activity was affected by

PMSF, EGTA and aprotinin.

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