Acta Biotechnol. 13 (1993) 2, 131-139 Akademie Verlag

Microbial Protein from Leguminous and Non-Leguminous Substrates

SHARMA, S . , MADAN, M.*

Centre for Rural Development and Appropriate Technology Indian Institute of Technology Hauz Khas New Delhi-110016, India

Summary

The biodegradation of leguminous and non-leguminous organic materials by Pleurotus sajor-cuju and P. ostreutus was studied. Comparisons were made between mushroom yield on both types of substrates. The conversion percentage from dry substrate to fresh mushroom (biological efficiency) was determined. Mushrooms were analyzed for their protein content, carbohydrates, percentages of ash, fat content and crude fibre. The energy value of fruit bodies was computed. The yield obtained from leguminous substrates was significantly high as compared to that yield which was obtained from non-leguminous substrates. The lowest yield of P. sujor caju (log & 0.3 g) and P. ostreatus (6.5 g & 1.2g) was obtained using the Bougainuilleu substrate and the highest one (519g & 16g, P. sujor-cuju and 4888 & 18g, P. ostreatus) using nitrogen- fixing leguminous Leucuena leucocepholea. The nitrogen content in fruit bodies was found to be higher with leguminous substrate than with non-leguminous ones.

Introduction

In India people suffer from malnutrition, especially because of protein deficiency. Huge quantities of horticultural and agricultural waste could be used for the cultivation of protein-rich food mushrooms. These have the ability to transform nutritionally value- less wastes into highly acceptable nutritive food [2,7, 13, 14, 171. Although mushrooms have far too often been regarded only as a delicacy, it would be more realistic to look upon them as food that can do much to relieve protein deficiency in developing coun- tries. They contain 30-40% protein on a dry weight basis and thereby surpass many other foods in terms of the protein content. The Pleurotus sajor-caju and P . ostreatus are Oyster mushrooms. The Oyster mushroom have been cultivated in some European and many other countries and information is available on culture techniques [6, 10, 11, 16, 201. RANGASWAMY et al. [16] demonstrated that this mushroom can also colonize unsterilized plant residues. KURTZMAN [12] has given a comprehensive review of the possibilities of nitrogen fixation by Pleurotus. The natural substrates for Pleurotus sp. (Oyster mush- room) have been listed by many workers [ I , 3, 15, 191. In the present study, leguminous and non-leguminous biomasses were used as the sub- strates for the cultivation of Pleurotus sajor-caju (also known as the Angel mushroom) and Pleurotus ostreatus. The aim of this paper is to use the biomass (waste) available

*Corresponding author

132 Acta Biotechnologica 13 (1993) 2

locally for the production of protein-rich food and to compare the leguminous (nitrogen- fixing) and non-leguminous substrates for the optimum yield of mushrooms. Pleurotus ostreatus is highly compatible with P. sajor-caju. All the substrates supplemented with paddy straw ( 1 : 1) were also tested. This paper describes methods for the cultivation of Oyster mushrooms (P . sajor-caju and P. ostreatus) using different kinds of waste as a substrate and reports the yields and nutritional values of this mushroom on different substrates.

Methods

Cultivation Methods

For the growth of P. sajor-caju and P. ostreafus, substrates selected for experimentation are repre- sented in Tab. 1. All leguminous and non-leguminous substrates were used separately as well as in combination with paddy straw in a ratio of 1 : 1. Paddy straw was kept as the control in both cases.

Spawn Preparation

The Pleurotus sajor-caju spawn was prepared on wheat grains and the P. ostreatus spawn on paddy grains. The spawns used in this study were prepared on autoclaved wheat and paddy grains, respectively, and incubated at 20 f 2°C for 15-20 days in glass bottles. The schematic process is as follows: 1 kg wheat and paddy grains is soaked over night and washed; boiled for 3-5 minutes ; added (2%) CaC03 + CaS04 (only in the case of P. sajor-caju); and then filled into 5 half litre bottles. These were filled to 75% of their total volume; plugged with cotton; sterilized at 15psi. for 50-60 min- utes; added inoculum and kept at 20 f 2°C for 15-20 days.

Cultivation Method for P . sajor-caju

20 fresh dry substrates (10 leguminous and 10 non-leguminous) were chopped into pieces (4-5 cm). These were soaked in hot water (60 "C) for about 3-4 h. The extra water was drained off. The wet substrate (in three replicates) was mixed with 8% spawn and 1 YO gram powder to provide

Tab. 1. Symbolic representation of treatments

Leguminous substate Symbol Non-leguminous substrate Symbol ~~

Phaseolus mungo straw L1 Oryza sativa straw N1 Cicer arietinum straw L2 Triticum aerinum straw N2 Phaseolus aurius straw L3 Cyamopsis tetragonoloba straw N3 Leucaena leucocepholea L4 Jatropha curcas (stem) N4 (thin branches + pod waste + seed coat) Cajanus cajan (branches) Acacia nilotica (thin branches) Prosopis julipora (thin branches) Mimosa pudica (thin branches) Cassia saemea (thin branches) Cajanus indica (branches)

LS Saccharum munja (leaves + stem)

L6 Lantana camara (branches)

L7 Pinus longifolia (needles)

L8 Morus alba (thin branches)

L9 Ricinus cornmunis (branches)

L10 Bougainvillea sp. (stem)

N5

N6

N7

N8

N9

N10

SHARMA. S., MADAN, M., Protein from Leguminous and Non-Leguminous Substrates 133

a nitrogen source. The substrate spawn mixture was placed in polythene bags. In each case 500 g of substrate was taken. These bags were provided with holes for proper aeration. The spawned bags were kept in a well ventilated hut at a temperature of 20-25 "C. The bags were opened after 18 days when the mycelium had covered the substrate. The spawn run substrates were placed on a shelf and within 7- 10 days of opening pin heads of mushroom appeared on all sides. These young mush- rooms attained the normal size in about 6-7 days when the first crop of mushrooms was harvested from each of the bags.

Cultivation Method for P . ostreatus

All 20 substrates (leguminous as well as non-leguminous) were pasteurized at 80 "C for 6 h. After cooling, the substrates were inoculated with P. osfreatus grain spawn and then incubated for about 14 days at 20-22 "C and 100% RH. The substrates were then put into plastic bags and fruiting took place. Crops were harvested in abundance.

Analytical Methods

Moisture:

The moisture content was determined by drying the fruit bodies to a constant weight at 105 "C [9].

Total Nitrogen :

The nitrogen content was determined by a Micro-KJELDAHL method [18]. The factor 6.25 was used to calculate the crude protein [5].

Crude Fats:

Crude fats were determined gravimetrically after a continuous extraction of the dried sample for 6 h with anhydrous ether (A.O.A.C., 7.056, 1980).

Carbohydrates:

The soluble carbohydrates were extracted with distilled water. The residue was refluxed with diluted hydrochloric acid (A.O.A.C., 8.019, 1980) for 2.5 h to solubilize starch; this was described as the "insoluble carbohydrates" [4].

Crude Fibre and Ash:

Crude fibre was determined by the method by SINGH and PRADHAN [ 181 where the dried sample was first made fat free and then treated with acids and bases. The obtained residue was then dried to a constant weight at 100 "C, and then ignited at about 600 "C to get ash. Crude fibre was the loss in weight due to ignition. Biologicuf Eflciency - The biological efficiency was calculated using the relation:

Wt. of fresh mushrooms harvested Wt. of dry substrate taken

Biological efficiency % = x 100

Results and Discussion

The average yields of P . sajor-caju and P. ostreatus are reported in Tab. 2 and 3, respec- tively, from three replicates grown on leguminous and non-leguminous substrates using the polythene bag method. The cultivation was continued for about 45 days during

Tab.

2a.

Yie

ld o

f Ple

urot

us s

ajor

-caj

u an

d Pl

euro

tus

ostr

eatu

s on

legu

min

ous

subs

trate

s e

W

P

Trea

tmen

t Y

ield

in c

rops

To

tal y

ield

M

oist

ure

Bio

logi

cal

Sym

bol

[gl

kl

[%I

effic

ienc

y ["/I

PS

I PO

I PS

I1

Pol

l PS

III

POIII

Ps

Po

Ps

Po

Ps

Po

LI

L2

L3

L4

L5

L6

L7

L8

L9

L10

345

266

85

300

292

125

323

240

114

328

281

142

218

285

153

216

232

190

223

278

172

201

204

67

312

185

58

301

229

104

Ps - P

leur

otus

saj

or-c

aju

Po - P

leur

otus

ost

reat

us

Tabl

e 2b

. Ana

lysi

s of

var

ianc

e (T

able

2)

91

68

10

160

75

3 17

1 43

6

162

49

45

57

49.2

41

20

8 40

12

41

34

52

93

92

41

13

1 13

2.5

80

92

23

32.5

498 f 21

50

0 f 20

.9

480 f 22

.6

519 f 16

42

0.2 f 25

.8

446 f 19

.2

429 f 23

.6

360 f 13

.4

502.

5 f 16

.9

428 f 6.

4

367 f 10

45

5 f 12

.1

417 f 23

48

8 f 18

.0

383 f 11

.2

352 f 1

3.4

371

f6

33

8 f 12

39

6 f 17

35

3.5 f 14

91.2

91

.4

90.3

91

.1

92.3

92

.4

90.2

3 91

.01

90.4

90

.09

92.3

92

.09

90.8

90

.98

91.8

92

.01

91.1

92

.03

91.3

2 91

.85

99.6

10

0 96

103.

8 84

.04

89.0

2 85

.08

72

100.

05

85.6

73.4

91

.0

83.4

97

.6

76.6

70

.4

14.2

67

.6

79.2

70

.7

Sour

ce

DF

Su

m o

f squ

are

Ps

Po

Mea

n sum

of s

quar

e F

valu

es o

bser

ved

PS

Po

Ps

Po

b 5

Rep

licat

es

2 1 1

3.70

17

18.6

56

.85

859.

3 0.

19

11.9

4 10

9830

.0

7697

.40

1220

3.3

25.8

8 16

9.58

Tr

eat m

en t

9 69

1 14.

66

Tota

l 29

74

568.

20

I 128

44.0

Er

ror

18

5339

.84

1295

.4

296.

65

71.9

6 8

Ps - P

leur

otus

saj

or-c

aju

For 9

and

18

degr

ees o

f fr

eedo

m

Po - P

leur

otus

ost

reat

us

Expe

cted

F v

alue

: At 0

.05

leve

l =

2.4

6 L

W

\o

\o

w

h)

At 0

.01

leve

l =

3.6

0 h

- v

Tabl

e 3a

. Yie

ld o

f Pl

euro

tus

sajo

r-ca

ju a

nd P

leur

otus

ostr

eatu

s on

non

-leg

umin

ous

subs

trat

es

Trea

tmen

t Y

ield

in

crop

s To

tal y

ield

M

oist

ure

Bio

logi

cal

kl

kl

["I ef

ficie

ncy

PSI

POI

PSII

P

oll

PSII

I PO

III

Ps

Po

Ps

Po

Ps

Po

[%I

NI

N2

N3

N4

N5

N6

N7

N8

N9

N10

350

240

75

325.

5 23

1 53

33

9 20

5 18

29

3 27

5 92

28

5 26

3 52

I43

134

5 30

2 25

5 80

33

8 25

0 42

-

-

-

10

6.5

- Ps

- P

leur

otus

saj

or-c

aju

Po - P

leur

otus

ost

reat

us

Tab

. 3b.

Ana

lysi

s of

var

ianc

e (T

ab. 3

)

43

64

23

28

70

33.5

22

34

12

.2

88.4

45

5.

8 48

11

.6

8.9

-

38.5

11

.6

85

48.8

39

.9

45

21.2

12

.5

-

-

-

- -

-

489 f 19

44

8.5 f 22

39

1 f 18

38

9.5 f 1

1 34

8.6 f 14

188.

5 f 7

430.

8 f 20

40

1.2 f 19

10

f 0.

3

-

306f

14

292.

5 f 18

23

9.2 f 18

36

9 f 1

3 31

9.9 f 18

145.

6 f 9

379.

9 f 17

30

7.5 f 21

6.

5 f 1

.2

-

90.3

91

.8

97.8

61

.2

89.9

90

.4

89.7

58

.5

90.1

90

.6

78.2

47

.84

91.8

91

.97

77.9

73

.8

90.6

91

.3

69.7

2 63

.98

89.3

90

.01

37.7

29

.12

90.4

91

.1

86.1

6 75

.98

90.3

90

.07

80.2

4 61

.5

91.0

2 91

.9

2.0

1.3

-

-

-

-

Sour

ce

DF

Sum

of

squa

re

Ps

Po

Mea

n su

m o

f sq

uare

Ps

Po

F

valu

e ob

serv

ed

Ps

Po

Rep

licat

es

2 15

37.5

17

26.5

9 76

8.75

86

3.29

10

.32

8.81

Tr

eatm

ents

9

8676

82.1

5 52

554.

51

9640

9.12

58

39.3

9 12

95.1

2 59

.60

Erro

r 18

13

39.9

9 17

63.4

9 74

44

97.9

7 To

tal

29

8705

59.6

4 52

9044

.59

Expe

cted

F v

alue

: R

epor

ted

in T

ab. 2

(Ana

lysi

s of

var

ianc

e)

vl K > U

P, 3 a r m 5 5'

0

E

v,

I

Tab.

4. C

hem

ical

ana

lysi

s of P

leur

otus

sujo

r-cu

ju a

nd P

. ost

reat

us o

n di

ffere

nt n

on-le

gum

inou

s sub

stra

tes

W

o\

Subs

trate

C

rude

fibr

e C

arbo

hydr

ate

[%I

Ash

Fa

t N

itrog

en

Cru

de

I”/I

[%I

[”/.I

prot

ein

(sol

uble

+ in

solu

ble)

I

I1

1%1

[”/.I

Ps

Po

Ps

(I)

(11)

Tota

l

N1

N2

N3

N4

N5

N6

N7

N8

N9

N10

I -

sol

uble

11

- in

solu

ble

13.2

1 11

.02

25.4

19

.6

45.0

12

.6

11.9

3 26

.3

18.7

45

.0

12.8

12

.03

25.8

20

.3

46.1

13

.4

11.6

24

.0

18.9

42

.9

13.6

12

.26

26.1

19

.4

45.5

13

.01

12.0

9 25

.9

20.6

46

.5

12.4

11

.21

24.6

18

.2

42.8

13

.0

11.1

0 25

.0

19.7

44

.7

12.9

12

.0

26.3

20

.6

46.9

12

.51

11.2

6 24

.0

18.4

42

.5

34.9

33

.3

36.8

38

.6

35.5

32

.9

36.3

34

.6

37.4

35

.8

(11)

27.3

26

.4

25.5

27

.3

28.6

25

.4

26.6

27

.7

26.8

25

.7

Tota

l

62.2

59

.7

62.3

65

.9

64.1

58

.3

62.9

62

.3

64.2

61

.5

..

Ps

Po

Ps

Po

Ps

Po

Ps

Po

6.7

5.2

1.92

1.

00

4.48

4.

32

26.0

627.

0 6.

3 5.

0 1.

80

0.98

4.

6 5.

2 28

.07

32.5

6.

4 5.

2 1.

83

1.10

4.

5 5.

9 28

.1

36.8

6.

1 5.

0 1.

63

0.81

5.

1 5.

4 31

.8

33.7

6.

61

5.1

1.71

1.

11

4.3

5.18

26

.8

32.3

6.

2 5.

3 1.

93

0.99

5.

2 5.

08

29.6

33

.2

5.9

5.1

1.90

0.

93

5.0

5.73

31

.253

5.8

5.99

5.

12

1.64

0.

89

4.9

5.64

30

.6

35.2

6.

6 5.

3 1.

78

1.02

4.

4 5.

3 27

.5

33.0

5.

89

5.13

1.

81

1.16

4.

8 5.

6 30

.0

35.0

c1

w

N

(A

z 2-

21

Tab.

5. C

hem

ical

ana

lysi

s of

Pleu

rofu

s saj

or-c

aju

and P. os

frea

lus o

n di

ffer

ent l

egum

inou

s sub

stra

tes

.5 Su

bstra

te

Cru

de fi

bre

Car

bohy

drat

e [%

I A

sh

Fat

Nitr

ogen

C

rude

pr

otei

n vl

(s

olub

le +

inso

lubl

e)

[%I

[%I

[%I

I1

l”/l

[%I

I

L1

L2

L3

L4

L5

L6

L7

L8

L9

LIO

I -

solu

ble

11-

inso

lubl

e

-.

Ps

Po

Ps

Po

z (I)

(11

) To

tal

(I)

(11)

Tota

l Ps

Po

Ps

Po

Ps

Po

Ps

Po

2-

13.3

12

.4

25.0

3 19

.3

44.6

33

.6

25.9

59

.5

5.83

5.

30

1.73

0.

93

5.25

6.

00

32.8

37

.5

F E 12

.4

11.3

23

.06

20.1

43

.7

32.9

26

.3

59.2

6.

25

6.1

1.32

1.

02

5.31

5.

96

33.1

37

.2

12.6

13

.3 1

13.0

1 12

.52

11.4

11

.63

12.3

1 13

.7

11.6

12

.3

12.1

0 11

.41

10.9

10

.6

11.8

12

.59

25.6

27

.3

26.2

25

.17

24.1

3 23

.9

26.9

25

.8

18.6

43

.2

35.7

26

.7

62.4

6.

20

5.92

1.

56

0.86

18

.8

46.1

35

.4

28.8

64

.2

5.69

5.

11

1.69

1.

03

19.9

46

.1

37.9

28

.4

66.3

6.

13

5.82

1.

48

1.06

20

.6

46.3

38

.2

29.2

67

.4

6.18

5.

06

1.53

1.

00

20.0

44

.3

36.3

23

.4

59.7

5.

96

5.13

1.

82

0.91

21

.3

45.2

35

.7

24.9

60

.6

5.43

5.

00

1.79

0.

90

19.6

46

.5

36.2

26

.61

62.8

5.

03

5.04

1.

64

0.96

20

.6

46.4

35

.4

27.8

63

.2

6.05

6.

00

1.71

1.

12

5.27

6.

10

5.00

5.

93

4.98

5.

84

4.82

5.

70

4.93

5.

91

5.06

5.

89

4.79

6.

03

5.21

5.

29

32.9

38

.1

3 1.2

3 37

.04

31.1

2 36

.50

30.1

2 35

.62

30.8

1 36

.9

31.6

2 36

.5

29.9

3 37

.68

32.5

6 33

.06

138 Acta Biotechnologica 13 (1993) 2

Tab. 6. Energy value of fruit bodies of Pleurotus sajor-caju and Pleurotus ostreatus on leguminous and non-leguminous substrates

Substrate (leguminous)

Energy value Substrate (non-leguminous)

Energy value

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

Ps Po 281.3 354.63 287.73 355.93 280.68 369.09 289.58 375.30 287.53 382.96 286.17 384.77 282.01 355.02 287.66 357.68 287.43 370.51 294.49 361.42

N1 N 2 N 3 N4 N5 N 6 N7 N8 N9 N10

Ps Po 273.34 340.35 279.25 344.09 282.55 367.27 277.13 371.84 275.62 363.13 289.00 339.37 277.53 365.75 281.63 361.32 283.92 364.63 272.24 359.70

which three crops were harvested. The maximum yield of fruit bodies was obtained in the first crop on all the substrates used here. The “biological efficiency”, e.g. the conversion percentage from dry substrate to fresh mushroom, was calculated in each case. The data were analyzed statistically. Tab. 2 expressed the significant highest yield of P. sajor-caju (5199 g f 16 g) and P. ostreatus (4889 g f 18 g) on L. leucocepholea and the lowest yield on Mimosa pudica (P. sajor-caju, 360 g f 13.4 g and P . ostreatus, 338 g f 12 g). In com- parison of Tab. 1 with Tab. 2, it can be seen that the yield of fruit bodies of both kinds of mushroom was higher on leguminous substrates than non-leguminous ones. It is a well known fact that nitrogen-fixing plants are the major source of all nitrogen that enters the nitrogen cycle in many natural ecosystems. Nitrogen-fixing plants (e.g. leguminous) do not depend solely on soil nitrogen but also on fixed nitrogen through symbiotic micro- organisms (Rhizobhm) that live in root nodules and convert atmospheric nitrogen into a usable form. Substrates rich in usable nitrogen may be a factor in enhancing the mush- room yield on leguminous plants. These results are all supported by ZADRAZIL [20] who found that the supplementation of wheat straw with nitrogenous substrates (soybean and alfalfa) increased the mushroom yield. Nitrogen-fixing substrates were found beni- ficial for both the mushrooms, P. sajor-caju and P . ostreatus. The leguminous substrates also changed the nitrogen content in fruit bodies of P. sajor-caju and P. osrreatus (Tab.

However, P . sajor-caju can rapidly produce fruit bodies under very extensive culture conditions [l I], the first “flush”(crop) being finished after 3-4 days. The quality of fruit bodies depends on the growth conditions. P . ostreatus requires a low temperature (18- 20 “C) as compared to P . sajor-caju (20-25 “C). The energy value of fruit bodies was cal- culated and listed in Tab. 6 . The low yield of P. ostreatus may be attributed to the slightly high temperature (20-22 “C) during the period of experimentation. Furthermore, ZADRAZIL [20] reported that P. sajor-caju converts maximally 25% of the lost organic matter into fruit bodies. This economical yield coefficient (yield of fruit bodies/loss of organic matter) is rela- tively lower than that of Agrocybe aegerita, Pleurotus sp. “Florida” and P. ostreatus.

Acknowledgements

The technical help of Sh. PRABHAT is acknowledged.

4-5).

Received October 19, 1992

SHARMA. S., MADAN. M., Protein from Leguminous and Non-Leguminous Substrates 139

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