INTRODUCTION:

The word keratinophilic means "keratin loving". The biggest group of organisms that can utilize keratin as the sole source of carbon and nitrogen are known as keratinophilic fungi. Keratinophilic fungi are minute organisms that cannot be seen by the naked eye. These are generally considered as soil saprophytes. The soil represents the main reservoir of fungi. Soil that is rich in keratinous material is most conductive for the growth and occurrence of keratinophilic fungi. Keratinophilic fungi have the unique ability to degrade keratinous substrates, e.g., hair, horns, hooves, feathers and nail. The fungi which degrade these substrates completely are termed as keratinolytic. In the soil, these fungi live in their teleomorphic (sexual) stages in the form of cleistothecia, whereas in keratinized material (host) they live in an anamorphic (asexual) stage in which they develop only a very simple morphology. Most of these fungi belong to families Arthrodermataceae and Onygenaceae, order Onygenales in Ascomycetes. The Arthrodermataceae and Onygenaceae are unusual in that majority of them are associated with birds and mammals. Keratinolytic members of fungi that occur commonly in soil as keratin decomposers generally belonging to groups-the Deuteromycetes and the Ascomycetes of the Kingdom Eumycota. These groups include both saprophytic and pathogenic fungal strains. When there is ample keratin substrate available in soil, these fungi multiply by asexual means by producing enormous numbers of conidia (aleuroconidia, arthroconidia). When the keratin substrate is depleted, however, the fungi reproduce by sexual means and form characteristic fruiting bodies called ascomata. The members of the order Onygenales are frequently isolated from burrow soils indicating that burrows are excellent habitats for these fungi during hot summers as the animals inside them are a regular source of keratin and moisture¹.

Keratin molecules are organized with various protein and cementing substances in more or less keratin rich structure. A keratinic substrate, such as a hair fragment, placed in the soil will be colonized by many micro-organisms, including fungi, which collabrate in using various components differentially and progressively according to their complexity. Keratin are insoluble fibrous protein derived from ectoderm and are poorly biodegradable. These proteins which belong to the scleroprotein groups are compounds that are extremely resistant to the action of physical, chemical and biological agents. Mechanical stability and high resistance to proteolytic degradation of keratin are due to their disulfide bonds, hydrogen bonds, salt linkages and cross linkings¹.

There are two kinds of keratin:

α- keratin: These contain most of the common amino acids, but they are primarily rich in cysteine residues and therefore disulfide bridge rigid brittle forms in horns and nails contain upto 22% cysteine, soft, flexible forms in the skin and in the hair and wool contain between 10 and 14%.

β-keratin: These lack both cysteine and cystine but are rich in amino acids with short side chains, especially glycine, alanine and serine, they are found in fibres of spider and silkworm, in scales and in the claws and beaks of the bird^2.

Common Habitats of Keratinophilic Fungi:

· Cattle shedsb. Animal burrows

· Garbage D. Sewage

· Bird's nest F. Barber's hair dumping area

· Poultry sheds H. Herbivore or carnivore dung

· Public places like parks, schools, marketplace etc.

Keratinophilic fungi also include dermatophytes, which cause diseases of the skin and its appendages. Several keratinolytic dermatophytes survive in the soil, in addition to their clinical habitat. Currently, almost all the habitats of the world have been surveyed for the presence of keratinophilic fungi. These fungi by virtue of their ability to colonize epidermal appendages, may become source of sanitary danger to human health. The dermatophytes have been divided into three ecological groups: Geophiles, Zoophiles and Anthropophiles³

Molecular methods over conventional methods:

The conventional methods of detection and identification of fungi have mainly relied on culture isolation and subsequent observations of morphological traits. These methods are time consuming, laborious, and may require day to weeks for isolation by culture. In addition, not all the fungal species are cultural on a given medium, which leads to analysis that may not accurately reflect the true fungal community in a fungal sample. With the advent of PCR, inexpensive DNA sequencing and a relatively large data bank of rDNA sequences, it is now possible to more objectively characterise and identify fungal species. However, due to the immense diversity in fungi, true group and species-specific detection can be difficult to achieve. We apply PCR based detection techniques of sequence polymorphisms in the internal transcribed spacer I and II region of the rDNA genes as a means of fungi identification³

APPLICATIONS:

Industrial Importance:

Keratinase producing microorganisms have the important industrial application in fermentation technology. Submerged fermentation of poultry waste by microorganism producing keratinase helps in the conversion of non-soluble keratin (feather) into soluble protein or polypeptide. These protein by product may be used as animal and livestock feed, and as leather filling agents. Keratinase has also emerging application in dehairing process in leather industry instead of sodium sulphides and also used as a detergent to remove strains on cloth. Valorization of keratin containing wastes like feathers from poultry farms and hair from leather industries may have the potential indevelopment of non-polluting processes. The scope of this work is to degrade the poultry feather wastes (insoluble protein) to soluble protein. By immobilization the keratinases have been used for tenderization of hard keratinous substances and removal of hair from leather⁴.

Several studies have been showed that soils are important sources of dermatophytes and keratinophilic fungi. The presence of dermatophytes in soil (especially parks) can be a reservoir for infection in human beings.

Keratinophilic fungi are natural colonizers of keratinic substrate. Some are keratinolytic and play an important ecological role in decomposing a keratin, the insoluble fibrous proteins. Because of the tight packing of their polypeptide chains in helix structure and their linkage by disulphide bridges, they are poorly biodegradable.⁴

Two main forms of attack have been identified:

a) Surface erosion.

b) Radial penetration.

In surface erosion the sequence of degradation proceeds as the level of keratinsation (the cysteine crosslinks) of the components of the keratinic matrix increases.

In radial penetration on the other hand speciallized hyphae can penetrate like a drill through the matrix, irrespective of the degree of keratinisation⁵

Medical importance.

Keratinophilic fungi along with dermatophytes are responsible for various cutaneous mycoses. Dermatophytes require keratin for growth. These fungi can cause different types of tinea in humans. The majority of the fungi producing diseases in human beings and animal exist freely in nature as soil saprophyte. Dermatophytes are spread by direct contact from infected people (anthropophillic organisms), animals (zoophillic organisms), and soil (geophillic organisms) and indirectly from fomites. As suggested by Toshibo et al. (1989), Keratinase may find applications in controlling skin diseases^5,6

OBJECTIVE:

1. Isolation of keratinophilic fungi from different soil samples using different keratin substrates.

2. DNA isolation of keratinophilic isolates.

3. Data analysis.

MATERIAL AND METHODS:

1. COLLECTION OF SOIL SAMPLES

Soil samples were collected from different habitats of Jammu region in an Autoclaved paper using sterilized spatula showed in table 1.

Table1: Soil samples from different Habitats.

S. No	Site of Collection (JAMMU)	Habitat
1.	Rajouri	Spring
2.	Rajouri	Clay
3.	RS pura	Burrows
4.	Akhnoor	Religious place (JHIRI)

Figure-1

Figure-2

Figure-3

1.1 Preparation of Soil Samples:

500 gm soil samples were air dried, crushed to make fine powder and sieved using muslin cloth. 50 gm of soil samples were taken in each autoclaved plate by using a sterile spatula.

1.2 Sterilization of Hair, Feathers and Wool

Hair, feathers and wool were used as substrates before the defating was done by soaking the hair, feathers and wool in a chloroform and methanol in the 1:1 ratio for 10 minutes and later rinsed 4-5 times in distilled water and then finely air dried. Cleaned dried baits were sterilized by autoclaving at 100°C at 15 lbs pressure^9,10

1.3 Isolation of Keratinophilic Fungi by Keratin Bait Technique (Vanbreuseghem. R 1953).

>20gm of each soil sample was placed into a sterile Petri dish and baited with sterlized small pieces of hair, wool and feather.

>Each petridish was moistened with 5-10 ml sterile distilled water and incubated at room temperature for up to 10-15 days at 37°C^[10,11]

>Plates were examined periodically for any mycelial growth on the baits.

> Isolation of fungi was done by taking colonies from baits and transfering them to SDA plates supplemented with chloramphenicol.

>Subculturing was continuously done on SDA plates until pure colonies were isolated.

1.4 Maintenance of Pure Cultures

All the fungal cultures were maintained on SDA slants at 4°C for further use and preservation of cultures.

1.5 Morphological Identification of Isolates

Slides were prepared by mounting fungal culture in lactophenol cotton blue. Microscopic identification of fungal isolates was done by studying various the shape of hyphae, presence or absence of morphological fearures such as micro or macroconidia, shape and position of macro and micro conidia on hyphae.^12,13

2. MOLECULAR CHARACTERIZATION OF FUNGAL ISOLATES

2.1 Genomic DNA isolation

> For the isolation of DNA fungal cultures were revived on SDA plates and incubated at 30°C for 5 days.

>Poured plates was streaked with a fungal mass taken from preserved slants for the isolation of genomic DNA. 125 ml SDA was inoculated with fungal isolate and keep under shaking condition at 30°C for 6-7 days to obtain large amount of biomass. Cells were harvested by filtering the broth with muslin cloth.

>Harvested cells were washed with autoclaved water and then air dried in the folds of filter paper. Genomic DNA was isolated from fungal biomass for following the protocol of Saghaimarrof et al (1984) with slight modification. About 1-2 gm dried fungal mycelial mass was grounded in precooled mortar pestle by using liquid nitrogen.

> The mycelia powder was transferred to centrifuge cup containing 10 ml of preincubated at 65°C of lysis buffer ie. (2% CTAB, 100mMtris Cl, 20mMEDTA, 1.4M Nacl at pH8).

>Centrifuge cups were incubated at 65°C for an hour with constant swirling after every 10 minutes. After incubation equal volume Chloroform: Isoamyl alcohol (24:1) was added and the content were gently mixed for 10 mins. Then, centifuged at 8000g for 10 mins at 4°C.

>After centrifugation supernatant was carefully transferred into another sterile centrifuge cup with cut tips. Then, double the amount of chilled ethanol was added to the supernatant and mixed gently.

>DNA was spooled out and then washed with 70% ethanol. Again, centrifuged at 5000rpm for 5 minutes at 4°C. After centrifugation pellet was air dried and dissolved in TE buffer¹³

3. PURIFICATION AND QUANTIFICATION OF DNA SAMPLES

3.1 RNAse treatment:

DNA was purified by incubating 50μl of DNA sample with l μl of RNAse at 37°C for two hours.

3.2 DNA quantification:

DNA was quantified on 0.7% Agarose gel by comparing with standard DNA of known concentration. Standard DNA used was uncut lambda DNA (300ng/ul, 150ng/μl, 75ng/μl)¹⁴

Table 4: Identification of Fungal Isolates from Different Soil Sample.

S. No.	Fungal accession	Identification
1	S1	Cylindrocarpon
2	S2	Cylindrocarpon
3	S3	Fusarium
4	S4	Fusarium
5	S5	Fusarium
6	S6	Fusarium
7	S7	Fusarium
8	S8	Fusarium
9	S9	Fusarium
10	S10	Aspergillus
11	R1	Aspergillus
12	R2	Aspergillus
13	R3	Aspergillus
14	R4	Colletotrichum
15	R5	Unidentified
16	R6	Aspergillus
17	R7	Colletotrichum
18	R8	Colletotrichum
19	2D	Fusarium
20	9D	Penicillium
21	B1	Paecilomyces
22	B2	Paecilomyces
23	B3B	Paecilomyces
24	B4	Paecilomyces
25	B5	Paecilomyces

RESULTS AND DISCUSSION:

The soil samples were collected from different habitats of Jammu. Fungal isolates were isolated from soil by Keratin-bait technique. All the soil samples were found to be positive for the presence of keratinophilic fungi. Total 43 fungal cultures were isolated from soil sample. Out of 43 pure cultures, 30 cultures were selected for further work. All the fungal isolates were streaked on SDA plates, most of them showed phenotypic differences on the basis of cultural characteristics such as texture, surface pigmentation (Table 2). Isolated keratinophilic fungi were identified microscopically using lactophenol cotton was done by studying blue stain. Microscopic identification of the isolate’s presence or absence, shape and position of micro and macroconidia. Few could not be identified due to lack of sporulation.^15,16

Out of thirty cultures, there were two Cylindrocarpon which were followed by seven Fusarium, five Paecilomyces, six Aspergillus, two Colletotrichum, one Microsporum, one Pencillium and six were unidentified (Table 5). The frequency of Fusarium was higher in spring soil followed by Aspergillus and Paecilomyces. The maximal frequency of these fungi may be the result of the presence of domestic animals¹⁷. In the present study, most of the fungal isolates isolated from different soil samples are known to be human pathogenic and are known to cause various skin diseases by many workers (Mahmoudabadi 2008).

A specialized group of fungi that colonizes keratinaceous substrates such as human hair, skin, nails, feathers, hooves and horns have been studied extensively in India since several of these are cause of human and animal diseases. Soil is the reservoir of such forms that are distributed in genera Colletotrichum, Paecilomyces, Chrysosporium, Epidermophyton, Aspergillus, Microsporum, Myceliophthora and Fusarium.¹⁸

The relative distribution of keratinophiles depends on the frequency of animals visiting a particular habitat but for species such as the genus Fusarium is a widely distributed phytopathogen found in a broad range of hosts.The current fungal taxonomic system has been identified by macroconidia and microconidia in the asexual stage, the morphological character of chlamydospore, hostrange, and secondary metabolites. However, the plasticity and intergradation of the phenotypic traits offered difficulty in identifying the filamentous fungi.¹⁹

For these reasons, the molecular biological method has been recently introduced in Fusarium systematics and the molecular variation at the DNA level has been studied in many works. The ribosomal DNA (rDNA) repeat unit contains spacer regions. Each repeat unit consists of a copy of 18S-5.85 and 28S-like rDNA and two spacers, the internal transcribed spacer (ITS) and intergenic spacer (IGS). The rDNA genes have been employed to analyze major evolutionary events because it is highly conserved, whereas the rDNA internal transcribed spacer (ITS1 and ITS2) was more variable so it has been used for the investigation of the species-level relationship. Thus, the ITS region has been used in classifying many other fungi species because of its systematic and taxonomic usefulness.²⁰

Table 5: Morphological identification of cultures on the basis of colour, texture, reverse and shape of spores (21)

S. No.	Accession No	Colour	Texture	Reverse Colony Characteristics	Spore Shape	Identification
1	S1	White	Cottony	Light Brownish	Oval	Cylindrocarpon
2	S2	White	Cottony	Light Brownish	Fusiform	Cylindrocarpon
3	S3	Orange	Cottony	Dark Orange	Microconidia Oval, macroconidia	Fusarium
4	S4	Orange	Cottony	Dark Orange	Macroconidia, oval	Fusarium
5	S5	Buff colour	Cottony	Dark Brownish	Oval, macroconidia	Fusarium
6	S6	Off white	Cottony	Orange	Oval, macroconidia	Fusarium
7	S7	Off white	Cottony	Orange	Oval, macroconidia	Fusarium
8	S8	Off white	Cottony	Orange	Oval, macroconidia	Fusarium
9	S9	Brown	Powdery	Light Brown	Globose Rough walled	Aspergillus
10	S10	Light	Powdery	Light Brownish	Oval, fusoid smooth Walled	Paecilomyces
11	B1	Light Pinkish	Powdery	Light Yellow	Oval, fusoid smooth Walled	Paecilomyces
12	B2	Light Pinkish	Powdery	Light Yellow	Oval, fusoid smooth Walled	Paecilomyces
13	B3	Light Pinkish	Powdery	Light Yellow	Oval, fusoid smooth Walled	Paecilomyces
14	B4	Light Pinkish	Powdery	Light Yellow	Oval, fusoid smooth Walled	Paecilomyces
15	B5	Light Pinkish	Powdery	Light Yellow	Globose Rough walled	Aspergillus
16	B6	Brown	Powdery	Light Brownish	Globose Rough walled	Aspergillus
17	B7	White	Powdery	Light Yellow	Microconidia	Microsporum
18	B8	White	White	Orange	Microconidia	Unidentified
19	B9	White	Velvety	Brownish	Microconidia	Unidentified
20	B10	Yellow	Velvety	Orange	Globose Rough walled	Aspergillus
21	R1	Light Brown	Powdery	Light Yellow	Globose Rough walled	Aspergillus
22	R2	Light Brown	Powdery	Light Yellow	Globose Smooth walled	Aspergillus
23	R3	Light Brown	Powdery	Light Yellow	Globose Smooth walled	Colletotrichum
24	R4	White	Cottony	Light Orange	Globose Smooth walled	Colletotrichum
25	R5	White	Cottony	Light Orange	Globose Smooth walled	Colletotrichum
26	R6	White	Cottony	Light Orange	Globose Smooth walled	Aspergillus
27	R7	White	Cottony	Orange	Globose Rough walled	Colletotrichum
28	R8	White	Cottony	Orange	Globose Rough walled	Colletotrichum
29	2D	Off White	Cottony	Orange	Microconidia	Fusarium
30	9D	Green	Cottony	White	Round conidia	Penicillium

Figure-4

Figure-5

Aspergillus:

The hyphae are well developed, profusely branched, septate, hyaline and their cells are multinucleate. Some cells of somatic hyphae give rise to conidiophores that arise singly and terminate in a bulbous head, the vesicle. According to the species a large number of conidiogenous cells (sterigmata) in one or two layers, are produced over the entire surface of the vesicle. Layers of sterigmata-bearing conidia are typically called phialides. As the phialides reach maturity, they begin to form conidia at their tips in chains. Aspergillus colonies appear to be variously coloured due to pigmentation of the abundant conidia²²

Penicillium:

The mycelium consists of hyaline, septate and highly branched hyphae. Conidiophore is simple, erect, long, branching at the tip to form a brush-like structure, technically known as Penicillus. Each branch of the conidiophore ends in a group of phialides that bear long conidial chains. The conidia are globose to ovoid, and pigmented, imparting characteristics colour to the colony.²³

Fusarium:

Is a filamentous fungus widely distributed on plants and in the soil. It is found in normal mycoflora of commodities such as rice, bean, soyabean and other crops. Hyaline septate hyphae, conidiophores, phialides, macroconidia, and microconidia are observed microscopically. Phialides are cylindrical, with a small collarette, solitary or produced as a component of a complex branching system. phialides on unbranched or branched Macroconidia are produced from conidiophores. They are two or more-celled thick walls, smooth and cylindrical or sickle-shaped. Macroconidia have a distinct basal foot cell and pointed distal ends. They tend to accumulate in balls or rafts. Microconidia on the other hand are formed on long or short simple conidiophores. They are one-celled smooth, hyaline, ovoid to cylindrical and arranged in balls.²⁴

Paecilomyces:

It is a cosmopolitan filamentous fungus which inhabits the soil, decaying, plants and food products. Paecilomyces is usually considered a septate, phialides, conidia and chlamydospores are hyaline hyphae, conidiophores, observed. Conidiophores are often branched and carry the phialides at their tips. The phialides are swollen at their bases and tapers towards their apices. Conidia are unicellular, hyaline to darkly coloured, smooth or rough, oval to fusoid and form long chains.²⁵

Colletotrichum:

It is a subcuticular, epidermal or subepidermal and may be either separate or confluent. Conidiophores are hyaline to brown, septate branched at the base. Conidia are hyaline, unicellular, falcate or lunate (sickle shaped) or cylindrical, muticate or with the apex prolonged into a simple cellular appendage, produced from philadesenteroblastically.²⁶

SUMMARY:

In the present work 30 fungal isolates were isolated from soil samples. Fungal isolates were identified by examining gross morphology of fungal colonies followed by microscopic visualisation. The genomic DNA of fungal isolates was isolated following the protocol of Saghai and Maroof et al with slight modification. Eukaryotic based ITS specific primers ITSI and ITS 4 were used amplify ITSI, 5.88 and ITS2 regions. PCR amplification resulted in products of 500 bp. PCR was also performed to confirm specificity of ITS1 and ITS4 primers by using primer pair ITS1 and ITS4 to amplify ITS region. The reason for targeting ITS regions is that it is a multicopy gene that apart from importing a greater threshold for detection, also exbits sequence variation unique for specie determination of fungi. ITS offers added advantage over other molecular targets for determining the species. Amplified DNA was cleaved with restriction endonucleases and evaluated electrophoretically in a process referred to a postanalysis variation in the size and number of restriction fragments detected by agarose gel electrophoresis has been used for identification and characterization of various fungi.

BIBLIOGRAPHY:

1. Buchman TG, Rossier, Meriz WG, Charache P. Detection of Surgical Pathogens by In Vitro DNA Amplification. Rapid Identification of Candida albicians by In Vitro Amplification of A Fungus Specific Gene. Surgery. 1990; 108:338-346.

2. Chen Y.C. Eisner J.D. Kattar M.M. Barret S.L. Cookson B.T. Identification of Medically Important Yeast Using PCR Based Detection of DNA Sequence Polymorphism in The ITS 2 Region of rRNA Gene. J.Clin Microbiol. 2000; 38: 2302-2310.

3. Deivasigama B and Alagappan K.M. Industrial Applications of Keratinase and Soluble Proteins Fom Feather Keratins. J. Environ Biol, 2008; 29: 933-936.

4. Deshmukh SK. Incidence of Keratinophilic Fungi from Selected Soils of Kerala State (India). Mycopathologia. 2002; 156:177-181.

5. Faggi E, Pini G, Campisi E, Bertellin C. Difonzo E, Mancianti F. Application of PCR To Distinguish Common Species of Dermatophytes. J. Clin Microbiol, 2001; 39:3382- 3385.

6. Graser Y. et al. Identification Of Common Keratinophilic Fungi Using PCR. J. Brizallian Dermatol. 1998; 138:576-582.

7. Irum F, Suhail M, Abro H. Keratinophilic fungi From the Soil of District, Jamshoro, Sindh, Pakistan. Pak J. Bot. 2007; 39: 1377-1382.

8. Iwen P.C., Freifeld, Bruening TA, Hinrichs. Use Of ITS Regions as Molecular Targets to Detect and Identify the Human Fungal Pathogens. Med. Mycol. 2002; 40 :87-109.

9. Kaul, S and Sumbali G. Keratinophilic Fungi Isolated from Poultry Farm Soil of Jammu, India. Mycologist. 2000; 14: 89-91.

10. Kumari GR, Mahrora S, Rao PS. Prevalence of Non Keratinophilic Fungi in The Soil. Ind J. Med. Microbiol. 2005; 23:144-145.

11. Lee Y Mi, Choi Y K, Min BB. PCR-RFLP and Sequence Analysis Of The rDNA it’s Region in the Fusarium species. J. Microbiol, 20003; 8: 66-73.

12. Llorens A, Hinojo MJ. Logrieco Jimenez M. Characterization of Fusarium spp. Isolates by PCR-RFLP Analysis of The Intergenic Spacer Region Of The rRNA Gene (r DNA). Int. J. Food Microbiol. 2005; 106: 297-306.

13. Mahmoudabadi AZ and Zarrin M. Isolation Of Dermatophytes and Related Keratinophilic Fungi from Two Public Parks in Ahvaz Jundishapur J. of Microbiol. 2008; 1:20-23.

14. Moallaci H. Zaini F. Mahmoudi M. Hashemi J. Isolation of Keratinophilic Fungi from Soil Samples Of Forests And Farm Yards. Iranian J. Publ Health. 2006; 35: 62-69.

15. Orr GF. Keratinophilic Fungi Isolated from Soils by Modified Hair Bait Technique. J. Med Mycol, 1969; 7:129-134.

16. Plaza GA. Brigmon RL, Whitman WB, Ulfig K. Rapid DNA Extraction for Screening of Filamentous Fungi Using PCR Amplification. Polish J. Environ. Studies. 2004; 13: 315-318.

17. Papini, Mancianti. R. Grassotti F, Cardini G. Survey OfKeratinophilic Fungi Isolated from City Park Soils of Pisa, Italy. Mycopathologia, 1998; 143: 17-23.

18. Saghai M, Solimn M, Jorgesen AR, Allord WR. Ribosomal DNA Spacer Length Polymorphism in Barley Menedels Inheritances Chromosomal Location and Population Dynamics. J. Pro. Acad. Sci. U.S.A. 1984; 81:8014- 8018.

19. Saidi SA, Das P, Sikdar A. Keratinophilic fungi of poultry and their environment in India. Indian J. Comp Microbiol Immunol Infect Dis. 21:49-55

20. Schrwaz P. Bretagn S. Gracia Hermoso D. Lortholary. Dromer O and Dannaoui, E. Molecular Identification of Zygomyctes from Culture and Experimentally Infected Tissue. J. Clin Microbiol. 2006; 44:340-341.

21. Sharma Rahul. Rajak RC. Keratinophilic fungi: Natures Keratin Degrading Machines. Resonance. 2003.

22. Shukia P, Shukla CB, Kango N, Shukla A. Isolation and Characterization of a Dermatophyte, Microsporumgypseum from Poultry Farm Soils of Rewa (Madhya pradesh) India. Pak J. Biol Sciences. 2003; 6: 622- 625.

23. Singh. Mishra A. Kushwaha RKS. Dermatophyte. Related KeratinophilicAnd Opportunistic Fungi in Indoor Dust of House and Hospitals. J. Ind Med. Microbiol. 2009; 27: 242-246.

24. Santos, R.M.D, A.A.P. Firmino, C.M. Sa Felix C.R. Current Microbial. 1996; 33: 364- 370.

25. Tripathi N, Khuswaha RKS. Indian Keratinophilic Fungal Flora in Fungi Biodiversity and Biotechonlogy. 2005; 31-62

26. Vanbreuseghem. R. Keratin Digestion by Dermatophytes: A Special Diagnositic Method Mycologia. 1953; 44: 176-182.

27. Jai Godheja, S. K. Shekhar, D.R. Modi. Biodegradation of Keratin from Chicken Feathers by Fungal Species as a means of Sustainable Development. Asian J. Pharm. Tech. 2014; 4(2): 69-73.

28. Bachir Ben Seghir, Iham Ben Amor, Ahlam Beki, Imane Souici, Hadia Hemmamia, Zeghoud Soumeia, Salah Eddine Laouini, Abdelkrim Rebiai. Preparation and Optimization of Keratin from Sheep wool using response Surface Methodology. Asian J. Research Chem. 2020; 13(3): 157-162.

29. Maarb S. Al-maoula Ahmed S. Dwaish. Anti-Dermatophytes Activity of some Algal Extracts Isolated from Baghdad City-Iraq. Research J. Pharm. and Tech. 2018; 11(12): 5449-5454.

30. Hemashree T, Prasunna Sree G, Sakthiselvan P. Synthesis of Keratin Nanoparticle and Characterization using FTIR. Research J. Pharm. and Tech. 2019; 12(6): 2664-2668.

31. Venkatesh Kamath B, Hawa Hashim, Kamesh Kumar Keshri, Swati Tripathy, Subrahmanyam. V. M. Strain improvement of a new Keratinolytic Fungi. Research J. Pharm. and Tech. 2021; 14(2): 843-845

32. Amit Chauhan, Pratima Singh, S K Shukla. Establishment of Individuality and determination of Inheritance from hair keratin phenotypes in North Indian Population. Research J. Pharm. and Tech. 2021; 14(2): 869-872.

33. Amber Vyas, Vishal Jain. Luliconazole promising drug for Dermatophytes: A Review. International Journal of Technology. 2023; 13(2):79-4

34. Suneetha V. Kerazyme Triggers the Production of Mosquitocidal Toxins. Research J. Pharm. and Tech. 2014; 7(11): 1315-1318.

35. Limce Thampi, Rani K Kuriakose, Aswani Balakrishnan, Muhsena P M, Sona Grace Nelson, T M Fathima Lulu, Veena Jayan. Cosmetological competence of nail patches incombating onychomycosis-An approach to improve the quality of life. Research Journal of Topical and Cosmetic Sciences. 2021; 12(2): 125-1

36. Srinidhi K R, V M Subrahmanyam, Angel Treasa Alex, Jesil Mathew A, Venkatesh Kamath B. Role of Nano-formulation in the treatment of Onychomycosis. Research J. Pharm. and Tech. 2020; 13(1): 453-455.

Received on 08.05.2024 Modified on 10.06.2024

Research J. Science and Tech. 2024; 16(3):181-188.

DOI: 10.52711/2349-2988.2024.00027