5. material and methods - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/10043/10/10_chapter...

Material and Methods

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5. MATERIAL AND METHODS

For the present investigation exploration tours were undertaken to different

localities of North West India in the monsoon period during the years 2007 to 2011 so

as to collect the material and make field observations. The field study and collection

trips were undertaken to Mandi, Chailchock, Barot, Bada Gram, Hamirpur, Kullu,

Manali, Shimla, Karsog, Cheog, Narkanda, Kufri, Chail, Solan, Rajgarh, Nahan,

Nouhradhar, Baijnath, Dharamshala, Kangra and Palampur in H.P.; Chakrata,

Mussoorie, DehraDun and Dhanoulti in Uttrakhand and Patiala, Rajpura, Fatehgarh

Sahib, Ropar, Hoshiarpur, Chattbir and Chandigarh in Punjab (Map 1). As a result of

these trips a total of 152 collections were made during the monsoon period which is

quite favourable for the collection purpose due to humid and warm climatic

conditions for their growth. Local collection forays were undertaken throughout the

year, however, the collections from distant localities were made during monsoon

season only. Many of the localities were visited time and again to understand the

ecological succession amongst different mushroom genera. The material and methods

with respects to the collection, taxonomy and identification of mushrooms, raising of

pure culture, biochemical analysis and molecular characterisation of these fungi has

been outlined in the ongoing account. The details about the composition of different

media, buffers and abbreviations used has been given in Appendix I, II and III

respectively. Information about research papers published has been given in Appendix

IV.

5.1. Collection of the material

Extensive fungal forays were undertaken to the various localities in North

West India during 2007 to 2011 for collection of material. Only fresh, clean and

healthy materials were collected in their all stages of development in abundant


43

quantity. The collected fruit bodies were wrapped in sheets in such a way that

collections can be rolled up inside with the ends twisted closed or standing up. The

fruit bodies with insect or worm holes were discarded. Field notes including details

about locality, altitude, collection number, date of collection, habitat, soil type,

association with forest type, photograph and other characters which are likely to be

lost during transportation were recorded on a note paper which was further tagged to

the paper packet containing the respective collection. Due precaution was taken to

avoid mixing of materials during transportation. Small, fragile and delicate specimens

were carefully kept above the stout and larger specimens in the collection bag so as to

protect them from any damage due to mutual mechanical pressure. Ethno-mycological

information was also collected by filling questionnaire, personal observation and

interviews with local, experienced persons and informants during monsoon season.

Amongst interviewees, about 30 - 40 % were aware about the ethno-mycological uses

and majority of them were the elders.

5.2 Taxonomy

5.2.1. Macroscopic characters

The field characters pertaining to gross morphology, shape, colour and size of

pileus, stipe, location, texture, disposition and consistency of various structures of the

fruit body, etc. were noted down on the field key (Atri et al., 2005 a). The colour

terminology of Kornerup and Wanscher (1978) was used for recording the colour of

spore prints and various carpophore parts. Delicate and fragile specimens were

worked out on priority basis followed by fleshy and viscid carpophores. Tough and

leathery ones were attended at the end as in delicate fragile mushrooms there is a

possibility of many characters being lost.


44

5.2.2. Spore deposit

The spore prints were taken in the temporary laboratory set up at each

collection site. Mature cap of the carpophores was removed by cutting the stipe near

the point of its confluence with the pileus with a clean knife and placed on a piece of

paper, with gills facing downwards. The spore print paper and cap was covered with a

bowl or petriplate after placing a water soaked cotton plug alongside the inverted cap

so as to maintain the humidity around the cap. The arrangement was then allowed to

stand for half an hour to about more than an hour depending upon the consistency of

the pileus. Larger and fleshy specimens usually took less time while smaller thin and

delicate specimens took more time for good spore deposit. The colour of the spore

deposit so obtained was noted on the field key (Atri et al., 2005 a) after comparing it

with the standard colour chart (Kornerup and Wanscher, 1978) immediately after it

was obtained. Spore print bearing paper of individual mushroom was then dried for

about 10 minutes at room temperature and finally wrapped in the standard wrapper

especially designed for the purpose. The wrapped spore print containing packets were

properly numbered and further stacked vertically in the labelled cardboard boxes.

These were then stored in dark, cool, and dry place. Crystals of 1 - 4 para-

dichlorobenzene were also placed in card boxes so as to check insect infestations.

5.2.3. Drying and preservation

After finishing spore prints and field notes / photographs, the specimens were

preserved for further microscopic study and onward deposition in the herbarium. For

this purpose, the collected material was divided into two unequal halves. The smaller

part of the collection was preserved in the liquid preservative1 (Hawksworth et al.,

1995) for further microscopic investigations and the major portion of the collection

was hot air dried in a wooden drier especially designed for this purpose (115 x 45 x

125 ml Rectified alcohol + 5 ml Formaline + 70 ml Distilled water


45

45 cm). The larger and stout specimens were placed in the lower and middle

chambers of the drier whereas the delicate and smaller ones were placed in the upper

chamber. Specimens were dried slowly at a temperature not exceeding 45ºC. The time

taken for drying the specimens varied depending upon the texture of the carpophores.

After drying, the material was finally packed in the moisture proof cellophane paper

packets. To avoid insect infestation small sachets of 1 - 4 para-dichlorobenzene

crystals were placed in these cellophane paper packets containing dried mushrooms

(Smith, 1949). Each packet containing desired sample was further wrapped in

standard white paper wrapper and the relevant data pertaining to each collection was

pasted on the packets. The standard herbarium packets (15 x12 cm) were finally

arranged in the especially designed cardboard boxes (38 x17 x 13 cm) in a vertical

fashion so as to avoid any damage to them due to mutual pressure. All the collections

examined during the present study have been deposited in the Herbarium of Botany

Department, Punjabi University, Patiala (PUN)1.

5.2.4. Macrochemical colour reactions

Macrochemical reactions were performed on the various parts of the

carpophores by using various dyes, stains and reagents as given below.

Melzer’s reagent

2

Melzer's regent is used to stain fungal tissue and exosporial ornamentation.

Typically three reaction types are common when stained with it. Amyloid or Melzer's-

positive reaction, in which the material reacts and gives blue to black colour,

pseudoamyloid or dextrinoid reaction, in which the material upon staining turns

brown to reddish-brown and inamyloid or Melzer's-negative reaction, in which the

1PUN: This is an abbreviation alloted to the Herbarium of Botany Department, Punjabi University, Patiala, by

the International Bureau for plant taxonomy and nomenclature of the International association for plant

taxonomy, Netherlands (Holmgren & Keuken, 1974).

21.5 g of Potassium iodide + 0.5 g Iodine + 20 ml Distilled water + 22.0 g Chloral hydrate

http://en.wikipedia.org/wiki/Amyloid_(mycology)

http://en.wikipedia.org/wiki/Dextrinoid


46

tissues do not change colour, or react faintly to become yellow-brown. Reaction with

Melzer's reagent is important in the taxonomy of light spored taxa.

Potasium hydroxide (KOH 10%)1

It is a standard chemical used to revive the dried material of all the groups of

Agaricales including lepiotoid and termitophilous mushrooms for anatomical study.

Cotton blue2

It is a hyphal stain used for staining the thin sections of mushrooms and light

coloured spores. Some spores and tissues which absorb it and turns blue and show a

contrast with the interior of tissue the reaction is called as cyanophilous while the

negative reaction is described as acyanophilous. The stained tissues can be easily seen

under the microscope as this dye is readily absorbed by the hyphal walls. The

laticiferous elements stain deep in this dye. Staining of agarics with cotton blue is of

immense taxonomic significance.

Cresyl blue3

This dye gives metachromatic reaction with the spores of light spored families

in which endosporium turns purplish, pinkish or reddish as is the case in tribe

Leucocoprineae of family Agaricaceae. This feature differentiates members of

Leucocoprineae from other tribes of the family.

Lactophenol4

It is used for mounting the mushroom sections. It can be used as such or for

clarity 1% solution of cotton blue lactophenol is prepared and used as mountant.

110.0 g KOH + 100 ml Distilled water

20.05 gm Cotton blue + 30 ml Lactic acid

31 gm Cresyl blue in 90 ml Distilled water

41gm Cotton blue+ 100 ml Lactic acid (25 ml Lactic acid + 25 ml Phenol + 25 ml Glycerin + 25 ml

Distilled water)


47

Congo red (2%)1

It is used for staining light spored agarics for clarity of sections.

Acetocarmine (1%)2

It is used on the basidia of light spored agarics to see the siderophilic reaction.

On the basis of siderophilic reaction of basidia tribe Lyophyllaceae is differentiated

from other tribes of family Tricholomataceae. In former the basidia show

carminophilic reaction i.e. on its application siderophilous granules are seen in the

basidia of members of tribe Lyophyllaceae which differentiates it from members of

other tribes.

Phloxine (2%)3

It is used for clarity of interior structure of hyphae and germpore in spores of

tribe Leucocoprineae of family Agaricaceae.

5.2.5. Microscopic observations

It includes the microscopic study of various carpophore parts as gills,

basidiospores, pileus, stipe, etc. The basidiospores for the study purpose were taken

from the spore deposit on the glass slide in a mounting medium. If the spore prints

were not available then the spores were obtained by teasing the gills. For revival, the

spores were treated with a drop of absolute alcohol, then dilute KOH and finally

washed. The various characters of spores with respect to their shape, size, thickness of

wall, ornamentation and apicular details, etc. were noted and the size of the

basidiospores was measured under oil immersion with the aid of ocular micrometer.

Anatomical details of various carpophore parts were examined by cutting free hand

sections from wet/dry materials. Dried material was softened for section cutting by

12 gm Congo red +100 ml Distilled water

245% Acetic acid + 1g Carmine

32 g Phloxine +100 ml Distilled water


48

soaking it for a short period of time in absolute alcohol so as to drive the air out and

then revived in 10% KOH. The revived material was finally washed in water and

embedded in pith for section cutting. After cutting the sections, these were stained in

cotton blue and mounted in lactophenol. Observations for internal details with respect

to cuticle, basidia, cystidia, hymenophoral trama, context, clamp connections, etc. was

done under oil immersion lens. The measurement of each carpophore part was taken

and recorded. Microphotography of the internal details was done under Nikon Eclipse

80 i microscope. Camera Lucida drawings of the worked out taxa with respect to their

microscopic details have been drawn under oil immersion lens. The magnification of

each drawing has been mentioned at appropriate place on the plate of individual

taxon. On the plates the morphology of the carpophores have been drawn to their

natural size.

5.3 Raising of the pure culture

The glassware used during the course of study was first washed with tap water

and subsequently dipped in chromic acid mixture (100 g Potassium dichromate + 400

ml Distilled water + 600 ml concentrated Sulphuric acid) for overnight (Tuite, 1969).

Then it was washed thoroughly with soap solution and tap water. Final washing was

given with distilled water and then dried in an oven maintained at 170ºC for 1 hour.

After drying, the flasks and test tubes were plugged tightly with sterilized cotton

plugs. Before use, the glassware was again sterilized in an autoclave at 15 lbs pressure

for 30 minutes. Along with this inoculation needles, pipettes, forceps, and cork borer,

etc. were also sterilized.

The pure cultures of some of the mushrooms, namely Termitomyces radicatus,

T. heimii, T. mammiformis, Macrolepiota rhacodes, M. dolichaula, Lepiota humei and

Leucocoprinus cepaestipes, were raised on the Potato Dextrose Agar (PDA) and Malt

Extract medium by inoculating small portion of carpophore flesh taken from the point


49

of confluence of stipe with the pileus aseptically. Through repeated subculturing the

cultures were purified. Cultures of only those species were raised which were

nutritionally and nutraceutically important. The composition of the media used for

raising the cultures has been given in Appendix- I.

5.4. Identification

Identification of the presently investigated taxa was done by consulting

relevant literature. Some of these publications are by Pegler (1977, 1983, 1986),

Dennis (1952), Smith (1949), Heim (1942 a, b, c, 1977), Sundberg (1971 b, 1989),

Arora (1986), Heinemann (1968, 1973, 1975), Sands (1970), Westhuizen and Eicker

(1990), Smith and Sundberg (1979), Vellinga (2001, 2007) and Atri et al., (2005 b),

etc. Besides this some of the identified collections were confirmed by comparing

them with already submitted collections available in the Herabarium of Botany

Department Punjabi University, Patiala (PUN). All the cultures are deposited in the

GenBank of Directorate of Mushroom Research, Chambaghat, Solan (H.P.). For

taxonomic treatment of the order Agaricales, and various families and genera

investigated, Singer (1986) and Kirk et al. (2008) has been followed. For

nomenclature purposes, International Code of Botanical Nomenclature (McNeill et

al., 2006) has been followed. During investigation many new taxa has been named.

Latin diagnosis for the new taxa proposed will be provided at the time of publication.

5.5 Nutritional and nutraceutical evaluation

Some of the edible lepiotoid and termitophilous mushrooms have also been

analyzed for the presence of proteins, carbohydrates, crude fat, crude fibers, moisture

content, ash content, phenolic compounds, flavonoids, carotenoids, alkaloids,

vitamins, mineral elements and heavy metals in mgs/100g of the sample following

standard biochemical techniques. To assess the nutritive values, fruit bodies were

oven dried at 40°C to constant weight and then ground into fine powder and stored for

further studies.


50

5.5.1 Protein content (AOAC), 1990

Procedure:-

Two gram mushroom sample was digested with conc. H2SO4 (25 ml) using

digestion mixture (Potassium sulphate and Copper sulphate 10 %). The contents were

diluted and volume made to 100 ml. Then 5 ml of aliquot was distilled with NaOH

(40%). Liberated ammonia was trapped in HCl (0.01N) containing methyl red

indicator and then titrated with .01 N NaOH. Nitrogen present in the sample was used

to calculate the crude protein present by using factor 6.25.

Titrate volume x 00014 x Volume made

Nitrogen (%) = ---------------------------------------------------------- × 10

Aliquot taken x Wt. of sample

5.5.2 Estimation of crude fiber (Maynard, 1970)

Two gram of ground material was defatted with petroleum ether. After

defatting, it was treated with 200 ml of diluted H2SO4 for 3 minutes with bumping

chips. The suspension was filtered through linen cloth and washed with water until it

became acid free. Then, the residue was subjected to alkali digestion by boiling with

200 ml of NaOH solution for 30 minutes. Filtered again with linen cloth and first

washed with 25 ml of boiling 1.25% H2SO4, then washed three times with 50 ml of

water and 25 ml of alcohol. The residue was removed and transferred to ashing dish

(pre-weighed dish W1). The residue was dried for 2 hr at 130±2°C. The dish was

cooled in a desiccator and weighed (W2). It was ignited for 30 minutes at 600±15°C

and cooled in a desiccator and reweighed (W3). The crude fiber content was

determined using the following formula.

Loss in weight on ignition (W2 - W1) - (W3 - W1)

Crude fiber (%) = -------------------------------------------------------------- × 100

Weight of the sample


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5.5.3 Crude Fat (AOAC 1995)

Transferred dried sample after moisture determination to a thimble and

plugged the top with cotton. Poured 75 ml or more of anhydrous ether through the

sample in the tube into the flask. Extracted the sample for 16 hrs or longer on a water

bath. When the ether has reached a small volume, poured it into a small dry

previously weighed beaker. Rinsed the flask thoroughly using several small portions

of the ether. Evaporated the ether on a steam bath at low heat then dried at 100 ºC for

1 hr and finally cooled and weighed.

Calculations:- Wt. of ether soluble material

Crude fat (%) = --------------------------------------- X 100

Wt. of sample

5.5.4 Ash (AOAC 17th

Edition 2003)

Test portion of 5 to 10 g of the sample was heated slowly over the flame in 50

to 100 ml silica crucible at 100°C until water is expelled. Placed the dish in a furnace

at 525°C and left it until white ash is obtained. Cooled and moistened the ash with

water, dried on steam bath and then on hot plate. Re-ashed at 525°C to constant

weight.

Ash % = (Final crucible wt – Initial crucible wt X 100)/Sample weight

5.5.5 Determination of moisture content (AOAC 17th

Edition 2003)

5.0 g of dried sample was taken in a moisture dish and placed in a hot air-oven

at 105 ± 2°C for 1 hr. The dish was covered, cooled in a desiccator and weighed. The

process of heating, cooling and weighing was repeated until the difference in mass

between two successive weighing was less than 1 mg.

100 × (M1-M2)

Percentage of moisture = -------------------

(M1-M)


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Where M1 refers to Mass in g of the dish with the material before drying; M2 refers to

Mass in g of the dish with the material after drying; M refers to Mass in g of the

empty dish.

5.5.6 Carbohydrate

Total carbohydrate amount was calculated by employing the following formula

100 - (g of Moisture + g of Protein + g of Fat + g of Ash + Fiber).

5.5.7 Assessment of energy value

Carbohydrate, fat and proteins, which act as the source of energy that is

interchangeable in terms of energy according to their specific chemical compositions.

The energy values were calculated by the method of Crisan and Sands (1978) using

the following formula.

Energy value (Kcal/100g) = Protein × 4 + Fat × 9 + Carbohydrate × 4

5. 5.8 Mineral and heavy metal estimation

For this purpose 1g of the mushroom sample was digested in 15 - 20 ml of 1:3

Perchloric acid and Nitric acid solution. The sample was kept for overnight and the

very next day it was heated slowly by gradually increasing the temperature from 30ºC

to 60ºC. The heating process was continued till the formation of precipitate. To the

white powder residue left in the flask, distilled water was added depending upon the

range of that very mineral. The water was added drop wise with continuous stirring so

that the minerals left in the powder residue are completely dissolved in the water and

then it was filtered with the help of Whatman filter paper. During filtration 15 - 20 ml

of water was used for dissolving the minerals and subsequently the volume was raised

to 25 ml. Atomic Absorption Spectrophotometer (Perkim Elmer) was used for

quantitative estimation of minerals. For this purpose the standard range was fed in

software and depending upon the range, dilutions were done and volumes calculated

according to the numbers of dilutions made. The conversion of the values so obtained


53

in the required quantity, subsequent calculations to determine the amount of mineral

in the sample was done.

5.5.9. Phenolic Compounds

Sample preparation

The fruiting bodies were air-dried in a Lyophilizer (Ly-Christ Alpha1-2) and

powdered before analysis. The dried samples (5 g) were extracted by stirring with 100

ml of methanol at 25 ±ºC at 150 rpm for 24 hrs. and filtered through Whatman No. 4

paper. The residue was then extracted with two additional 100 ml portions of

methanol, as described earlier. The combined methanolic extract was evaporated at 40

±ºC to dryness and redissolved in methanol at a concentration of 50 mg/ml, and stored

at 4 ±ºC for further use.

Determination of total phenolic compounds

Phenolic compounds in the mushroom extracts were estimated by a

colorimetric assay, based on procedures described by Singleton and Rossi with some

modifications (Singleton and Rossi, 1965).

Procedure: For this purpose, 1 ml of sample was mixed with 1 ml of Folin and

Ciocalteu’s phenol reagent. After 3 minutes, 1 ml of saturated sodium carbonate

solution (20 g dissolved in to 100 ml of distilled water) was added to the mixture and

adjusted to 10 ml with distilled water. The reaction was kept in the dark for 90

minutes, after which the absorbance was read at 725 nm in Analytikijena 200 - 2004

spectrophotometer. Gallic acid was used to calculate the standard curve (0.01 – 0.4

mM; Y = 2.8557X- 0.0021; R2 = 0.9999) and the results were expressed as mg of

gallic acid equivalents (GAEs) per g of extract.


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5.5.1.0 Flavonoid contents (Yoo et al., 2008)

Procedure: For the determination of flavonoid contents, the methanol extract sample

(250 μl) was mixed with 1.25 ml of distilled water and 75 μl of a 5% NaNO2 solution.

After 5 minutes, 150 μl of a 10% AlCl3.H2O solution was added. After 6 minutes, 500

μl of 1M NaOH and 275 μl of distilled water was added to the mixture. The solution

was mixed well and the intensity of pink colour was measured at 510 nm. Chatequin

was used to calculate the standard curve (0.022–0.34 m M; Y = 0.9629X-0.0002; R2

=

0.9999) and the results were expressed as mg of chatequin equivalents (CEs) per g of

extract.

5.5.1.1 Estimation of β-carotene and lycopene (Barros et al., 2007 c)

Procedure: For β-carotene and lycopene determination, the dried methanolic extract

(100 mg) was vigorously shaken with 10 ml of acetone–hexane mixture (4:6) for 1

minute and filtered through Whatman No. 4 filter paper. The absorbance of the filtrate

was measured at 453, 505 and 663 nm. Contents of β-carotene was calculated

according to the equations (Barros et al., 2007 c) of β-carotene (mg/ 100 ml) = 0.216

× A663 - 0.304 × A505 + 0.452 × A453 and lycopene (mg/100 ml) = -0.0458 A663 + 0.372

A505 - 0.0806 A453. The results were expressed as μg of carotenoid/g of extract.

5.5.1.2 Alkaloids estimation (Maxwell et al., 1995)

Procedure: The alkaloids were extracted from 5 g of each of the dried powdered

mushroom samples using 100 ml of 10 % acetic acid, which was left to stand for 4

hrs. The extracts were filtered to remove cellular debris and then concentrated to a

quarter of the original volume. To this concentrate, 1 % Ammonium solution was

added drop-wise until the formation of precipitate. The alkaloids thus obtained were


55

dried to a constant weight at 65 ºC in an oven. The percentage of alkaloids was

calculated by using formula.

Weight of residue

Percentage alkaloids % = ------------------------------------ × 100


5.5.2 Estimation of Vitamins

5.5.2.1 Estimation of retinol (Vitamin A) (Indian pharmacopoeia, 1996)

Procedure:- For this purpose 3 g of sample, 5 ml of 50% (w/v) potassium hydroxide

solution and 50 ml of ethyl alcohol were added and refluxed in a water condenser for

1 hr. The solution was then cooled and transferred to a 500 ml separator, to which 50

ml of hexane was added and shaken vigorously for 5 minute resulting in the formation

of two separate layers. The organic layer was passed through sodium sulphate into a

200 ml volumetric flask while the aqueous layer was shaken 3 times by taking 30 ml

hexane each time. All the organic layers were pooled together and diluted to 200 ml

with required amount of hexane. The absorbance in UV spectrophotometer at 325 nm

was recorded. Using the factor 1830, the vitamins A content was calculated which is

expressed as International units (IU), which primarily refers to their biological

potency.

Sample absorbance x 200 x 1830

Vitamin A (IU)/100g sample = ------------------------------------------- × 100


5.5.2.2. Estimation of Thiamine (Vitamin B1) (Indian Pharmacopoeia, 1996)

i) Preparation of buffer solution

To 6.8 g of potassium di-hydrogen phosphate, 8 ml of 1 M sodium hydroxide

solution was added and diluted with 1000 ml with water.


56

ii) Preparation of dye solution

To prepare it, 0.06 g Bromothymol blue was dissolved in 100 ml of

chloroform.

iii) Preparation of standard solution

For this purpose Thiamine hydrochloride RS (100 mg) was dissolved in 100

ml of water.

Working standard solution

One ml of stock was diluted with 100 ml of sample buffer for preparation of

sample solution. To 10 g of sample powder, 100 ml of buffer was added and filtered

through Whatman filter paper.

Procedure: 10 ml of sample solution and working standard solution were taken in

two different dry separating funnels. 10 ml of chloroform and 10 ml of dye solution

were added to both of the solutions and shaked for 2 minutes continuously. Then,

these were allowed to stand for 5 minutes with occasional shaking. The chloroform

layer was collected by passing it through sodium sulphate anhydrous. The readings

were taken at 420 nm using Shimadzu UV 118 spectrophotometer. Chloroform was

used as blank.

SAA × STW × 1 × 10 × 10 × 1 × STP

Thiamine HCl (mg)/ = ---------------------------------------------- × 100 ×1000

100g of sample STA × 100 × 100 × 1 × SAW × 10 × 10

Where SAA represents sample absorbance; STA represents standard absorbance;

SAW represents sample weight, STP represents standard purity while STW represents

standard weight.

Determination of thiamine

Thiamine (mg) /100 g of sample =

Molecular wt. of thiamine (300.77)

Thiamine HCl (mg) /100 g of sample × -----------------------------------------------

Molecular wt. of thiamine HCl (337.2)


57

5.5.2.3 Estimation of riboflavin (Vitamin B2) (Indian Pharmacopoeia, 1996)

Procedure: To 5 g of sample powder, 150 ml of water and 5 ml of glacial acetic acid

were added. The solution was boiled for 5 minutes and then cooled. To this, 30 ml of

1.0 M sodium hydroxide solution was added and diluted to 550 ml with water. The

solution was filtered and absorbance was measured at 444 nm in Shimadzu UV-1201

spectrophotometer. Water was used as blank.

SA 500 1

Riboflavin (mg) /100 g of sample = ------ × ----- × ----- 100 × 1000

328 SW 100

Where 328 represents molar extension coefficient; SA represents sample absorbance;

while SW represents the sample weight.

5.5.2.4 Estimation of Ascorbic acid (Vitamin C) (Indian Pharmacopoeia, 1996)

a) Preparation of metaphosphoric acetic acid solution (MPAA)

To 15 g of metaphosphoric acid, 40 ml of glacial acetic acid was added and

diluted with 100 ml of water.

b) Preparation of 2, 6-dichlorophenol indophenol solution

2, 6-dichlorophenol indophenols salt (0.05 g) was diluted with 100 ml of water

and the solution was filtered.

c) Preparation of standard solution

Stock solution

To 0.05 g of L-ascorbic acid standard, 20 ml of MPAA solution was added

and diluted with 250 ml water.

d) Preparation of sample solution

To 10 g of sample powder, 20 ml of MPAA solution was added and then it

was diluted with 500 ml water. Subsequently the solution was filtered through filter

paper.


58

Procedure: - To 10 ml of standard stock solution, 5 ml of MPAA solution was added

and titrated against 2, 6 – dichlorophenol - indophenol solution till the appearance and

persistence of pink colour for 10 seconds. The titration was completed within 2

minutes. The titer value was noted. Sample solution (100 ml) was taken and same

procedure was repeated.

SAV x STV x 10 x 500 x 1x STP

= -------------------------------------------------- × 100

STV x 250 x 1 x SAW x 100 x 100

Where SAV refers to sample titre value; STV refers to standard titre value; STW

refers to standard weight; SAW refers to sample weight and STP refers to standard

purity.

5.6 Enzymes assay

5.6.1 Colorimetric detection of Cellulase enzymes

Activity of cellulase enzymes was detected by dye staining of carboxymethylcellulose

(CMC).

Composition of Cellulose basic medium (CBM) (g l-1

in DW H2O)

C4H12N2O6 5

KH2PO4 1

MgSO4.7H2O 0.5

KCl 0.5

Peptone 2

Yeast Extract 0.2

Agar 16

C19H42N.Br 1%

pH 6.5

Ascorbic acid

(mg)/100g of sample


59

Procedure: CBM medium was supplemented with 2 % w/v low viscosity CMC and

1.6 % w/v agar and then autoclaved and inoculated with test fungus after pouring. The

test fungus was incubated at 25±1˚C in darkness and allowed to grow for 4 - 6 days

until the colony diameter reached 30 mm. The plates were then flooded with 1 % w/v

aqueous solution of hexadecyltrimethyl ammonium bromide solution for 2 minutes.

The added solution was then poured off and activity was observed as clear zone

indicating degradation of CMC by the production of extracellular enzyme i.e.

Celluloase or CMCase.

5.6.2 Screening of Ligninolytic enzymes through spot test.

5.6.2.1 Spot test for Laccase (g l-1

in DW H2O) (Saparrat et al., 2000)

Regents used:-

Glucose 10

KH2PO4 2

MgSO4 0.5

CaCl2 0.1

C6H10O4 2.2

(NH4)2C4H4O6 0.5

Yeast 0.2

ABTS 0.2

Agar-Agar 16-18

pH 5

Procedure: Medium was autoclaved and poured in to sterilized petriplates, which

were then inoculated with test fungus after pouring. The test fungus was incubated at

25˚C in darkness. Laccase activity was observed as greenish blue coloration around

the inoculum showing the presence of Laccase. The pH of the medium was adjusted

at 5 before autoclaving at 15 psi for 20 minutes.


60

Extracellular enzymatic activity was measured by the colour intensity of the

medium. Plates were observed once a day for three consecutive weeks.

5.6.3. Extracellular Ligninolytic Enzyme Essay

For this purpose Wheat straw extract liquid medium and Czapek Dox liquid

medium were used. The composition of both these media has been provided below:

Wheat straw extract liquid medium (g l-1

in DW H2O)

Wheat straw 50

Glucose 10

PH 7

Czapek Dox liquid medium (g l-1

in DW H2O)

Glucose 10

K2HPO4 2

MgSO4 0.5

FeSO4 10 mg

KNO3 3g

Yeast extract 0.2 g

pH 7

The production of extracellular enzymes was carried out in the wheat straw

extract liquid medium and Czapek Dox liquid medium. The pH was adjusted to seven

before autoclaving at 15 psi for 20 minutes. 150 ml flasks containing 50 ml liquid

medium were inoculated with 5 – 8 days old five mycelium bits (5 mm in diameter).

Three replicates of flasks of both the media were incubated in static condition in BOD

incubator under shaking condition in a rotator shaker at 150 rev/minute at 30ºC. The

cultures were harvested at the 5th

, 10th

and 15th

day of incubation. Each sample was

centrifuged (10,000 g for 10 minute) at 4ºC. The supernatant of liquid culture was

used for enzyme assay. The enzymatic reactions were carried out in triplicate and


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determined using a double beam Perkin Elmer Lambda 12 UV/VIS

spectrophotometer. All the enzyme activities were measured at room temperature (20

± 2ºC). The enzymatic activity was expressed as International units (U) defined as the

amount of enzyme required to produce 1 mol product min-1

and expressed as U-1.

5.6.4 Estimation of extracellular protein

Protein concentration was determined following Bradford method (1976).

Protein content in the sample was determined from standard curve and the amount of

protein µg ml-1

was calculated. The assay reagent was made by dissolving 100 mg

coomassie blue G-250 in 50 ml of 95% ethanol. The solution was then mixed with

100 ml of 85% Ortho-phosphoric acid (OPA) and made up to 1 liter by adding water.

The reagent was filtered through Whatman no. 1 filter paper and then stored in an

amber bottle at room temperature. The reagent was used after 24 hrs.

Bovine serum albumin (BSA) was used as the protein standard because it is

inexpensive and readily available in pure form. BSA was dissolved in 100 ml 0.15 M

NaCl solution (100 μg/ml for the microassay). Plastic and glassware cuvettes used in

the assay were absolutely clean and detergent free. Quartz (silica) cuvette was not

used, as the dye binds to this material (Bradford, 1976).

For microassay, 900 μl protein reagent was added to the 100 μl of the sample

gently so as to avoid foaming which results in poor reproducibility. Absorbance was

measured at A595 of the sample and standard against the reagent blank (1 ml). The

absorbance of each sample was measured after 2 and 30 minutes of addition of

protein. The amount of protein in the sample was determined from standard curve and

the amount of protein μg ml-1

was calculated.


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5.6.5 Estimation of laccase an extracellular enzyme (Han et al., 2005.)

Laccase activity was measured following the oxidation of ABTS (є420, 36,000

M-1 cm-1). Took one plastic cuvette and added 950 µl of sodium malonate buffer and

50 µl of ABTS. Took another cuvette and added 950 µl of buffer, 50 µl of ABTS and

50 µl of sample. Adjusted the factor 555.5 in UV/VIS spectrophotometer. Observed

the absorbance of enzyme substrate complex. Repeated steps 2-3 for all the samples.

Vt x 106

Formulae for calculating factor: -----------------

E x L x Vs

Where Vt refers to total reaction volume; E refers to e value of nm; L refers to

path length and Vs refers to Sample enzyme volume.

5.7 Molecular characterization

Molecular characterization of some of the mushroom samples has been done

by following methods:-

Reagents used:-

(a) CTAB (Cetyl trimethyl Ammonium bromide) buffer – 100 ml (b) TAE 50X-

100 ml

(c) T:E (10:1) buffer solution- 100 ml (d) Sodium acetate (3M) 24.6g/100 ml

(e) Chilled Ethanol (100%) (f) Ethanol (70%) (g) Cholorform: Isoamyl alcohol (24:1)

(h) Phenol: Chloroform (1:1) (i) RNase (10 mg/ml) (j) Proteinase K (10 mg/ml)

All reagents were prepared in double distilled water

5.7.1 Methodology for DNA isolation: For molecular characterization, the genomic

DNA was extracted from the fruit bodies of collected specimens of few of the taxa.

a. DNA isolation (Van Kan et al., 1991)

All the isolates were grown in liquid shake culture on Malt broth medium in

100 ml Erlenmeyer flask containing 25 ml of medium at 25°C for five days. The total

DNA was extracted from 100 mg of fungal mycelium crushed with micro-pestle in

conical micro-centrifuge tubes with liquid nitrogen. The dried powder of the


63

individual carpophore was added in an extraction buffer containing Tris-HCl (200

mM) at pH 7.5, NaCl (250 mM), EDTA (25 mM) and SDS (0.5%), and treated with

1μg/μl ribonuclease A. During incubation at 65°C for 20 minutes, the cell suspension

was mixed thoroughly by inverting the eppendorf tube several times. To it was added

0.5 ml of pure equilibrated phenol. After thorough mixing it was allowed to stand for

15 minutes followed by addition of 0.5 ml of chloroform: isoamylalcohol mixture

(24:1). Again after thorough mixing it was allowed to stand for 15 minutes and

followed by centrifugation for 20 minutes at 12,000 rpm. The upper aqueous phase

was removed carefully without taking any of the interphase material and transformed

it into a fresh eppendorff tube. Again 0.4 ml of chloroform: isoamylalcohol (24:1)

mixture was added to it and mixed by inversion by centrifugation at 12,000 rpm for

10 minutes. The supernatant was transferred into a new tube followed by addition of

0.54 volumes of isopropanol so as to precipitate the DNA. After thorough mixing by

inversion, it was left for 15 minutes at room temperature and then centrifuged at

15,500 g for 10 minutes. Supernatant was discarded and the DNA pellet so obtained

was washed with 70% ethanol so as to remove salts. After centrifuging for 5 minutes,

the supernatant was removed and the pellet was dried for 10 - 15 minutes. The DNA

pellet was resuspended in 300 μl of 0.2 M ammonium acetate and was left to stand for

overnight at 4°C. Afterwards, DNA was precipitated by adding 600 μl of ethanol. The

DNA pellet so obtained after centrifugation was washed with 70% ethanol and dried

under vacuum. Then the pellet was suspended in 25 μl of MQ water.

5.7.2 DNA quantification

The quantification of the genomic DNA content was done with UV-vis

spectrophotometer of Hitachi model U-1500 by measuring optical density at 260 nm


64

and 280 nm following strict instructions in order to remove contaminants, which

otherwise can hamper in manipulation of DNA.

5.7.3 Electrophoresis of DNA on agarose gels

DNA was loaded on agarose gel (0.7% w/v) prepared in 0.5 × TAE, pH 8.0

using a 6x loading dye. Ethidium bromide (0.5 μg/ml) was added to stain the gel prior

to pouring. The DNA was then electrophoresed at 3 volts/cm for 45 - 60 minutes and

visualized on a U.V. transilluminator. The composition of TAE is given in

Appendix II.

5.7.4 . Spectrophotometric quantification of DNA

The concentration of extracted DNA in suspension was estimated by

spectrophotometric measurement at A260. Double-stranded DNA suspensions gives an

OD of 1.0 at a wavelength of 260 nm using a cuvette with 1 cm light path which is

treated equal to a concentration of 50 μg/ml in terms of quantity. The quality of the

DNA was evaluated by measurement at A260/A280. Ideally, for pure DNA the

A260/A280 ratio should be 1.8 - 2. Any variation from this ratio of absorbance will

indicate impurity in the isolate DNA. Ratios (A260/A280) less than 1.8 indicates protein

or phenol contamination, while ratios greater than 2.0 indicates the presence of RNA

along with.

5.7.5 Ethidium bromide fluorescent DNA quantification

DNA was migrated electrophoretically in an agarose gel containing ethidium

bromide (0.5 μg/ml). The quantity of DNA was visually determined with reference to

a known DNA concentration of lambda phage (Fermentas, USA) by comparing the

intensity of fluorescence.


65

5.7.6 Amplification of internal transcribe spacer (ITS) region

Internal transcribe spacer (ITS) region of the rDNA from genomic DNA was

amplified by PCR using the primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and

ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) (White et al., 1990). The 50 μl reaction

mixture for PCR amplification was prepared which contained 10 ng DNA, 1x PCR

buffer, 1.5 mM MgCl2, 0.2 mM of each dNTPs, 0.5 μM of each primer and 2.5 units

of Taq DNA polymerase (Fermentas, USA). Amplifications were performed in a

thermal cycler (Perkin Elmer, USA) with an initial denaturation step of 94ºC for 5

minutes followed by 35 cycles of 94ºC for 1 minute, 54ºC for 1 minute and 72ºC for 1

minute and a final extension of 72ºC for 10 minutes. Control containing no DNA

template was included for the presence of contamination of reagents and reaction

buffer. Aliquots (5μl) of amplification products were electrophoresed on a 1.5%

agarose gel and visualized on a UV transilluminator. Success of amplifications done

was confirmed by agarose gel (0.8% w/v) electrophoresis and ethidium bromide

staining.

5.7.8 Sequencing

The ITS of nrDNA sequences of all the isolates were compared with those

available in GenBank databases using BLAST program (Altschul et al., 1997). The

sequences of ITS products and nrDNA large subunit were aligned to minimize the

number of inferred gaps. The sequences were edited with BioEdit 5.0.6 (Hall 1999)

and aligned using MAFFT v 6.240 with other sequences obtained from GenBank. All

sequences were submitted to NCBI database.


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5.8 Statistical analysis

For each mushroom species three samples were analysed and also all the

assays were carried out in triplicate. The results are expressed as mean values and

standard deviation (SD). These were analyzed using one-way analysis of variance

(ANOVA). Significance was accepted at P≤0.05.

5. material and methods - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/10043/10/10_chapter...

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