- The project analyzed the cultivation of three Pleurotus species (P. ostreatus, P. florida, P. pulmonarius) with and without bacterial supplementation. - Biochemical analysis showed protein, sugar, flavonoid, polyphenol, and tannin content varied between the species and were generally higher with bacterial supplementation. - Antioxidant activity, as measured by DPPH and ABTS inhibition, was also highest in species supplemented with bacteria, suggesting the bacteria help increase the nutritive value of Pleurotus mushrooms.

1. Biochemical Analysis of Fruiting Bodies of some
Pleurotus spp. Developed with and without External
Bacterial Supplementations
SUBMITTED BY
PINKI PAUL
ROLL NO.-PG/BOT/20/0015
REGISTRATION NO:1231721400027 OF 2020-2022
P.G Department of Botany
Barasat Govt. College

2. CONTENTS
Introduction
Objectives
Common Species
Bioactive Components
Medicinal Properties
Role Of Endophytes
Materials
Cultivated Species
Methods
Result
Discussion
Conclusion
Reference

3. INTRODUCTION
Oyster mushrooms or commonly known as Pleurotus.Oyster
mushroom (Pleurotus spp) is preferable all over the world for its
good taste and easy production methodology. Not only the great
taste but it has numerous medicinal properties. Fruiting bodies of
this mushroom contains so many pharmacological compounds like
polysaccharides, polyphenols, flavoonoids, alkaloids etc.The fruiting
bodies of three Pleurotus species was developed in presence of some
endophytic bacterial isolates.

4. OBJECTIVES
• Comparison of Biochemical Components of Different Pleurotus spp.
• Determination of Antioxidant activity of different Pleurotus spp
• Determination of Endophytic Bacterial Effect on Biochemical Analysis

5. List of some commonly cultivated species of
Pleurotus for fruiting bodies
P. sajorcaju
P. florida
P. pulmonarius
P. ostreatus
P. citrinopileatus
P. eryngil
P. cornucopiae
P. floridanus
P. sapidus
P. djmor
These species of Pleurotus mushroomare uses generally
cultivated for better production. Moreover these mushrooms
are economically important for the oyster mushrooms
cultivators. These species are mostly cultivated because of
their easy and low costing production technologies than
others mushrooms. Not only that these mushrooms have
faster growth rate in comparison to others.

6. Bioactive Compounds Isolated From Oyster Mushrooms
Types of Bioactive
Compounds
Immunomodulating
Amino Acids
Polysaccharides
Terpenoids
Beta-glucans
Lectin
Ergosterol
Protein
Glucans

7. MEDICINAL PROPERTIES OF Pleurotus sp.
The medicinal use of oyster
mushrooms has
pharmacologically active
Compounds. Such as
antimicrobial, antiviral,
and so many others are
described below, in this
review of
literature.(Lindequist
et.al.2005)
Biological source Activity/Use
P. sajorcaju Antibacterial and antifungal activity
P. florida Antibacterial activity and reduce solid tumors in mice
P. pulmonarius Reduce solid tumors in mice
P. ostreatus Cytotoxic, apoptotic antihepatoma andantitumor activity
Lower cholesterol levels,plasmacholesterol reducing
activities
P. citrinopileatus Reduce metastatic tumor nodules in mice
P. eryngil Cytotoxic against leukaemia, antibacterial and antifungal
activity
P. cornucopiae Antihypertensive activity
P. floridanus Antibacterial activity

8. Role of Endophytic Bacteria In Biochemical Components
• Endophytic bacteria considered mutualistic and mediate nutrients
availability to facilitate the growth and competitiveness of the host
(Zhang, et al. 2020).
• Endophytic compounds represent a wide range of organic
molecules including terpenoids, peptides, carbohydrates,
aromatics, hydrocarbons and others and it seems that these
compounds may have a role in the host microbe relationship.

9. Some Important Biochemical Components From Endophytes
• Ambuic Acid
• Hydrocarbon
• Terpenoids
• Phenazines
• Phenylpyrroles
• Siderophores
• Hydrogen cyanide

10. Mushroom Species
Pleurotus pulmonarius (
1805)
Pleurotus florida (3308)
Pleurotus ostreatus
(1802)
Bacterial species:
PJ1, PJ2, PP, FS2, OP
Chemicals
Sodium carbonate
Sodium hydroxide.
Copper sulfate
Sodium potassium tartarate
Folin-Ciocalteau reagent
Bovine serum albumin (BSA)
Dinitro Salicylic acid (DNSA)
Dextrose sugar
70 % Methanol
Sodium nitrite
Aluminum chloride
Catechol
Gallic acid
Tannic acid
DPPH, ABTS
MATERIALS

11. Cultivated Pleurotus spp in Laboratory
Pleurotus ostreatus (1802)
Pleurotus florida (3308) Pleurotus pulmonarius ( 1805)

12. Methods For Biochemical Analysis of Pleurotus spp.
Protein Estimation
Reducing Sugar
Estimation
Flavonoid
Estimation
Polyphenol
Estimation
Tannin Estimation
DPPH Inhibition
ABTS Inhibition

13. O.D at 660nm
2 ml Lowry C
950 μl
dH2O.
50 μl aquous
extract
Of fruit bodies
Blue colour
30 mins Dark
Incubation
200 μl 1N Folin
– Ciocalteau
15 mins
incubation
Protein Determination

14. . Yellow to Red
colouration
5 mins boiling
water bath
1.5 ml
DNSA
450μl
dH2O
50 μl aquous
extract
O.D at 540nm
Reducing sugar Determination

15. 50μl methanolic
extract
Flavonoid Determination
50μl 70%
methanol
75μl
NaNO2
6 mins
Incubation
150μl 10%
AlCl₃
5 minutes
Incubation
750μl 1M
NaOH
15 mins
Incubation
Final volume
2.5 ml by dH2O
Pink
Colouration
O.D at
510 nm

16. 2 mins
Incubation
Polyphenol Determination
50μl methanolic
extract
50μl 70%
methanol
400μl
dH2O
500μl 2%
Na₂CO₃
Dark
Green colour
500μl Folin-
Ciocalteau
Final
Volume 2.5ml
by dH2O
30 mins
Dark
Incubation
O.D at
720 nm

17. 50μl
methanolic
extract
500μl Folin-
Ciocalteau
1 ml 35 %
Na₂CO₃
3ml dH2O
50μl 70%
methanol
30 mins
Incubation
O.D at 750
nm
Tannin Determination

18. 50 μl methanolic extract
50 μl 70 % methanol
20mins Dark Incubation
2 ml DPPH
50 μl dH2O
O.D at 517 nm
Violet colour of
DPPH fade away
Antioxidant Determination
2 ml ABTS
Sky colour of
ABTS fade away

19. .
Inhibition of Antioxidant activity, = [(A0 – A1)/ A0]×100
Where A0 = O.D of control/ blank Set
A1 = O.D of the tested sample

20. Result:
Experiment sets Protein
(μg/g)
Sugar
(μg/g)
Flavonoid
(μg/g)
Polyphenol
(μg/g)
Tannin
(μg/g)
DPPH
%
ABTS
%
3308 Control 35 0.3 1.3 9.4 48.5 78.07 99.28
Sub+Spn+FS2 63.9 18.6 5 10.4 81.9 59.03 96.22
Sub+FS2+Spn 160 45.5 10 21.6 128 46.05 99.28
Sub+FS2
+
Spn+FS2
172.7 42.5 9.5 6.8 62.8 24.8 97.12
1802 Control 26.2 8.6 3. 6 50 52.98 97.3
Sub+Spn+OP 97.8 7.6 4.2 8.7 66.6 49.51 99.64
Sub+OP+Spn 119.6 10.4 2.2 9.3 67.7 64.32 99.82
Sub+OP
+
Spn+OP
12 13 3.9 8.9 51 27.88 97.48
1805 Control 79 12.1 7 14.7 89.5 42.69 97.48
Sub+Spn+PP 72.6 32.9 5.7 11 84.4 3.65 99.64
Sub+PP+Spn 107.1 32 4.3 11.8 84.1 9.71 99.96
Sub+PP
+
Spn+PP
116.2 63.8 3.9 11.4 91.2 1.05 98.82
Sub+Spn+PJ1 89.7 61.9 5.8 12.2 81.7 71.53 99.28
Sub+PJ1+Spn 93 19.5 5 7.7 67.7 45 98.74
Sub+PJ1
+
Spn+PJ1
121.2 28.5 3.1 10.5 71.8 14.51 97.84
Sub+Spn+PJ2 89.7 10.5 3.5 6.8 54.8 68.46 97.3
Sub+PJ2+Spn 61.4 21.3 4.8 12 74.1 75.57 96.58
Sub+PJ2
+
Spn+PJ2
169.4 33.8 5.7 9 76.8 64.61 94.42
Sub+Spn+PJ1+PJ2+PP 52 11.5 5.3 12.8 95.6 67.98 86.87
Sub+PJ1+PJ2+PP
+
Spn+PJ1+PJ2+PP
132.2 4.7 2.3 9.8 75 58.84 95.36
Determination of various
Biochemical Components
of different Pleurotus spp.

21. 0
20
40
60
80
100
120
140
160
180
200
Protein Content
Comparison of Three Pleurotus spp With & Without Bacterial Supplementation

22. 0
10
20
30
40
50
60
70
Sugar Content

23. 0
2
4
6
8
10
12
Flavonoid content

24. 0
5
10
15
20
25
Polyphenol content

25. 0
20
40
60
80
100
120
140
Tannin content

26. 0
10
20
30
40
50
60
70
80
90
Antioxidant activity by DPPH

27. 80
85
90
95
100
105
Antioxidant activity by ABTS

28. SUMMARY
• Pleurotus spp. are rich in some biochemical components. The measurements of these compounds
were given in this project(Table 5)
• Protein concentration is high in P. florida,moderate in P. pulmonarius and lowest in P. ostreatus
with bacterial supplementation.
• Reducing sugar is maximum in P. pulmonarius with bacteria PP and P. ostreatus is moderate with
OP strains and minimum sugar is present in P. florida without bacteria.
• Flavonoid content is maximum in P. florida with FS2 bacteria, moderate in P. pulmonarius with
bacterial supplementation and minimum in P. florida without bacterial supplementation.
• Polyphenol Content is high in P. florida with FS2, moderate in P. pulmonarius with bacterial
supplementation and minimum in P. ostreatus without bacteria.
• Tannin content maximum in P. florida with FS2, moderate in P. pulmonariuswith OP and minimum
in P. ostreatus without bacteria.
• Antioxidant activity was measured by the % of inhibition by DPPH and ABTS solutions.
• Maximum inhibition by DPPH in P. florida without bacteria, moderate in P. ostreatus with bacteria
and minimum in P. pulmonarius with bacterial supplementation. Lowest inhibition by ABTS in P.
pulmonarius ,maximum in P. ostreatus and in P. florida.

29. CONCLUSION
Present analysis on Pleurotus spp cultivation and comparison of different
strains with or without bacterial supplementation. The results generally proven
that the concentration of different biochemical compounds are gradually
increased with bacterial supplementations than without control sets of
experiment. Thus it can be assumed that these bacteria are involves in
increasing the nutritive value of Pleurotus spp.

30. ACKNOWLEDGEMENT
It is my great pleasure to express my regards and gratitude to Dr. Nirmalendu
Das, Associate Professor, Department of Botany, Barasat Government College
for his continuous help , valuable suggestions, constant guidance and helpful
advice to carry out my project work with all kinds of help. It is my great
fortune to having such a great advisor to guide in my project. I would also
greatful to Chandana Paul, Registered Ph.D Scholar of our department for her
continuous help.
.
Pinki Paul

31. REFERENCES
• Ahmed, S. A., J. A. Kadam, V. P. Mane, S. S. Patil and M. M. Baig.2009.Biological efficiency and
nutritonal contents of Pleurotusflorida (Mont)singer cultivated on different agroresidues. Nat.Sci. 7:
44-48.
• Chin-A-Woeng, F.C T, Bloemberg V.G and Lugtenberg B. J. J. (2003) Phenazines and their role in
biocontrol By Pseudomonas bacteria. New Phytologist.157: 503–523.
• Das N, Pasman B, Mishra S, Bhattacharya B, Sengupta C.(2007) Comparative Studies of Antibacterial
Properties of Three Pleurotus Species (Oyster Mushroom). Nat Sci; 10: 178-183.
• Fernando WGD, Nakkeeran S, Zhang Y (2005) Biosynthesis of antibiotics by PGPR and its relation in
biocontrol of plant diseases. In: Siddiqui ZA (ed) PGPR: biocontrol and biofertilization.Springer,
Dordrecht, pp 67–109
• Krishna S, Usha PTA. (2009) Hyoglycaemic effect of a combinationof Pleurotus ostreatus, Murray
Koenigii and Aegle marmelos in diabetic rats. Indian J Anim Sci 79: 986-987.
• Lindequist U, Niedermeyer T. H. J., Ju¨lich W.D. The Pharmacological Potential of
Mushrooms.Rev.eCAM 2005;2(3)285–299doi:10.1093/ecam/neh107.
• Patel Y, Naraian R , Singh V.K.2012. Medicinal Properties of Pleurotus Species (Oyster Mushroom): A
Rev.World J. Fungal & Plant Biol., 3 (1): 01-12.
• https://www.google.co.in/