Standardization, Characterization and Isolation of Trichoderma-Silver Nanoparticle-A Pharmaceutical Approach in Field of Nano-Medicine

Silver is a well known antimicrobial agent. It is utilized in many antimicrobial and medication. The Trichoderma and its Metabolites have also been meant for antimicrobial activity against the various bacterial and fungal strain. In the present investigation Nanoparticle of silver is prepared by chemical method, green synthesis using plants and biosynthesis using microbes. Trichoderma secondary metabolite prepared by solvent extraction method from Trichoderma harzianum which act as a capping and reducing agent. The biosynthesised silver Nanoparticles were characterized by UV-Vis spectroscopy and TEM. UV-Vis spectra of silver Nanoparticle and trichoderma extract showed absorption spectra at 420nm & 430 nm respectively while the fused nanoparticle with Trichoderma secondary metabolite showed absorption spectra at 415 nm corresponding to the surface Plasmon resonance of silver Nanoparticle. It was determined the nanoparticle showed absorption spectra at 415 nm and morphology as spherical with size range 8 to 24 nm and providing good antimicrobial activity as Trichoderma silver fused nanoparticle against many microbial strain, so it can be prepared for pharmaceutical approach against the infectious disease caused by clinical pathogenic organisms. Original Research Article Gupta and Gaur; JPRI, 33(48A): 79-91, 2021; Article no.JPRI.74691 80


INTRODUCTION
Nanotechnology is basically the science that deals with the matter at the scale of 1 billionth of a meter. Means 10¯⁹m=1nm. Nanoparticle is having very important role in both biotechnology and pharmaceutical industries. Nanoparticle is the most fundamental component in the fabrication of a nanostructure. Basically the size of a Nanoparticles range between 1 nm to 100 nm. The metallic nanoparticles have different physical and chemical properties rather than its bulk form of metal. Also Nanoparticles of various metals have unique electrical, optical & biological properties. Due to preserving the qualities, play a very vital role in pharmaceuticals order too [1,2,3].
These Silver Nanoparticles can be prepared by microbiological synthesis, chemical method and green synthesis i.e using plant. For green synthesis plant like Aloe barbadensis is used. Also many microbes is used for synthesis of silver Nanoparticles like Trichoderma viridae [4,5]. Other microbes used are Fusarium oxysparum, Alternaria alternate,Aspergillus terrus, Trichoderma virens, Trichoderma ressei, Trichoderma aspergillum,Phoma glamerata,Trichoderma longibrachitum. For production of silver nanoparticle by chemical method like silver nitrate is used [6,7,8] Pharmaceutical approach of these Silver Nanoparticle formed by green synthesis using Aloe barbadensis have been observed having antimicrobial activity against clinical pathogen like E. Coli, S. Typhi and many other organisms by agar well diffusion. So, it's the novel tool in the study of nanotechnology.
Silver nanoparticle prepared by chemical method using nutrient broth silver nitrate and trichoderma extract showed a good sensitivity against Pseudomonas, Rhizopus with Slight sensitivity against Proteus and Fusarium species [5,3].

LITERATURE REVIEW
The word Nano is referred to a Greek word which means dwarf or something which is very small. Nanotechnology also shortened to nanotech is the use of matter on an atomic, molecular and supramolecular scale. Nanoscience quantum leap in almost every field of science and nanotechnologies make life calm in this era [2,5].
The Nanoscience and nanotechnology represent an expanding research area and involve structures, devices and system with novel properties and function due to the arrangement of their atoms on the 1-100 nm scale. This field arouse much awareness and controversy in early 2000's and led to start of commercial application of nanotechnologies [1,2].
There is a difference between Nanoscience and nanotechnology. Nanoscience is the study of structure & molecules on a scale of nanometer ranging between 1 to 100 nm and in case of nanotechnology, technology is one that utilizes it in practical application such as devices, textiles, human health care etc [4,3].

Silver: Properties & Use
Silver is a chemical element with Ag as its symbol and belong to 11 th group in the periodic table and its atomic number is 47. Silver is lustrous, soft, very ductile and malleable metal. Silver have many application as in photography, dentistry, cutlery, mirror, as catalyst in oxidation reaction, coins and jewellery, In electrical & electronic industries for items such as printed circuits and computer [3,7].
In the field of nanotechnology and Nanoscience, it is used in form of silver nanoparticle and are increasingly used in various field including medical, food, healthcare, consumer and industrial purposes due to their unique physical and chemical properties [9]. These include optical, electrical and thermal, high electrical conductivity and biological properties [10].
As silver exhibits a size and shape dependent optical effect known as localized surface Plasmon resonance (LSPR) at the nanoscale the ability to synthesize silver nanoparticles in different shape vastly increase ability to tune their optical behaviour [11].
So also it is well said sometimes a small things make a big impact. Ag*NP have a unique electrical, thermal, optical & biological properties [12,13]. It is the reason that silver Nanoparticle has been introduced in practical application like molecular diagnostics and a trial of its fused version with natural antimicrobial agent is undergoing research to find out the pharmaceutical approach in the field of clinical sciences [14]. The basic chemistry behind Ag*NP is when this nanoparticle is in solution, molecules associated with the nanoparticle surface to establish a double layer of charge that stabilize the particle and prevent aggregation [15,16].
Silver nanoparticle are being used in numerous technologies and incorporated into a wide array of consumer products and take benefits of their desirable optical conductive and antibacterial properties [14,17]. Ag*NP have diagnostic application used as biological tags for quantitative detection, antibacterial application like Ag*NP are incorporated in apparel, footwear, wound dressing & bactericidal & bacteriostatic capability [16].
Advances in field of Nanoscience and nanotechnology have changed the way we diagnose, treat & prevent various diseases in many aspects of a human life. Ag*NP can be prepared by green synthesis that is plant extract like Aloe barbadensis, biological synthesis that is microbes & chemical synthesis like by using silver nitrate [1,2]. This Ag*NP also have antibacterial activity. When zeta potential determined by double beam UV-Vis spectrophotometer and FT-IR was measured, it was determined to be 194. Yellow pigment is secreted in PDA and this yellow pigment is basically secondary metabolites. Microscopically conidiophores is branched with wider phialides and enlarged in middle also cylindrical. It is saprophytic belong to Ascomycota [19,20]. As per Gams and Bisset, Trichoderma comes under imperfect fungi. It is good spored, soil borne of have potential of degradation as not involved in sexual reproduction and can perform decomposition [21,22].
Trichoderma is also used as biological fungicides. The secondary metabolites produced by Trichoderma are kojic acid, citric acid and acetic acid. All of these work as a biocidal antibiotic against clinical bacterial & fungal pathogens [13,18] The Trichoderma have many useful properties, it control plant pathogenic fungi and play major role in nitrogen fixation which is important for plant growth. Trichoderma uptake the nutrients and promote the root & shoot growth and improve soil texture and help in control of phytopathogenic disease [4,11].
Due to the use of chemical method for protection of plant from phytopathogen have lead to decrease in product quality and soil texture. So to increase in demand of ecofriendly product replacement of chemical pesticides is done with biosynthesised based pesticides [1] The trichoderma species attack host by coil around the host hyphae and produce hydrolytic (fungitoxin) enzymes like chitinases, glucanases, proteases, peptaibol antibiotics and thus lead to phytopathogen control by biological method [2,8].
For preparing Ag*NP, as given by Chikdu et al (3)0.1 M silver nitrate was prepared in water and distributed as 10 ml,15 ml & 20 ml of Ag*NO3 in 3 small beaker and kept in magnetic stirrer for 15 min at 65°C. To it 1 ml plant extract was added drop wise in all the 3 beaker of Ag*NO3 with continuous stir. The above solution was kept in stirrer for 15 min & colour change was observed to reddish brown which indicate synthesis of Ag*NP & formation of Aloe barbadensis extract-Ag fused nanoparticle. This was characterised UV-Vis spectroscopy for analysis of NP, the synthesised Ag* Nanoparticles showed highest absorbance peak at 420 nm [ Konigii secret Protein and enzyme that act as reducing agent and capping agent. The biosynthesised Ag*NP have the absorbance of 413 nm by U-Vis spectroscopy and size of nanoparticle is 8-24nm and morphology as spherical when determined by TEM [12].
The biosynthesis of nanoparticle by using fungi is more advantageous rather than plant because this fungus produce more protein that lead the increase production of nanoparticle and also provide longer stability. Also extracellular synthesis of Ag*NP is found in fungi like Fusarium oxysporum, Aspergillus fumigates, Neurospora crassa [23,24].
Ag*NP also perform application in the field of biolabelling, antimicrobial filters and bactericidal activity. Trichoderma kongii prepared Ag*NP treated against S.typhimurium, 45 µg/ml inhibit its growth [9]. Trichoderma first came into known as antimicrobial agent by Christian Hendrik Persoon and its quality is it is fast growing [12,14].

F. Preparation of silver nanoparticles-For
the preparation of silver nanoparticles two stabilizing agents, sodium dodecyl sulphate (SDS) and sodium citrate were used. For the synthesis of silver nanoparticles, silver nitrate solution (from 1.0 mM to 6.0 mM) and 8% (w/w) sodium dodecyl sulphate (SDS) were used as a metal salt precursor and a stabilizing agent, respectively. Hydrazine hydrate solution with a concentrate ranging from 2.0 mM to 12 mM and sodium citrate (1. 0 mM to 2.0 mM) were used as reducing agents. Citrate of sodium was used as stabilizing agent at room temperature. The transparent colorless solution was converted to the characteristic pale yellow and pale red colour, when citrate of sodium was used as stabilizing agent. The occurrence of colour was indicated by the formation of silver nanoparticles. The silver nanoparticles were purified by centrifugation. To remove excess silver ions, the silver colloids were washed at least three times with deionized water under nitrogen stream. A dried powder of the nanosize silver was obtained by freeze-drying [16,14,17,18,21].  [25,26,19,22].

H. Characterization of prepared nanoparticles via UV-absorption spectra and Transmission electron microscopy (TEM) -
The formation of Ag*NPs and TR-Ag*NPs by the bioreduction of Ag+ to Ag0 was easily monitored using UV-Vis spectroscopy. The scanning was performed in the range of 200-700 nm. The morphology and size were determined by TEM [27,20,23,24,28]. I. Determination of antimicrobial activity of Trichoderma-Ag* fused nanoparticles against the pathogens-The antimicrobial activity of Trichoderma extract and Trichoderma fused silver nanoparticles was determined at different concentrations against local isolated pathogenic cultures viz. Pseudomonas aeruginosa, Bacillus subtilis, Micrococcus luteus, Leuconostoc mesentroides, and Aspergillus niger by well diffusion method [29,[30][31][32][33][34][35]

RESULTS
The present study showed the isolation and characterization of Trichoderma spp. culture isolated from soil. Further the culture was determined taxonomically and was determined as Trichoderma harzianum and was denoted as per the accession no-NCFT.9153.17. The culture photograph is shown in Fig. 1.
On microscopic and taxonomic identification of trichoderma culture the colony was observed as green colour with compact conodiophore. Conidia is compact or loose tuft in shades of green. . On microscopy septate hyaline hyphae seen along with branched phialids, were hyaline, flask shaped and inflated at the base. Colony is green colour with sweet or coconut smell. Thus the culture was identified as T. Harziamun (Fig.  2).
Extraction of secondary metabolite extract from T. harziaum: T. harzianum extracted prepared and filtered by solvent extraction method to determine secondary metabolite. And then this solvent extract was further vacuum dried, to evaporate the solvent and obtain the powder form having secondary metabolite for further identification and preparation of nanoparticle in fusion with silver Fig. 3. The GC-MS was done to identify the secondary metabolite and it was found to be kojic acid, citric acid & acetic acid Fig. 4.

Preparation & Characterization of Trichoderma fused Silver Nanoparticle (TR-Ag NPs)
In the present investigation Tr-Ag*NPs were produced (Figs. 5 & 6). The particle size was determined by SEM. And the absorption spectrum was determined at 200-700 nm. It was found the TR-Ag*NP were very fine shape and size having optimal size 20 nm same as in case of green synthesised nanoparticle or T.viridae synthesised nanoparticle and are having slight rough spherical structure.
The UV absorption spectra of the fused nanoparticle had the maximum wave length 415 nm (Fig. 7).
On determination of antimicrobial activity of trichoderma harzianum solvent extract (Fig. 8a) and Trichoderma silver fused nanoparticle (Fig.  8b) against clinical pathogen gave significant result. Also the antimicrobial sensitivity was observed more in case of trichoderma silver fused nanoparticle against clinical pathogen rather than sensitivity test with Trichoderma extract only.       (Table 3a) Show more zone of inhibition rather than TR extract alone (Table  3b).

DISCUSSION
When green synthesised Ag*NPs was prepared and fused plant Aloe barbadensis extract silver fused nanoparticles finally formed was known by colour change to reddish brown whereas when Trichoderma harzianum secondary metabolite silver fused nanoparticle formed was known by colour change to greenish colour of solvent extract which was fused by silver. Also, a per research T.konigii secrete protein amd enzymes which act as a reducing agent and capping agent [9]. AgNP synthesised by T. konigii also gave a comparable good result as antimicrobial agent against clinical pathogen and it was also found the absorbance by UV spectroscopy as 413 nm which was approx same range as 415nm for the T. harzianum silver fused nanoparticle and also the absorbance UV spectroscopy by Aloe barbadensis extract silver fused nanoparticle as 420 nm. Same in the case of T. viridae, it also has the ability of biosynthesis of Ag*NP and when the T. viridae silver fused nanoparticle is reacted against clinical pathogenic organism give the remarkable anti microbial sensitivity report. Even this is sensitive toward MRSA strain too.
Thus the green synthesised and biosynthesised Ag*NP and the Trichoderma extract when fused together give a remarkable result against many clinical pathogenic strain and can be a good scope for diagnostic & pharmaceutics in the field of nano-medicine.

CONCLUSION
Thus as per the different studies done, these trichoderma silver fused nanoparticle have the properties of antibiotics and play an important role in pharmaceutics in the field of nanoscience and have a great role in nanotechnology. Silver nanoparticles have been used for wound healing and enhance tumour killing effect of anti cancer drugs. The biosynthesis of Nanoparticles by using fungi is biocompatible, biodegradable and ecofriendly advantageous rather than chemical or plant based. Ag*NP synthesis because these fungus produce more protein that lead the increase production of Nanoparticles and also provide long stability. It can thus be used to prepare potent biocidal product against clinical pathogen.
Further different-different dose based study is required to determine and analyse the antimicrobial sensitivity produced of Ag*NP produced by different method is green synthesis, microbial synthesis and chemical synthesis of Ag*NP and its AST against same pathogenic strain at different doses.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.