Вы находитесь на странице: 1из 30

“Wood Ear Mushrooms Of West

Dissertation submitted for the partial fulfilment of project (Molecular and


Bengal”
applied Mycology and Plant Pathology) for the degree of Master of Science
in Botany

Susmita Sett
Roll Number: 91/BOT/171004
Registration Number: 613─1221─1246─14

Under the supervision of


Prof. Krishnendu Acharya
Department Of Botany, University Of Calcutta
Genus: Auricularia

The genus Auricularia, belonging to the family Auriculariaceae, was established by


Bulliard in 1787.

Auricularia is a cosmopolitan saprophytic basidiomycetious jelly group of fungi


usually found in association with decaying wood & it popularly named as wood ear
mushroom.
Kingdom Fungi
 It has gelatin like consistency.
Auricularia is a phragmobasidious group Division Basidiomycota

of fungus with three transverse septation.


Class Agaricomycetes,
It is fourth most edible cultivated
mushroom worldwide and popular as low Order Auriculariales
calorie dietary food with good source of
Family Auriculariaceae
various amino acids, crude fibres and
Type species: Auricularia mesenterica
biologically active metabolites. (Dicks) Pers.
Literature Review

 World scenario:
MycoBank database (http://www.mycobank.org) has 175 specific or infraspecific records in Auricularia, while the
number in Index Fungorum (http://www.indexfungorum.org) is 165.

Table:1 distribution of different species across the world


 Indian scenario:
 According to the earliest report four species reported in “Fungi of India” viz A. epitricha, A. mesenterica, A. rugosissima, A.
vespertilis (E. J. Buttler & G. R. Bisby 1931).
 Seven species (A. delicata, A fuscosuccinea, A mesenterica, A peltata, A polytricha, A tenuis, A auriculae judae) reported in
“Manual of Indian Edible Mushrooms” from West Bengal (R. P. Purakayashtha 1985).
 Four species viz. A. delicata, A. polytricha, A. mesenterica, A. auricula from Manipur (Singh S.M. 2008).
 Six species viz A. polytricha, A. auricular-judae, A. cornea, A. mesenterica, A. delicata, and A. auricula repoted from different
parts of India (Sohi & Upadhyay 1990, Singh 2008, Mukherji & Manoharachary 2010).
 A. olivaceous a new species was reported from North India ( Kumari et al. 2013).
Auricularia spp. Mushrooms used as food:

 Several species of Auricularia cultivate world


wide and consumed.

A.auricula-judae , A. fuscosuccinea, A. delicata, A.


nigricans, A. tenuis, A. mesenterica (Boya
2011).

• A. auricula-judae known as “Black food of


china” due to high melanin content.

Beneficial property:
 Low fat high calorie diet with high amount of
fibre. A. mesenterica contain 76%
carbohydrate, 15% protein, 0.80% crude fat,
3.92% fibre, 3% ash content.

 Rich source of amino acids viz.glutamic acid,


lysine,alanine,serine, threonine (Sekara et al
2015).

 Novel functional food AHP Auricularia mushrooms


(polyasaccharide of A. auricula, polyphenols of used in soups, salads,
Hawthorn & Puneria radix) active against various Chinese dishes.
dyslipidemia (Luo et al. 2009).
Medicinal properties of Auricularia sp.

 Antioxidant
• Ethanolic extract of A. auricula activate nitric oxide synthase enzyme
(Acharya et al. 2004).
• Poolysaccharide of A.auricula increased SOD & glutathione activity (Lin et
al. 2013).
 Antimicrobial
• Melanin of A. auricula showing antibiofim activity against E.Coli (Bin et al.
2012).
• Inhibit Violecein production which effect the quorum sensing of
Chromobacterium violeceum (Zhu et al. 2011)
 Antitumour
• β – D glucan of A. auricula induces apoptosis (Ma et al. 2010).
 Prebiotics
• ethanolic extract of A. auricula-judae promote activity of Lactobacillus and
inhibit pathogenic strain (Swagnagn et al. 2018).
 Anti-coagulant
• A. polytricha suppress platelet aggregation (Hokma & Hokma 1981)
 Anti-inflammatory
• Polysacharide of A. auricula-judae having anti-inflammatory effect (Ukai et
al. 1983).
 Immunomodulatory
• Proteins isolated from A. polytricha shows immunodulatory property (Sheu
et al 2004)
Study area: The state West Bengal

 Located at the eastern part of the


India, in between 27°13'15"‒
21°25'24"N latitudes and 85°48'20"‒
89°53'04" E longitudes.
 It is the only state in India where
Himalayas are in the north and Sea is
at the south, with both plains and
plateaus covering the remaining
region and seems to be a miniature of
India.
 The climate varies from tropical
savannah in humid subtropical.
Barring the mountainous parts of
Darjeeling and Jalpaiguri, the entire
states experience a warm tropical
monsoon climate.
 The summer temperatures in the
states ranges between 26˚c to 43˚c
and the winter temperatures ranges
from 10˚c to 19˚c.
 North Bengal receives the highest
rainfall, 200–400 cm. In the coastal
area rainfall is about 200 cm, in the
Ganga plain and in the central part of
OBJECTIVE OF THE STUDY

Main objective of the study is to explore diversity of macro-fungal


genera Auricularia of West Bengal based on different macro and
micro morphological characters.

Identification and characterization of different species of


Auricularia collected from various parts of West Bengal & provide a
artificial key of the studied genus.

Phytochemical profiling of two specimens measuring the


bioactive metabolites and antioxidant potential.
MATERIALS AND METHODS
1.Collection of sample from
the field with proper
photograph & field notes
2.Properly dry the sample with
heat
3.Documentation of
macroscopic characterization
of the macrofungi
4.Study the microscopic
characters of the specimens
Sample.ressurect after 5.Preparation of complete
soaking into water description sheet of the
studied macrofungi
6. Identifiy the macrofungi
with the help of literatures &
keys
7.Prservation of the studied
macrofungi

Data sheet for the characterization of


Auricularia species using the internal
anatomy of the basidiocarp (created by
Paola González-Colón). Type A refers to
species that have a medulla and eight
zones. Type B refers to species that
lack a medulla and have only six
Dried & preserved sample zones, where the Superior and Inferior
Laxa are absent.
Identifying Characters of Auricularia species

ZP

• Cross section through ZC


basidiomata showing eight
ZSS
hyphal zones, i.e., Zona Pilosa
ZLS
(ZP), Zona Compacta (ZC), Zona
Subcompacta Superior (ZSS),
Medulla
Zoma Laxa Superior (ZLS),
Medulla layer, Zona Laxa Inferior
ZLI
(ZLI), Zona Subcompacta Inferior
(ZSI), Hymenium (Scale bar=100
mm).
ZSI

Hymenium
A B C
• Abhymenial hairs (A) •Monomitic type of hyphal system; generative
(scale bar 100 µm) hyphae thin walled, hyaline, highly branched &
clamped (B-C) ) (scale bar 5 µm)

•Smooth, thin
walled, hyaline,
allantoid
basidiospores
(D) (scale bar 5
µm)

•Phragmobasidi
a with three
transverse
septations (E-F)
(scale bar 10
D µm) E F
1.
Taxonomy
Identification of six species of Auricularia with detailed macro-
microscopic features based on standard keys and literatures
(Lowy 1951, Montoya- Alvarez et al. 2011 ,Looney et al. 2013).
1 2

Fig:1 Auricularia fuscosuccinea Fig:2 Auricularia cornea Ehrenb. (Scale


(Mont.) Henn. ( Scale Bar 10mm). Bar 20mm)
Macro-microscopic features: Macro-microscopic features:
•Basidiomata, gregarious, sessile, orbicular, •Basidiomata, gregarious, substipitate,
3.2−4.5 cm wide and 2−3 mm thick; orbicular, 1.7–2.9 cm wide and 1.5–2 mm
abhymenial surface reddish orange (7B6); thick; abhymenial surface pilose reddish
hymenium smooth brownosh orange (6C3), brown (9D4) ; hymenium smooth greyish
smooth. violet (19D4).
•Presence of disticnt eight zones; zona pilosa •Presence of distinct eight zones; zona pilosa
7.74−25.8 µm wide, hair 21.52−34.4 × 181.83−314.07 µm wide, hair 60.34−349.11 ×
3.44−5.17 µm. 4.31−8.18 µm.
•Medulla distinctly present. •Medulla distinctly present.
•Basidium cylindrical, 30.17-56.03 × 4.3-5.1 •Basidium cylindrical, 30.14-51.72 × 4.3-5.1
µm in diam. With three transverse septations. µm in diam. With three transverse septations.
•Basidiospores measuring 8.6-11.32-13.76 × •Basidiospores measuring 10.6-13.93-17.24 ×
3.87-4.9-6.02 µm with mean Q value of 2.26. 4.31-5.77-7.75 µm with mean Q value of 2.46.
Fig:3 Auricularia fuscosuccinea (a)
Fig:4 Auricularia cornea (a) Cross
Cross section of basidiomata
section of basidiomata showing
showing different zones, (b)
different zones, (b) Abhymenial hairs,
Abhymenial hairs, (c) Basidia, (d)
(c) Basidia, (d) Basidiospores, (e)
Basidiospores, (e) Clamp connections
Clamp connections of hyphae. (Bars:
of hyphae. (Bars: (a) 100 µm, (b˗e) 5
(a) 100 µm, (b˗e) 5 µm).
µm).
3 4

Fig:5 Auricularia delicata (Mont. ex. Fr.) Fig: 6. Auricularia mesenterica


Henn. (Scale Bar 20mm) (Dicks.:Fr.) Pers. ( Scale Bar 10mm)
Macro-microscopic features: Macro-microscopic features:

•Basidiomata solitary, sessile, orbicular, 2.6−5.6 •Basidiomata gregarious, sessile, effuse-


cm wide and 3−4 mm thick; abhymenial surface reflexed, 2.2−2.6 cm wide and 0.7−1.5 mm
reddish brown (8D7); hymenium reticulate, thick; abhymenial surface densely villose with
greyish ruby (12C6). concentric zones alternate with light brown
(7D4) and orange grey (5B2); hymenium with
• Presence of disticnt six zones; zona pilosa folded ridge greyish red (7B4), smooth.
25.86−47.41 µm wide, hair 25.86−47.41 ×
4.3−6.4 µm. • Presence of disticnt six zones; zona pilosa
• Medulla absent. 528.96−1520.79 µm wide, hair
•Basidium cylindrical, 34.48-40.94 × 4.3-6.46 396.72−1405.5 × 1.79−3.58 µm.
µm in diam. With three transverse septations. • Medulla absent.
•Basidiospores measuring 8.62-11.32-13.79 × •Basidium & basidiospores not observed.
4.31-4.97-6.46 µm with mean Q value of 2.
Fig: 7. Auricularia delicata (a) Cross Fig:8 Auricularia mesenterica (a)Cross
section of basidiomata showing section of basidiomata showing different
different zones, (b) Abhymenial hairs, zones, (b) Abhymenial hairs, (c) Clamp
(c) Basidia, (d) Basidiospores, (e) Clamp connections of hyphae. (Bars: (a) 100 µm,
connections of hyphae. (Bars: (a) 100 (b) 10 µm, (e) 5 µm.
µm, (b˗e) 5 µm).
5 6

Fig: 9. Auricularia nigricans (Sw.) Birkebak Fig:10 Auricularia Sp. (Scale Bar 20 mm).
Looney & Sánchez- Garcia (Scale Bar 10mm).
Macro-microscopic features: Macro-microscopic features:

•Basidiomata solitary, sub-stipitate, orbicular, •Basidiomata gregarious, sessile, orbicular,


2.2−3.5 cm wide and 0.8−1 mm thick; 1.7−3.5 cm wide and 0.5−1 mm thick;
abhymenial surface violet brown (10E6) densely abhymenial surface greyish violet (16E7) pilose;
pilose; hymenium smooth brownish red (10D6), hymenium smooth reddish brown (9E8), smooth.
smooth. •Presence of disticnt eight zones; zona pilosa
•Presence of disticnt eight zones; zona pilosa 11.42−57.12 µm wide, hair 40.94−97.3 ×
146.54−318.94 µm wide, hair 107.25−474.1 × 4.31−6.46 µm.
4.31−6.46 µm. •Medulla distinctly present.
•Medulla distinctly present. •Basidium cylindrical, 51.72-64.65 × 4.3-5.6 µm
•Basidium cylindrical, 30.17-34.48 × 4.3-5.1 µm in diam. With three transverse septations.
in diam. With three transverse septations. •Basidiospores measuring 8.62-9.57-12.93 ×
•Basidiospores measuring 9.48-12.54-13.79 × 4.31-4.69-6.89 µm with mean Q value of 2.04.
4.31-4.43-4.74 µm with mean Q value of 2.82.
Fig: 11. Auricularia nigricans, (a) Cross section of Fig:12 Auricularia sp. (a) Cross section of
basidiomata showing different zones, (b) basidiomata showing different zones, (b)
Abhymenial hairs, (c) Basidia, (d) Basidiospores, (e) Abhymenial hairs, (c) Basidia, (d)
Clamp connections of hyphae. (Bars: (a) 100 µm, Basidiospores, (e) Clamp connections of
(b˗e) 5 µm). hyphae. (Bars: (a) 100 µm, (b˗e) 5 µm).
Artificial key to the species of Auricularia:

1.Hymenium smooth.………………………............................................................2

1a. Hymenium reticulate…………………………………………………............ A.delicata

2. Medulla present .……………………………………………………..........………………. 3

2a. Mesulla absent ……………………………………………….......……… A. mesenterica

3. Abhymenial hairs up to 40 µm long …………………….......……. A. fuscosuccinea

3a. Abhymenial hairs more than 40 µm long ……………........…………………………4

4. Basidiospores up to 13 µm long………………………..........……………………….… 5

4a. Basidiospores more than 13 µm long …………….........………..……….. A. cornea

5. Basidia <50 µm long and hairs > 100 µm long …...........……………. A. nigricans

5a. Basidia >50 µm long and hairs <100 µm long ……….......…….... Auricularia sp.

 
2. Phytochemical profiling of two specimens A. delicata
& A. mesenterica using infusion, decoction &
hydroalcoholic extraction method measuring the
bioactive metabolites and antioxidant potential.

Dried specimen Powder made from dried


specimen
Bioactive profiling of different
extracts
(Martins et
al. 2015)

(Khatua et
al.2017)
Quantification of Bioactive Metabolites

Fig: 13. Determination of phenol, flavonoid and ascorbic acid content of infusion, decoction
and hydroalcoholic extract obtained from A. delicata & A mesenterica

Abbreviations: GAE: Gallic Acid Equivalent; QE: Quercetin Equivalent

• ẞ carotene, lycopene were detectecd in very scanty amount.


Determination of DPPH radical scavenging
activity
DPPH radical scavenging activity

C
10 µl
20 µl

30 µl
40 µl
Infusion Decoction Hydroalcoholic
extract

A B
Fig:14 Graph representing the working principle of DPPH assay(A),
Microtitre plate showing gradual discolouration of DPPH withincreasing
concentration of extract reflecting the radical scavenging activity (B)

Fig:15. Determination of DPPH radical


scavenging activity of infusion, decoction
Table 2: EC50value of DPPH radical scavenging activity of three and hydroalcoholic extract obtained from
different extracts of A. delicata and A. mesenterica. the values have A. delicata (a) infusion, (b) decoction, (c)
been expressed as mean±standard deviation. hydroalcoholic] & A. mesenterica (d)
infusion, (e) decoction, (f) hydroalcoholic].
Determination of ABTS radical scavenging
activity
(A) (B)
Control ABTS radical
scavenging activity

20 µl
50 µl

70 µl

100 µ l
Hydroalcoho
Infusion Decoctio
n
lic extract (a)

Fig16: Working principle of ABTS radical scavenging assay (A),


Microtitre plate showing gradual discolouretaion with increasing
concentration of extract depict the increase radical scavenging
activity (B).
EC50value (µg/ml) INFUSION DECOCTION HYDROALCOHO
LIC EXTRACT

45.03 ±0.022 35.10 ±0.034 56.69 ±0.061


A. delicata

A. 109.6 ±0.003 106.7 ±0.001 116.2 ±0.007


(b)
mesenterica
Fig:17. Determination of ABTS scavenging
Table 3: EC50 values of three different extracts of A. delicata and activity of infusion, decoction and
A. mesenterica. In ABTS radical scavenging activity. the values hydroalcoholic extract obtained from A.
has been expressed as mean±standard deviation. delicata (a) & A mesenterica (b),
Determination of Total
Antioxidant Activity

Fig: 19. Mechanism of total antioxidant activity


Total Infusion Decoction Hydroalcoho
Antioxida Fig:20. Determination of total antioxidant
lic Extract
nt Activity activity of infusion, decoction and hydroalcoholic
(µg/mg of
ascorbic extract obtained from A. delicata & A.
acid) mesenterica.

A delicata 0.00165 ± 0.000403 ± 0.00742 ±


0.002 0.003 0.003  This is a direct method to evaluate
reducing efficiency of antioxidant. It
A 0.00125 ± 0.00132 ± 0.00 0.00742 ±
mesenteri 0.001 0.003
is different from other method as
ca phosphomolybdenum complex is
formed without generations o free
Table 4: Quantitative estimation of total radicals.
antioxidant activity of three different extracts of A.
delicata and A. mesenterica. The values has been
expressed as mean ± standard deviation.
Conclusion
 The proposed work has been described Six taxa of the
genus Auricularia of which five of them are identified upto
species level and one upto genus level.
 Auricularia cornea is reported first time from West
Bengal.
 Quantitative estimation shows that A. delicata & A.
mesenteica both contain high amount of phenol, flavonoid
& ascorbic acids and showing promising antioxidant
activity.
 EC50 value of hydroalcoholic extract of A. delicata
showing highest DPPH & ABTS radical scavenging activity
although all the three extracts of both the specimens
showing positive results in antioxidant screening.
Paper communicated

 Saha, R., Sett S., Chatterjee T., Basak G., Roy A. &
Acharya K. Contribution to the macromycetes of west
bengal, India: 57–62. Research journal of pharmacy
and technology. 2019.
References
Acharya, K., Samui K., Rai M., Dutta B. B. & Acharya R. Antioxidant and nitric oxide synthase activation of Auricularia
auricula. Indian journal of experimental biology. 2004: 42; 538-540.

Hokma, Y. & Hokma J. L. In vitro inhibition of platelet aggregation with low dalton compounds from aqueous dialysates
of edible fungi. Research communications in chemical pathology and pharmacology. 1981:31; 177─180

Khatua, S., Dutta A. K., Chandra S., Paloi S. & Das K. Introducing a novel mushroom from mycophagy community
with emphasis on biomedical potency. PLoS ONE. 2017:12(5): e0178050.

Lowy, B. A Morphological Basis for Classifying the Species of Auricularia. Mycologia. 1951:43(3); 351 ̶ 358.

Lin, W. Y., Yang M. J. Hung L. T. & Lin L. C. Antioxidant properties of methanol extract of a new commercial gelatinous
mushrooms (white variety of Auricularia fuscosuccinea) of Taiwan. African journal of Biotechnology. 2003:12(43);
6210─6221.

Ma, Z., Wang J. & Zhang L. Evaluation of water soluble ẞ-D glucan from Auricularia auriccula-judae as potential anti-
tumour agent. Carbohydrate Polymers. 2010:80; 977─983.

Martins, N., Barros L., Santos-Buelga C., Silva S., Henriques M., Ferreira I. C. F. R. Decoction, infusion and
hydroalcoholic extract of cultivated thyme: Antioxidant and antimicrobial activities and phenolic chracteristics. Food
Chemistry. 2015:167; 131─137.

Montoya˗Alvarez, A. F., Hayakawa H., Minamya Y., Fukuda T., López ˗ Quintero C. A. & Franco ˗Molano A. E.
Phylogenetic relationships and review of the species of Auricularia (Fungi: Basidiomycetes) in Columbia. Caldasia.
2011:33(1); 55─66.

Nagata, M., Yamashita I. & Gakkaishi N. S. K. Simple method for simultaneous determination of chlorophyll and
carotenoids in tomato fruit.1992:39; 925─928.

Continued.......
Park, Y. K., Koo M. H., Ikegaki M., & Contado J. L., Comparison of the flavonoid aglycone contents of Apis mellifera
propolis from various regions of Brazil. Arquivos de biologia e tecnologia. 1997:40; 97─106.

Prieto, P., Pineda M. & Aguilar M. Spectrophotometric quantification of antioxidant capacity through the formation of a
phosphomolybdenum complex: specific application to the determination of vitamin E. Analytical Biochemistry.1999:
269; 337─341.

Sawagwan, T., Wanwipa W. & Lalit P. Study of prebiotic properties from edible mushroom extraction. Agriculture and
natural resources. 2018:52; 519─524.

Singleton, V. L., & Rossi Jr. J. A. Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents.
American Journal of Ecology and Viticulture. 1965:16; 144─158.

Sheu, F., Chien A. L., Chen Y. F. & Chin K. L. Isolation and characterization of an immunomodulatory protein (AAP)
from the Jew’s ear mushrooms A. polytricha. Food Chemistry. 2004: 87; 593─600.

Rekha, C., Poornima G., Manasa M., Abhipsa V., Pavithra D. J., Vijay, K. H. T. & Kekuda, T. R. P. Transactions
Ascorbic acid, total phenol content and antioxidant activity of fresh juices of four ripe and unripe citrus fruits.
Chemical Science. 2012:1; 303─310.

Ukai, S, Kiho T, Hara C, Kuruma I & Tanaka Y. Polysaccharides in fungi. XIV. Anti-inflammatory effect of the
polysaccharides from the fruit bodies of several fungi. Journal of Pharmacobiodyn. 1983:6(12); 983─990.

Zhu, H., He C. C. & Chu Q. H. Inhibition of quorum sensing in Chromobacterium violecium by pigments extracted from
Auricularia auricula. Letters in Applied Mycology. 2011: 52; 269─274.
ACKNOWLEDGEMENT
I would like to express my sincerest gratitude to my respected supervisor in this project, Prof.
Krishnendu Acharya, Department Of Botany, University Of Calcutta for his constant guidance
and encouragement which kept me inspired throughout the course of my work.

I would like to express my heartfelt gratitude to the Department of Botany, University of


Calcutta, for giving me the opportunity to finish my final semester project from this esteemed
institution.

I would like to convey my thanks to Miss Rituparna saha and Miss Sandipta Ghosh, research
scholar, Department of Botany, University of Calcutta, for their constant support and immense
patience.

I would also like to thank Dr. Khushi Mukherjee, Dr. Surojit Sarkar, Mr. Adhiraj Dasgupta, Mr.
Pranab Samanta, Miss Somanjana Khatua, Miss Sudeshna Nandi, Mr. Anik Sarkar, Mr. Entaj
Tarafdar, Mr. Diptosh Das, Mr. Mangal Biswas, Miss Juna Tamang, Miss Alisha Thapa
Research Scholars of Department of Botany, University of Calcutta, for supporting me and
helping me with this work.

I would like to take this opportunity to thank my classmate Gouri Basak, Sreeparna Sarkar,
Ipsita Bandopadhyay and my seniors Tribeni Chatterjee, Anirban Sardar for their help and
encouragement.

Finally, I express my heartfelt gratitude to my parents and other family members, without
whose blessings and help this work could not have been furnished within the stipulated time
frame.
Thank you

Оценить