6

Chapter 2 REVIEW OF LITERATURE

The Chinese mushroom has very old history. This mushroom has been used

as food for human beings since Cho dynasty about 3000 years ago in China.. It

was introduced in southeast Asian countries by overseas Chinese, (Baker, 1934

and Benemertio, 1936), since then, its cultivation has been conducted in various

countries outside the China, like Philippines, (Clora, 1937; Go, 1959), Malaysia

(Baker 1934; Sands 1935), Burma (Seth, 1944) and Thailand (Jalavicharama,

1950;Hashioka, 1962). The history of the Chinese mushroom cultivation is very

old. As far as its artificial cultivation is concerned it is believed that, it was begun

in Nanhua temple of Chaohsi, Kwantung province in southern China, almost 200

years ago, (Chang, 1977)

2.1. METHODS OF CULTIVATION

2.1.1. Fungus culturing

Munjal, (1973) reported that the productivity of spawn culture was related

to the formation of chlamydospores. He described that culture beds with dens

chlamydospores always produced a high yield of mushroom. Hence paddy straw

pieces mixed with 4% chalk powder were best suited for spawn production of

Chinese mushroom.

Delmas and Sun, (1984) reported the traditional method of Chinese

mushroom culture in humid tropical climate. For modern culture, a high cellulose

7

substrate having C/N ratio was required. It was sterilized after fermentation at

60°C for 24 hours. After spawning the temperature was required to be maintained

at 35°C-38°C for about 4 days, then reduced 30°C to encourage flushing and

raised again after flush but casing is not necessary.

Qumio, (1988) made attempts to recycle the spent rice straw residues both

from Volvariella spp. and Pleurotus spp. either producing another crop of the

same mushroom or producing another mushroom from same substrate, thus fully

utilizing the straw for production of edible mushrooms before using the spent

compost for feed.

Luh, (1996) described the cultural methods developed in Taiwan. He

reported that rice straw covered by compost was used as the medium for beds and

production started 10-12 days after inoculation.

2.1.2. Cultivation forms

Ho, (1972) suggested an economical plastic house with a bamboo frame

structure that for indoor cultivation of Chinese mushroom consisting of polythene

film of 0.1mm was lined inside this mushroom house and has a layer of sugarcane

leaves on top, to block solar radiation and minimize heat penetration during hot

summer .An electric blower with a one forth horse power and a polythene air duct

was used to provide ventilation.

8

Grahum and Yaung, (1974) observed that beds of 32 cm depth gave the

highest yield of 2.25Kg /100Kg of waste and maintained at a temperature of 35°C-

38°C slightly below the optimum from mycelial growth of Volvariella volvacaea,

throughout the cropping season .

Purkayastha et al., (1981) reported that orientation of paddy straw beds has

a profound effect on the fruiting bodies production of Volvariella volvacea helix

and tyre types of beds ere found to be more productive than the conventional criss-

cross type. Several wastes for Chinese mushroom were used as substrates. Among

them cotton and jute wastes were most and least productive, respectively,

supplementation gram flour up to 600g / bed augmented the production. Climatic

conditions during June and July in west Bengal appeared to be the most favorable

for the production of Volvariella volvacea.

Qumio, (1986) reported that water hyacinth could be used for growing not

only Volvariella mushroom but also Pleurouts and Auricularia. The mushroom

grows fast and yield more if grown in this substrate than on rice straw. It could also

be used as substrate for preparation of the spawn either alone or in combination with

other substrates such as rice straw and saw dust. All parts of the plant including roots

could be used in making beds for Volvariella mushrooms or in the preparation of

mushroom bags for Pleurotus and Auricularia.

Li, (1989) studied the cultivation of Volvariella volvacea on wheat straw in

fields after the wheat harvest. High yield of Volvariella volvacea and 20% increase

in yield of the subsequent wheat crop was achieved. He also described the techniques

9

concerning the treatment of wheat straw compost, cultivation management and pest

control.

Wang, (1990) observed the key points in the management of Volvariella

volvacea grown on rice straw in open fields in relation to the stages of spawn

production, mycelial growth and fruiting bodies formation.

Pan and Li, (1990) studied the construction and design of semi-underground

sheds. Cultivation techniques including the selection of high quality spores and

substrates, bed construction and the control of growth conditions were discussed.

Hua, (1990) grew Volvariella volvacaea strain V2 on a rice straw substrate

in an apple orchard or in shelters with conventional management. The former

method gave slightly higher yields and better quality fruiting bodies than the later.

Soil samples taken from around the trees 2 years after Volvariella volvacaea

cultivation showed improved organic matter, P and K contents. The orchard

environment which provided shade and high air humidity appeared to be

favourable for Volvariella volvacaea cultivation.

Nayak et al., (1990) reported the feasibility of growing paddy straw

mushroom Volvariella volvacea inside plastic tunnels was. Tunnels (4m x 1.7m x

0.085m) were prepared with transparent UV stabilized film (200) in 2 beds (0.9m

x 0.9m) were made one of the beds was covered with black LDPE (Low density

polyethylene) film (150) under the UV stabilized film. The average mushroom

yield at the end of 41 days after spawning was 2.7 kg/m2.

10

2.2. SOLID STATE FERMENTATION

Garo, (1964) used different substrates such as paddy straw dried banana stalks

and leaves, water hyacinth, wheat straw and sugarcane baggasse for mushroom

production. He observed only the beds made of sugarcane baggasse did not produce

mushrooms. The yield obtained from the beds of banana leaves was superior to the

yield obtained from any other substrate.

Gupta et al., (1970) tried wheat maize barley oat pearl millet, and sorghum

straw but the yield was very low as compared to that produced on paddy straw.

Before, 1970, paddy or rice straw was practically the only material used for

preparing the medium for commercial cultivation of mushroom under natural

condition.

Chen and Graham, (1973) grew Volvariella volvacea successfully on oil palm

pericarp waste. French material composed for five days before spawning gave the

highest yield of about 1.54Kg/100 Kg of waste. Bed temperature may be one of the

most important factors affecting the incubation period and duration of crop.

Chang, (1974) studied the cultivation of Volvariella volvacea on cotton waste

compost in plastic green houses. The compost was prepared by adding 4% rice or

wheat bran and 4-6% lime stone to cotton waste, after which it was soaked in water

and fermented for to 4 days .Production by this method was high and cropping was

also regular.

Graham, (1974) studied the performance of three field isolates collected from

oil palm bunch waste in Selangor and four isolates from Sarawak, Hong Kong,

11

Indonesia and Singapore, which were compared in two experiments. The Hong Kong

isolate yielded 5-6 Kg/100Kg of substrate and gave a maximum bed yield of over 3.1

Kg or 7.4 Kg/100 Kg of waste. The performance approached and surpassed yield

from paddy straw.

Madane et al., (1974) produced spawn of Volvariella volvacaea on paddy

straw mixed with 2.5% by weight of oat meal. Same results were obtained when

powered wheat or gram was substituted for the oat meal and when wheat or sorghum

straw or sugarcane bagasse was used instead of paddy straw.

Granhum, (1975) found a wide range of cultural characteristics and yielding

ability in single spore isolation from five cultures of Volvariella volvacaea. A

selected isolate from Hong Kong culture out yielded the parent culture by about

125% and one from a Sarawak culture out yielded its parent by about 199%.

Jablonsky, (1981) studied the substrates consisting of standard mushroom

compost prepared with 11 days at stage 1, and 8 days at stage II, horse manure

composed for 3 days and wheat straw treated in various ways. Half of each substrate

was treated for 4 hours at 900C (pasteurized) and the other half at 1200C for 1.5 hour

sterilized. Pasteurization of substrates produced higher yield than sterilization. The

highest yield was with chopped wheat straw. The addition of 3% CaCO3 reduced the

yield.

Qumio, (1981) reported that the following substrates supported very well

mycelial growth of Volvariella volvacea, rice straw, Ipil-Ipil leaves, sigadillas leaves,

new paper prints, coconut coir dust and banana bracts. It took 8 days for mycelium to

12

fill up the entire diameter of the Petri dish containing the substrate. This only

confirmed the fact Volvariella, unlike Agaricus species, could grow directly on un

composted substrates and therefore could be considered less specific in growth

requirements than the later mushroom.

Devi and Nair, (1987) observed that Volvariella volvacaea spawn prepared

out of 3-10 days old culture supported maximum sporocarp formation, when used for

spawning the beds. Maize and wheat grains supported good mycelial growth and

found to be suitable substrate for spawn preparation. At room temperature of

(28+4OC) the spawn remained viable with out a reduced in yield for about 20-60

days.

Alam and Khan, (1989) calculated growth percentage index of locally

crushed fresh mill bagasse with 25%, 10% and 5% molasses. The growth percentage

index in the first flush ranged between 305- 425%, in the second flush between 283-

300%, in the third flush between 220-240% and in the fourth flush it was 180%.

Adewusi et al., (1993) determined the biological value of 5 mushrooms

Chlorophyllum molybditis, Psathyrella atroumbonata, Termitomyces robustus,

Termitomyces striatus and Volvariella esculenta by using weanling rats and

observed that T. robustus and V. esculenta did not support growth at all.

Khan et al., (1994) tried dried water hyacinth for the cultivation of

Volvariella volvacaea and concluded that water hyacinth + cotton waste at the rate

1:1 gave maximum yield of Chinese mushroom followed by dried water hyacinth

alone and cotton waste alone.

13

Chiu et al., (1996) described that the cultivated strains of shiitake in China

were genetically very homogeneous, very like to the cultivated strains of Agaricus

bisporus and Volvariella volvacea. However, their collection of L. edodes, covers

an enormous geographical area, (approx. 1700 km N to S, 700 km E to W) and

results demonstrated that the shiitake industry in China depends on an extremely

small gene pool.

Salmones et al., (1996) reported the mycelial growth of two Mexican

strains of Volvariella volvacea (Bull.: Fr.) Sing., in 13 agro industrial wastes. He

used, banana leaves, bracts of pineapple crown, coconut fiber, coffee bran, coffee

pulp, corn cob, corn stover, orange peel, rice bran, rice straw, sisal bagasse,

sugarcane bagasse and wheat straw as substrate and evaluated mycelial growth,

mycelial thickness and pinhead formations. Fruiting bodies were obtained only

from one strain growing in bracts of pineapple crown, coffee pulp, rice straw and

sisal bagasse. Primordia were developed between 13 and 15 days. He recorded that

the highest biological efficiency was achieved on rice straw, 33.8%, while the

results obtained for coffee pulp, sisal bagasse and bracts of pineapple crown were

15, 7.8 and 6.2%, respectively. Chemical analyses of the substrates registered C/N

ratios of 33:1 to 80:1.

Cheung, (1997) evaluated the feasibility of using food waste, such as soya

milk residue, to produce nutritive fungal biomass. They produced edible

mushroom mycelia of Volvariella bombycina, Lyophyllum ulmarius and Pleurotus

citrinopileatus, were produced in liquid culture containing soya milk waste. They

14

observed similarities in the crude protein, lipid, ash and nucleic acid contents

between the mycelia and fruiting bodies. Differences were observed in the amount

of total dietary fiber and amino acid composition.

Reyes et al., (1998) described the nutritional and physical requirements for

the efficient mycelial colonization of Volvariella volvacea (Bull. ex. Fr.). Singer.

The investigation was limited to the evaluation of two commercial strains

(designated Vvc1 and Vvc2) and two wild strains (designated EAAC-0001 and

EAAC-0002) of V. volvacea from the Philippines. The four strains of V. volvacea

had varying preferences for carbon. Vvc1 preferred polysaccharides (starch and

cellulose), whereas Vvc2 grew luxuriantly at a relatively rapid rate in sugar

alcohol (sorbitol). The two wild strains preferred starch as a carbon source. In

terms of nitrogen utilization, soytone, peptone, and glycine supported efficient

mycelial colonization of the four strains. Efficient colonization of Vvc1, Vvc2,

and EAAC-0002 with dense mycelial growth was noted in mycological agar.

EAAC-0001, on the other hand, grew more efficiently in malt extract agar. The

Philippine strains of V. volvacea grew luxuriantly when incubated at 35°C and pH

8.0 under dark and sealed conditions. They concluded that under optimum

physiological conditions, Vvc1, Vvc2, and EAAC-0002 were fast-growing strains,

whereas EAAC-0001 was a moderately growing type.

Tonial et al., (2000) used industrial residues from cassava and potato starch

processing as substrates to produce the edible mushroom Volvariella volvacea

(Bull. Fr.) Singer. Three strains of V. volvacea (LPB 08, 59, 77) were grown in a

15

medium containing 2.4% (w/v) declassified potato flour and 1.6% (w/v) cassava

bagasse (PFCB) in petri dishes. Growth performance of cultures was evaluated by

measuring their rate of radial growth and biomass production. Strain LPB77

showed highest growth on PFCB agar medium. The liquid PFCB medium was

optimized with regard to residue composition, nitrogen source and pH The best

results were obtained after 8, days of fermentation in a medium containing 4.8%

(w/v) declassified potato flour and 1.2% (w/v) cassava bagasse, pH5,

supplemented with 0.1% (w/v) of KNO3, giving a C/N ratio of 30.

Philippoussis et al., (2001) cultivated ten selected wild and commercial

strains of Pleurotus ostreatus, Pleurotus eryngii, Pleurotus pulmonarius,

Agrocybe aegerita and Volvariella volvacea on three agricultural wastes, i.e.

wheat straw (WS), cotton waste (CW) and peanut shells (PS). They observed that

one commercial strain of V. volvacea presented higher growth rates when the

composted CW medium was used. Furthermore, earliness in the fructification of

P. ostreatus, P. pulmonarius and V. volvacea strains was promoted in CW

substrates. They detected positive correlation between cellulose content and

mushroom yield for V. volvacea strains.

Obodai et al., (2003) evaluated the biological efficiencies (yield of the

mushroom against the dry matter of the substrates) of two strains of the mushroom

Volvariella volvacea, V99 and VVO, by using banana leaves, cocoyam peelings

and oil-palm pericarp as substrates. They observed that primodia were after11-12

days on banana leaves. Both strains showed their highest production on banana

16

leaves, with biological efficiencies of 43 and 72%. V99 fruited on all the

substrates but VVO fruited only on banana leaves as it had mycogone infection on

the other substrates.

Akinyele and Akinyosoye, (2005) cultivated the mushroom, Volvariella

volvacea on various agro wastes and observed Maximum mycelia extensions in

cotton waste (98.23 ± 0.1 mm) and a combination of rice husk and cotton waste

(101.87 ± 0.4 mm). A decrease in moisture content resulted in significant increase

percentage crude protein content of mushroom-treated waste compared to the

untreated they concluded that changes in crude fiber and ash content of treated and

untreated wastes were not significant, mineral contents were observed to increase.

Phosphorus and potassium ion content also increased in mushroom-treated

samples.

Belewu and Belewu, (2005) studied the solid state fermentation of banana

leaves by lignin degrading mushroom (Volvariella volvacea) ,yield of fruiting

body and compositional changes of substrates were evaluated .The biological

efficiency was 5.21while the total weight of fruit yield was 2.5 kg.

2.3. PHYSIOLOGY

2.3.1. Effect of temperature

Alicbusan and Ela, (1967) reported that the bed should be pressed lightly

because it must reach to the temperature of 40°C to 45°C because this temperature

favourable for the mushroom production but not for the organisms present in the

bed.

17

Chang, 1972 reported the best temperature for the growth and fructification

of Chinese mushroom is 26°C to 30°C and needs bed temperature of 34 to 37°C.

Agarwala, (1973) reported that tropical mushrooms can grow at a

temperature up to 45C or even more and the minimum temperature should not go

below 25C in any case even for a short period. He concluded that at higher the

temperature faster growth and higher yield will be obtained. The cropping period

lasted 25-30 days under poor temperature conditions.

Samajpati et al., (1977) studied the cultivation of V. volvacea on paddy

straw beds where growth conditions were satisfactory from April to August but

highest yield was obtained in July when the optimum temperature was 32C and

humidity was about 85%.

Khan and Kusar, (1981) reported prospects and potential of mushroom as a

cottage industry in Pakistan. Variety of climatic conditions such as northern hilly

areas with high humidity and low temperature were suitable for temperature kind of

mushrooms where as plains of Punjab, Sindh, and Balochistan were suitable for

cultivation of tropical mushrooms in summer. Many agricultural wastes and

industrial waste in the form of straws, leaves and cotton and corn wastes were

available in large quantities.

Ramakrishnan et al., (1986) observed that use of transparent polythene

sheet to cover the bed showed higher yield than with black polythene sheet and he

also reported the optimum temperature for Chinese mushroom cultivation was 35

to 37.

18

Morris et al., (1988) observed the changes in morphology and viability of

20 species of fungi including Volvariella volvacea during freezing were examined

in relation to cooling rate and the presence of glycerol. They observed that the

morphological response of Phytophthora, Aschersonia and Volvariella differed

from other genera, with shrinkage occurring at all rates of cooling.

Frank, (1989) recommended measures to obtain high yield of V. volvacaea.

The best planting date was when both day and night air temperatures were 23°C.

Lime was sprayed on the straw to increase the pH value from 7.5 to 8.5. The spawn

was planted at an age of 18-20 days.

Wang and Li, (1989) studied the various deformities occurred in cultures of

V. volavcea including the production of empty mycelium, abnormal bodies formed

from the hyphae, deformed fruiting bodies and withering of young mycelium. They

suggested that these deformities could be avoided by the maintenance of a constant

growing temperature in the range of 22-28C.

2.3.2. Effect of light

Yau and Chang, (1970) obtained fruiting under 12 hours light. They

reported the light intensity of 50lux was optimum. Antonio and Fordyce, (1972)

reported an appreciable quantity of light (15 minutes full sunlight) was required

for the initiation of fruiting bodies of Volvariella volvacea. The average yield was

84g/Kg dry weight compost.

19

Khan, (1976) described that the light is not required for the Chinese

mushroom during the period of spawn running, it respond favorably to weak light

condition and ventilation

Chakravarty and Mallick, (1979) reported that growth of V. diplasia was

vigorous in complete darkness, intermediate in diffused light and slowest in full

light. Light passing through red or blue filters produced good growth but light

from green or yellow filters inhibited the growth.

Singh and Saksena, (1983) mentioned that in case of V. volvacea light

intensity showed no effect on mushroom yield but colour, texture and shelf life of

mushroom grown under dark and diffused light conditions was better than the

mushroom produced under bright light conditions.

Fasidi, (1996) described that Volvariella esculenta (Mass) Singer, is able to

grow at a temperature range of 20-40°C (optimum = 35°C) and pH range of 3-10

(optimum = 6.0) and a wide range of agricultural wastes for growth. Of these the

rice straw and induced the widest mycelial extension and rice bran produced the

highest mycelial density. The unfermented cotton waste compost produced the

highest fruit body yield.

Banik and Nandi, (2000) described that V. volvacea known as paddy straw

mushroom grows well in humid and tropical environment, the biological

efficiency of this was very low in comparison to oyster mushroom. Low

productivity was a big hurdle for its commercial exploitation but the traditional

substrate for this mushroom cultivation, with biogas residual slurry manure

20

increased its production by producing fruit bodies bigger in size and higher in

number and also increased the protein content significantly. The mineral nutrients

viz., P, Ca, K, Fe, Cu, Zn and Mn were also increased. So, they concluded that

supplementation of this bio manure for cultivation of tropical mushroom

Volvariella volvacea may be a step towards its successful commercial

exploitation.

Zervakis et al., (2001) determined the influence of environmental

parameters on mycelial linear growth of different mushroom crops including

Volvariella volvacea in two different nutrient media in a wide range of

temperature. V. volvacea grew faster at 35°C, Wheat straw, peanut shells and

particularly cotton gin-trash supported fast growth of V. volvacea.

Jonathan et al., (2004) studies were conducted on the effects of

temperature, pH, vitamins and plant hormones on the vegetative growth of

Volvariella esculenta (Mass) Singer. They observed that, this mushroom had its

optimum radial growth at 35°C with mycelial extension of 85.0 mm and the pH

that supported best growth was 6.0. Pyridoxine was the most utilizable vitamin

with mycelial dry weight of 123 mg/30 cm3. They also observed that among the

tested phytoharmones, 2, 4-D (10.0 ppm) stimulated the best growth of 150 mg/30

cm3 but, 0.1 ppm of GA3 supported poor growth (53.0 mg/30 cm3).

Akinyele and Adetuyi, (2005) studied the effect of temperature variations

on the growth of V. volvacea cultivated on various agricultural wastes singly and

in various combinations. A pH range of 5.5 to 8.5 recorded the maximum mycelial

21

yield and the highest mycelia weight was recorded at pH6.5 while poor mycelia

growth of the mushroom and the least mycelia weight was recorded at pH 2.0.

Highest mycelia growth of mushroom was recorded between 25°C and 30°C and

the least mycelial dry weight of 0.5 mg obtained at 10°C.

2.4. LIQUID FERMENTATION

2.4.1. Background

Anon, (1954) while working in the Syracuse University, New York,

conculede that it is possible to grow the highly priced mushroom Morchella

hortensis, without any problem and at very low cost in water culture under

constant motion.

Atacador, (1967) reported that out of five edible mushrooms cultivated in

liquid medium, V. volvacea gave the highest mycelial yield. The best medium for

its production had pH 5, and contained 4% urea, 4% sucrose and best propagation

period was 5 days to get maximum mycelial yield.

Kostadinov et al., (1972) described a method of producing Pleurotus

ostreatus mycelium in sub-merged culture which may be used as inoculation

material (spawn) for production of sporophores on a substrate of crushed corn

cobs. They also reported the most suitable nutritive medium was a combination of

cane molasses with supplementation of NH4NO3, NH4SO4 and Potassium

dihydrogen phosphate.

Lin and Hu, (1972) reported that sugarcane baggase was found to be

suitable and more economical for mushroom cultivation as straw compost. The

22

dried product was amended by the wheat bran (10%), ammonium dihydrogen

phisohate (1%), calcium chloride (5%) and water (60%) followed by heat

treatment at 55°C for 72 hours.

Bukhalo, (1982) selected some species and strains of edible fungi for

biomass production in submerged culture. Similarly Khan et al., (1982) reported

that Pleurotus can be can grown in cane molasses medium and resulting biomass

can be used as a source of protein rich food.

Garo and Neelakanton, (1982) investigated the production of single cell

protein by Aspergillus terrens by using alkali treated baggase as potential

substrate. They obtained maximum biomass protein content of 20% by

continuously shaking of culture for seven days at pH 4.

Quimio, (1984) mentioned potential of using coconut water, first time as a

liquid medium for mycelial production of Lentinus sajor caju and other edible

mushroom, secondly as a routine agar medium for the growth of edible

mushrooms. He observed that coconut water both from young and mature nuts can

support the mycelial growth of edible mushrooms. After first week of incubation,

12gm of dry weight of Lentinus sajor- caju mycelia was harvested from 100ml of

sterilized coconut water. When the coconut water was incorporated with agar

medium it performed almost equally to the PDA medium. Trials with V. volvacea

and some other mushrooms showed that coconut water agar medium can be used

as a routine laboratory medium in place of potato dextrose agar medium.

23

Mahmood, (1986) optimized the concentration of inorganic growth

nutrients for the production of mycelial protein from rice polishing with

Arachniotus spp. Maximum crude protein percentage was obtained in the shake

medium containing calcium chloride 0.001%, magnesium sulphate 0.001%, and

potassium dihydrogen phosphate 0.08%, after 72 hours of incubation at pH 4 and

30°C temperature.

Dey et al., (1995) investigated biosorptions of Pb2+, Cr6+, Cd2+ and Ni2+ by

using live and dead fungal mycelia. Of the four fungi, namely Polyporus

ostreiformis, Volvariella volvacea, Pleurotus sajor-caju and Phanerochaete

chrysosporium, they observed total biosorption was effected in 6 days up to the

Pb2+ concentration of 6 mg/l, with a specific uptake of 1.33 mg Pb2+ /g dry cell

mass. The removal of other three metals varied between 28.8-73.3% from a

medium containing 4 mg/l of each of the metals.

Cai, et al., (1998) studied that the edible straw mushroom, Volvariella

volvacea (V-14), produced β-glucosidase when grown in liquid culture on a

variety of carbon sources including cellulose, cellobiose, salicin, sorbose, lactose,

esculin, cotton wool, and filter paper. They purified two cell-associated β-

glucosidases, BGL-I and BGL-II, 32-fold and 23-fold, respectively, from extracts

of cellulose-grown mycelium. The enzymes were found to be homogeneous and to

have native molecular weights of 158 kDa (BGL-I) and 256 kDa (BGL-II) by gel

filtration. Both isozymes displayed relatively broad pH optima with maximum

reaction velocities recorded at pH 7.0 for BGL-I and pH 6.2 for BGL-II, and were

24

rapidly denatured at temperatures of 60°C and above. Isozyme activities were

adversely affected by several reported β-glucosidase inhibitors, various metal ions,

and lignin-derived aromatic acids and aldehydes. Glucose production from

microcrystalline cellulose by a commercial cellulase preparation was enhanced by

9.7% in reaction mixtures supplemented with BGL-II.

Chen et al., (2004) isolated a Laccase (lac1) from culture fluid of

Volvariella volvacea, grown in a defined medium containing 150 micro m CuSO4

subscript by ion exchange and gel filtration chromatography. RT-PCR analysis of

gene transcription in fungal mycelia grown on rice straw revealed that, apart from

during the early stages of substrate colonization, lac1 was expressed at every stage

of mushroom developmental cycle defined in their study, although the levels of

transcription varied considerably depending upon the developmental phase.

2.4.2. Liquid media used for fermentation

Edwards, (1954) observed the growth of edible fungi in sub-merged

culture, consisted of a liquid medium through which air was constantly bubbled.

The rapid growth of mycelium was obtained from one cream and one white

variety of cultivated mushroom Agricus compestris. He concluded that sub-

merged culture might be much cheaper than ordinary mushroom culture. The

mycelial biomass produced could be used for soups or flavouring of food items.

Block, (1959) concluded that yields were high and no special production

problems were encountered when mushroom mycelium were grown in sub-

merged liquid culture with carbohydrates and nitrogen compounds with mineral

salts.

25

Torver, (1968) worked out a technology for the cultivation of mycelium of

different species of higher fungi in liquid nutritive medium. He assumed that

mycelium of some mushroom species produced in liquid culture would be used as

spawn in mushroom cultivation.

Anthony, (1977) reported that fungi can be successfully propagated on a

wide variety of substrates. He found that protein contents of fungi were strain

dependant and influenced by the growth conditions.

Bukhalo et al., (1978) studied that Pl was successfully grown in liquid

nutrient medium containing 10% red clover extract, sucrose, peptone and mineral

salts on commercial scale. The fungus was cultivated on complex media

containing potato waste and molasses. The rate of mycelial growth was greater on

sub-merged culture than that in surface culture.

Nagaso and Yoshikawa, (1978) observed that mycelial growth and yield

was best with shaking at 100 cycles/minute rather than shaking at 90 or 120

cycles/ minute.

2.5. BIOCHEMICAL ANALYSIS

2.5.1. Nutrients contents

Tumwasorn et al., (1980) reported 11.5% to 12% ash in the straw of different paddy

varieties and crude fat contents of paddy straw before and after mushroom

production were reported to be 0.5% and 0.2%, respectively.

26

Vijaya nad Pandaya (1981) reported proximate composition of paddy straw

mushroom. The value for moisture fat, crude protein, carbohydrates, crude fiber and

ash were 92%, 0.21%, 2.19%, 1.15% and .99% respectively.

Khowala and Sengupta, (1985) purified the enzyme, endo-alpha-

mannanase, from culture filtrate of a mushroom Volvariella volvacea by acetone

precipitation, ion-exchange chromatography (DEAE-Sephadex), and gel-

permeation chromatographies on Bio-Gel P-300 and on Sephacryl S-200 columns.

They observed single protein band on sodium dodecyl sulfate-disc gel

electrophoresis at pH 6.8 and has a molecular weight of approx. 56,000. It has no

alpha- or beta-mannosidase activity and does not act on beta-gluco-or

galactomannan. The enzyme shows maximum activity on baker's yeast alpha-

mannan at pH 5.0 and at 55°C, and is fairly stable between pH 3 and 6 and

temperatures up to 50°C. Enzyme activity is inhibited by Hg2+, sodium azide,

iodoacetic acid, EDTA, and Ag+, in decreasing order.

Kalisz et al., (1986) concluded that Agaricus bisporus was grown on defined

liquid media with protein as sole source of carbon, nitrogen or sulpher and with these

nutrients supplied in the form of glucose, ammonium or sulphate. The culture

filtrates of Agaricus were tested for extracellular laccase activity. Constitutive

laccase production was observed under all conditions tested. However, Agaricus

laccase, though constitutive, was induced by protein and repressed by ammonium.

No detectable extracellular laccase activity was found in tested cultures of Coprinus

cinereus or Volvariella volvacea.

27

Kishida et al., (1989) concluded that A (1--3)-beta-D-glucan branched by

O-6 substitution (FCAP), obtained from the cold-alkali extract of the fruiting body

of V. volvacea, exhibited potent growth-inhibitory activity against implanted

tumors in mice. They suggested that the Volvariella glucan was structurally

heterogeneous with regard to the distribution of branches, having less branched,

moderately branched, and highly branched segments.

Cheung (1996) analysed the mycelia caps and stalks of fruiting bodies of four

edible mushrooms (Lentinus edodes, Lycophyllum shimeji, Pleurotus sajor-caju,

and Volvariella volvacea) for their total dietary fiber contents.The TDF cotents of

all of the mushrooms were considerably greater than those determined by using

the Uppsala method. Mushroom mycelia had higher TDF values than did the

fruiting bodies .The TDF composition of the mycelia and the TDF composition of

the caps and stalks of the mushroom fruiting bodies were similar. Sugar

composition reflected that β-glucans were the major fiber polysaccharide with

chitin, hemicelluloses, and polyuronides as minor ones.

Phutela et al., (1996) screened two strains of Volvariella diplasia (Vd IIHR

and Vd TNAU) and three of V. volvacea (Vv IARI, Vv MU and Vv TNAU) for

the production of cellulases and xylanases. They observed Vd IIHR exhibited

maximum activity of cellulases. This strain also showed maximum biomass

production (5.8 g I- broth) and yield potential (BE 8.6%). The xylanase enzyme

showed maximum enzyme activity (9.73 U mg-1 protein) in Vd TNAU strain. All

the enzyme activities were the maximum in culture filtrates after 8 d of growth.

28

Lin et al., (1996) crystallized Volvatoxin A2; an ion channel disturbed

cardiotoxic and hemolytic protein from the edible mushroom, V. volvacea, by the

vapor diffusion method using polyethylene glycol 4000 and ammonium sulfate in

sodium acetate buffer pH 4.6.

Zhi et al., (1998) purified a novel single chained ribosome inactivating

protein (RIP) from fruiting bodies of the edible mushroom Volvariella volvacea.

The mushroom RIP, designated volvarin, exhibited a potent inhibitory action on

protein synthesis in the rabbit reticulocyte lysate system. It also exerted a

deoxyribonuclease activity on super coiled SV-40 and demonstrated a strong

abortifacient effect in mice.

Wang et al., (1998) summarized existing information about mushroom

lectins, with an emphasis on those from the species which have been most

extensively characterized including various Agaricus species, Areanita

pantherina, Boletus satanas, Coprinus cinereus, Ganoderma lucidum, Flammulina

velutipes, Grifola frondosa, Hericium erinaceum, Ischnoderma resinosum,

Lactarius delerrimus, Laetiporus sulphureus, Tricholoma mongolicum and

Volvariella volvacea. Immunomodulatory and antitumour/cytotoxic activities have

been carried out on lectins from Agaricus bisporus, Boletus satanas, Flammulina

velutipes, Ganoderma lucidum, Grifola frondosa, Tricholoma mongolicum and

Volvariella volvacea.

Chiu et al., (1998) explained that recalcitrant nature, persistence and

toxicities of chlorophenols make them priority pollutants for treatment. He

29

compared the ability of various fungi including (Armillaria gallica, A. mellea,

Ganoderma lucidum, Lentinula edodes, Phanerochaete chrysosporium, Pleurotus

pulmonarius, a Polyporus sp., Coprinus cinereus and Volvariella volvacea), and

the spent mushroom substrate of P. pulmonarius (SMS) to remove

pentachlorophenol (PCP) using a batch cultivation system. All these fungi showed

active breakdown in addition to bio sorption as their PCP removal mechanisms.

Mau, (1998) irradiated fresh common (Agaricus bisporus) and high-

temperature mushrooms (A. bitorquis) with ultraviolet-C (UV-C) for 0, 0.5, 1, and

2 h at 12 °C. Fresh common, shiitake (Lentinula edodes), and straw mushrooms

(Volvariella volvacea) were irradiated with UV-B for 0, 0.5, 1, and 2 h at 12 °C.

After UV-C irradiation for 2 h, vitamin D2 contents in common and high-

temperature mushrooms increased from 2.20 and 4.01 µg/g of dry weight to 7.30

and 5.32 µg/g, respectively. After UV-B irradiation for 2 h, vitamin D2 contents in

shiitake and straw mushrooms increased from 2.16 and 3.86µg/g to 6.58 and 7.58

µg/g, respectively. The increase rates in shiitake and straw mushrooms were not as

high as in common mushrooms.

Yao et al., (1998) purified a novel single-chained ribosome-inactivating

protein (RIP) with a molecular weight of 29,000 from fruiting bodies of the edible

mushroom V. volvacea. The mushroom RIP, designated volvarin, exhibited a

potent inhibitory action on protein synthesis in the rabbit reticulocyte lysate

system with an IC50 value of 0.5 nM. It also exerted a deoxyribonuclease activity

on supercoiled SV-40 DNA and demonstrated a strong abortifacient effect in mice.

30

She et al., (1998) purified a novel lectin from the fruiting bodies as well as

cultured mycelia of the edible mushroom V. volvacea. They observed that the

lectin, designated as VVL, was a homodimeric protein and had no carbohydrate

moiety, and its hemagglutinating activity was inhibited by thyroglobulin. The

immunomodulatory activity was demonstrated by its potent stimulatory activity

toward murine splenic lymphocytes. VVL also enhanced the transcriptional

expression of interleukin-2 and interferon-γ by reverse transcriptase-polymerase

chain reaction. VVL possessed a molecular structure distinct from other

immunomodulatory proteins previously reported in the same fungus.

Cai et al., (1999) described that the edible straw mushroom, Volvariella

volvacea produced a multi component enzyme system consisting of endo-1, 4-b-

glucanase, cellobiohydrolyse, and glucosidase for the conversion of cellulose to

glucose. Biochemical analysis of different culture fractions in cultures exhibiting

peak enzyme production revealed that most of the endoglucase was present either

in the culture filtrate (45.8%) of the total or associated with the insoluble pellet

fraction remaining after centrifugation of homogenized mycelia (32.6%).

Cellobiohydrolyse distributed with 58.9% of the total enzyme present in cultural

filtrates and 31 % associated with the pellet fraction.

Whiteford, (2000) described the types, economic significance and methods

of production of the principal cultivated mushrooms are described in outline.

These organisms are all less than ideal for conventional genetic analysis and

breeding, so molecular methods afford a particular opportunity to advance our

31

understanding of their biology and potentially give the prospect of improvement

by gene manipulation. They also described the gene sequences isolated from the

paddy straw mushroom V. volvacea and many other mushrooms.

Isiloglu et al., (2001) determined the concentrations of Cu, Cd, Co, Ni, Mn,

Pb, Zn and Fe in 66 samples of mushroom fruiting bodies, representing seven

species, mainly all edible, were determined by atomic absorption

spectrophotometry. The mushrooms were collected from near roads and inner

parts of forest and lawns in Balikesir in the north western part of Turkey. The

results indicated that the Fe level in the species Volvariella speciosa (Fr.) Sing.

from near the road was the highest with a mean of 6990 mg/kg. The Cd was

accumulated mostly by Lactarius sanguifluus (Paulet: Fr.) Fr. and V. speciosa

from near road with a mean of 1.60 mg/kg.

Liu et al., (2001) studied the antiproliferative activity of a fungal lectin

(VVL) isolated from the mushroom, V. volvacea. It was observed that VVL did

not exert ribosome-inactivating activity or induce any changes in the expression of

cyclins A, D1, and E. However, it did activate the expression of cyclin kinase

inhibitors, namely p21, p27, p53, and Rb, in a dose-dependent manner. Flow

cytometric analysis demonstrated an accumulation of cells in the G2/M phase in a

time- and dose-dependent manner, indicating that VVL arrested cell proliferation

by blocking cell cycle progression in the G2/M phase.

Fu et al., (2002) studied the antioxidative potency of commercially

available mushrooms in Taiwan. The order suggested by him of inhibitory activity

32

of mushroom extracts on oxidation in emulsion system was Agaricus bisporus >

Hypsizigus marmoreus > Volvariella volvacea > Flammulina velutipes >

Pleurotus eryngii > Pleurotus ostreatus > Hericium erinaceus > Lentinula

edodes. In the thermal oxidative stability test, using lard, the order of antioxidative

activity of test materials showed similar tendencies, except for the extract of

Lentinula edodes.

Wang and Liu, (2002) isolated the lectin from the dried fruiting bodies of

the mushroom Agrocybe cylindracea a heterodimeric lectin with a molecular

weight of 31.5 kDa and displaying high hemagglutinating activity. The larger and

the smaller subunits resembled Agaricus bisporus lectin and fungal

immunomodulatory protein from Volvariella volvacea respectively in N-terminal

sequence. The lectin exhibited potent mitogenic activity toward mouse

splenocytes. The hemagglutinating activity of the lectin was inhibited by lactose,

salicyclic acid and inulin.

Ahlawat et al.,(2005) isolated and evaluated the six parent strains and 11

monosporous isolates from two strains of V. volvacea for their enzymes induction

level, substrate colonization and yield potential. They observed that all parent

strains preferred wild grass over wheat straw and paddy straw for vegetative

growth along with highest level of induction of β-glucosidase and xylanase.

Mushroom yield in parent strains was related to the level of various

lingocellulolytic enzymes produced on a substrate. Monosporous isolates and

33

parent strains produced more cellulases at 8 and 10 d, while xylanase, laccase and

polyphenol oxidase were more at 10 or 13 d growth on paddy straw.

2.5.2. Medical effects

Lin et al., (1974) isolated the cardiotoxic protein volvatoxin from

Volvariella volvacea and another cardio toxic protein isolated from the

Flammulina velutipes (Curt. ex Fries Sing), which is widely eaten in the Orient.

This protein is called flammutoxin. Flammutoxin has three biological activities

similar to those of volvatoxin, direct hemolytic action against human group ‘O’

blood cells; ability to cause a writhing reaction with a delay before onset; and

effect on the electrocardiogram at a dose of 0.25 mg per kg body weight, causing

depression of the ST segment and inversion of the T wave.

Fassold et al., (1976) isolated Volvatoxin A, present in the mushroom

Volvariella volvacea, causes a competitive, dose and time dependent inhibition of

the Ca2+ accumulating activity of a sarcoplasmic reticulum rich microsomal

fraction isolated from guinea pig ventricular muscle. They explained why

volvatoxin A increases the diastolic resting tension in heart muscle.

Kishida et al., (1992) purified a potent antitumor-active branched (1 → 3)-

β-D-glucan (VVG) from fruiting body of Volvariella volvacea. They observed that

conversion of the glucosyl groups substituted at 0-6 atoms of the (1 → 3)-linked

D-glucose residues into the corresponding polyhydroxyl groups gave significant

enhancement of the original activities, whereas deletion of the polyhydroxyl

groups resulted in a great reduction of the activity. When D-glucose residues of

34

the branches were modified to the 3,6-anhydro D-glucose residues. They observed

that the previous findings that, besides the conformation of (1 → 3)-β-glucan

backbone, the molecular shape and the distribution pattern of the substituted

groups located outside the backbone chains, must also play an important role in

exhibiting anti tumor action.

Chiu et al., (1995) described that the straw mushroom Volvariella volvacea

was one of the common edible mushrooms cultivated in Southeast Asian

countries. It has been reported to produce a hypotensive response in animals

including humans. An aqueous extract of the mushroom (SME) was prepared and

given through intravenous injections to normotensive rats. They examined the

effects of SME on the kidney function of water-loaded rats and on isolated tissue

preparations of the tail artery and right atrium. An IV injection of SME produced a

hypotensive effect in rats with an ED50 of 25 mg dry weight/kg body weight. This

hypotensive effect of SME was attenuated or blunted in the presence of

hexamethonium, phentolamine, pyrilamine and cimetidine suggesting the

involvement of the α-adrenergic component of the autonomic system and/or

histaminergic stimulation. They also observed that SME did not increase urinary

excretion nor sodium diuresis. It produced positive chronotropic and inotropic

effects on isolated right atria and induced contraction of isolated tail artery strips.

This latter contractile response was inhibited by antagonists of serotonin and α-

adrenoceptor, ketanserin and phentolamine respectively. Partial purification using

dialysis and liquid chromatography revealed that the hypotensive active

35

substances had molecular masses between 8000 and 12000 dalton. These

substances were heat stable and resistant to trypsin digestion. In view of the

similarity in blood pressure and cardiovascular response, SME might contain

serotonin-like substances.

Fasidi and Kadiri, (1995) studied the toxicological aspects of seven

Nigerian mushrooms, namely, Chlorophyllum molybditis (Mayer ex. fr.) Masse,

Cortinarius melliolens Fries, Lentinus subnudus Berk, Pleurotus tuber-regium

(Fries) Singer, Termitomyces robustus (Beeli) Heim, Tricholoma lobayensis Heim

and Volvariella esculenta (Mass) Singer. Amatoxin spot test and chromatographic

screening of the mushrooms revealed the absence of amatoxins and phallotoxins

because none of the mushroom extracts tested killed the experimental rats.

Cheung, (1996) fed male Sprague-Dawley rats with two semisynthetic diets

supplemented with 2% cholesterol and 1% β-glucan type extracellular

polysaccharide isolated from two liquid cultures of straw mushroom (Volvariella

volvacea) mycelium containing different carbon sources. They concluded that both

mycelial extracellular polysaccharides exhibited hypocholesterolemic activity in

rats with alimentary-induced hypercholesterolemia.

Fasidi and Akwakwa, (1996) studied the growth requirements of

Volvariella speciosa (Fr. ex. Fr.) Sing. were studied. They observed all the tested

carbohydrates except cellulose significantly enhanced mycelial growth and

Mannitol was the most utilized, followed in order by fructose and maltose. All the

organic and inorganic nitrogen sources investigated significantly improved

36

growth. Calcium, magnesium, sodium, potassium and zinc significantly enhanced

growth whereas hormones and vitamins did not.

2.5.3. Bioconversion of lignocellulosic wastes

Buswell et al., (1996) described that edible mushroom cultivation was one

of the most economically-viable processes for the bioconversion of many types of

lignocellulosic wastes. According to him Lentinula edodes, Volvariella volvacea

and Pleurotus sajor-caju were three important commercially cultivated

mushrooms which exhibit varying ability to utilize different lignocellulosics as

growth substrate he explained that V. volvacea, which preferred high cellulose-,

low lignin-containing substrates produces a family of cellulolytic enzymes

including at least five endoglucanases, five cellobiohydrolases and two β-

glucosidases, but none of the recognized lignin-degrading enzymes.

Rajor, (1996) described that the Sawdust, a bulky waste generated by wood

processing industries, has very few profitable and eco friendly uses and has a

problem of proper disposal. They observed that treatment with the fungus V.

volvacea and a dilute solution of urea converted sawdust from a phytoinhibitory

material to a phytostimulatory soil conditioner. Analyses of the major biopolymers

of sawdust after fungal treatment indicated the decrease in the levels of cellulose,

hemicellulose and lignin.

Chiu et al., (2000) described that mushroom cultivation was a direct

utilization of their ecological role in the bioconversion of solid wastes generated

from industry and agriculture into edible biomass, which could also be regarded as

37

a functional food or as a source of drugs and pharmaceuticals. They concluded

that, this was very true for Lentinula edodes, Volvariella volvacea, and

Ganoderma lucidum, which were commonly consumed in Asian communities but

are now gaining popularity worldwide. Besides the conventional method, strain

improvement could also be exploited by protoplast fusion and transformation.

Biodiversity is the key contribution to the genetic resource for breeding programs

to fulfill different consumer demands. Spent mushroom compost, a bulky solid

waste generated from the mushroom industry, however, could be exploited as a

soil fertilizer and as a prospective bioremediating agent.

Datta and Chakravarty, (2001) studied comparative utilization of

lignocellulosic componnts of paddy straw by Tricholoma lobayense and

Volvariella volvacea. They observed that T. lobayense degraded the lignin more

actively till the end of spawn run phase. However, V. volvacea could utilize

cellulose and hemicellulose throughout spawn run and cropping phases, but was

unable to utilize lignin at any stage.

Yadav et al., (2002) devised an optimized protocol for the bioconversion of

eucalyptus bark. It comprised: (i) mechanical reduction in bark size to 0.5-3.0 cm,

(ii) moistening to 60-65%, (iii) fortification with ligninase-rich fungus Volvariella

sp. (S-1) and 2% urea and (iv) maintenance of this composting mix under aerobic

and ambient condition for 14-15 weeks. The resulting bark soil conditioner (BSC),

with physico-chemical and microbial properties which would enrich soil

fertility/productivity.

38

2.5.4. Uptake of heavy metals Purkayastha and Mitra, (1992) observed the uptake of a few metals by V.

volvacea during submerged growth of the organism in sub lethal concentration of

each metal salt. They concluded that the uptake of Pb2+ and Hg2+ was 5 and 5.23

micrograms g-1 respectively while that of Cu2+ was 500 micrograms g-1 under

experimental conditions. Treatment of spawned substrate separately with different

metal salts showed maximum and minimum uptake of Pb2+ (100 micrograms g-1)

and Cd2+ (2.93 micrograms g-1) respectively by sporocarps. All metal salts at test

concentrations reduced biological efficiency of sporocarp production but markedly

by Co2+. Cd2+ and Co2+ were highly toxic to mycelia and sporocarps

respectively and the uptake of Cu2+ by mycelia and Pb2+ by sporocarps were

highest among the five metals tested.

2.6. GENETICS

Boekhout and Enderle, (1996) designated a neotype for Volvariella

gloiocephala (DC. Fr.) Boekhout & Enderle, to serve as a representative collection

for the current concept of this species, that generally was considered conspecific

with V. speciosa (Fr.: Fr.) Kummer.

Chiu and Moore, (1999) determined the electrophoretic karyotype of the

Chinese straw mushroom, Volvariella volvacea. They observed that haploid strain

V34 of V. volvacea has 15 chromosomes ranging in size from 1.4 to 5.1 Mb. No

chromosomal polymorphism in terms of size and number was seen in either of two

growth stages: vegetative mycelia or fruit-body gill tissues. They also prepared

39

DNA fingerprints by the arbitrarily-primed polymerase chain reaction. Variation

in DNA fingerprints was evident in protoplast regenerants derived from the same

vegetative mycelium. Thus the haploid mycelium of strain V34 was heterokaryotic

but the bulk genotype is stable during fruit body development. They overviewed

the known mechanisms to generate genetic variation and proposed a novel

mechanism that could account for the 1:1 segregation ratio of self-fertile to self-

sterile progeny regularly obtained from selfed Volvariella volvacea fruit bodies.

Ding at el., (2001) isolated an endoglucanase, EGI, from fluid of

Volvariella volvacea grown on crystalline cellulose by ion exchange and gel

filtration chromatography and preparative PAGE .Their work was aimed at

generating improved strains of V. volvacea with enhanced growth, substrate

conversion capacity and higher production capacity.

Guo et al., (2002) used PCR technique for amplifying THP gene in an

unknown vector with primer AFP1 and AFP2. Then THP gene was ligated to

pGEM T-Vector to be the plasmid pGTHP4. Then isolated and purified big

fragment containing promoter with the Agarose Gel DNA Extraction Kit, and also

purified the small fragment containing THP gene from 1% agarose gel with the

Agarose Gel DNA Extraction Kit. The big fragment and the small fragment were

ligated at Nco I digested cohesive-end. The ligation product was re-ligated to be

cyclic plasmid by addition to a specific adapter, resulting in the pCTH823, a

expression vectorof V. volvacea.

40

Shi et al., (2002) assessed aqueous extracts of the sporophores of eight

mushroom species for their ability to prevent H2O2-induced oxidative damage to

cellular DNA using the single-cell gel electrophoresis assay. They observed the

highest genoprotective effects with cold (20°C) and hot (100°C) water extracts of

Agaricus bisporus and Ganoderma lucidum fruit bodies, respectively. No

protective effects were observed with mushroom derived preparations (MDPs)

from Lentinula edodes, Pleurotus sajor-caju, and Volvariella volvacea. They

concluded that some edible mushrooms represent a valuable source of biologically

active compounds with potential for protecting cellular DNA from oxidative

damage.