In the recent years, synthesis of nanostructured materials has drawn the attention of scientists due to its unique physical and chemical properties. In order to produce specific sizes of nanoparticles, corresponding metal salts are reduced using various physical, chemical and biological processes(Golubeva et al. 2011; Mythili et al. 2018). Among various mode of nano synthesis biological synthesis has immense importance due to its flexible behaviour as electron donor and protection of nanoparticles along with non-polluting nature. Bio-conjugative material, such as protein, amino acids, biopolymers, carbohydrate etc. were extensively used in the field of nanoscience (Golubeva et al. 2011; Ramesh et al. 2016; Prasher et al. 2020; Espeche Turbay et al. 2021). Very recently, various researchers used plant extract, bacteria, fungi etc, as a green precursor for the synthesis of heavy metal nanoparticle (Saxena et al. 2012; Medda et al. 2015; Guilger-Casagrande and Lima 2019; Ren et al. 2019; Hamouda et al. 2019; Nilavukkarasi et al. 2020). Similarly, a large number of wastes were used including animal derived wastes like hair keratin, eggshell, feather, etc. were also used in nano synthesis (Liang et al. 2014; Lee et al. 2014; Sinha and Ahmaruzzaman 2015; Patnam et al. 2016; Wang et al. 2016; Gopiraman et al. 2017; Gao et al. 2019). But very limited literature are there on the animal derived fluid wastes like bile juices, blood etc. which contains steroidal amphipathic molecules such as –NH and –COOH group from fatty acids, cholesterol, proteins, phospholipids, bilirubin etc.(Nonappa and Maitra 2008; Rahmah 2021). Moreover, these materials may play vital role towards reducing the metals and subsequently stabilised the nanoparticles (Das et al. 2017; Jang et al. 2019). Animals derived waste products including bile is considered as complete waste in the slaughterhouses. However, use of bile is not new in the field of medicinal application. It was reported that black bear’s dried bile was used for jaundice treatment(Beuers et al. 2015).
Nanomaterials, an emerging tool can be effectively used as an antimicrobial agent to solve the current problem (Li et al. 2013; Banerjee et al. 2020; Neves et al. 2021). A large number of nanomaterials are being widely used, among them, silver nanoparticles has attracted attention, due to its wide range of applications, starting from antibacterial application, wound healing, food packaging, and textile industries etc.(Dankovich and Gray 2011; Valli and Vaseeharan 2012; Durán et al. 2016; Jin et al. 2018; Riaz Rajoka et al. 2020; Minh Dat et al. 2021). Various reports regarding antibacterial activity of AgNP against both Gram-positive and Gram-negative have been made (Lekeshmanaswamy and Anusiyadevi 2020; Neves et al. 2021). In spite of its high potential antibacterial activity, they show poor stability leading to aggregate in aqueous solution, thus, the antibacterial activity is lost (Ghosh et al. 2012; Esmaile et al. 2020; Thi Lan Huong and Nguyen 2021). Therefore, it becomes much important to hunt for novel biological route of silver nanoparticle synthesis which can overcome the above mentioned drawbacks. It is also evident that the size, shape and stability of the nanoparticle largely depends upon the interaction of reducing agent with metal ion, capping agent and metal nanoparticles (Mondal et al. 2019, 2021). Therefore there is urgent need to find a novel precursor for biological synthesis of silver nanoparticle which can overcome the above said drawbacks.
In the recent years biofilm formation has been considered as a major concern in the field of the drug resistance strategies (Abebe 2020). Biofilms are the association of microorganism usually bacteria or fungi, which can resist the antimicrobial agents as well as the immunity defence mechanism of human leading to infection and other fatalities (Khatoon et al. 2018). The associated microorganism for the biofilm formation usually in the healthcare equipment like catheters and other implants are namely, Escherichia coli, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Bacilus sonorensis (Sanchez et al. 2016; Yılmaz Öztürk et al. 2020; Mohan and Panneerselvam 2021). Biofilm formation takes place with a single microbial cell attachment on the surface of the substratum. After that, it starts forming the conditioning film formed due to self-produced (EPS) insoluble extracellular substance adhered due to electrostatic interaction (Abebe 2020). Multilayer colonization starts after cell to cell interaction and leads to the maturation of the biofilm. This formed bilayer acts as a barrier to the antimicrobial agents leading to failure of the drug therapy (Roy et al. 2017; Singh et al. 2017).
Additionally, morphological modification and controlled processes for AgNPs synthesis have gained attention in recent years. Various reports have been made on the process parameter optimization of silver nanoparticle synthesis to increase the yield of production based on the Surface Plasmon Resonance (SPR) data (Ren et al. 2019). Statistical optimization is a wonderful time reduction technique which is widely used to optimize the operational parameters in nano synthesis (Baghkheirati and Bagherieh-Najjar 2016; Sen et al. 2020; Mondal et al. 2021). Several methods are available for optimization such as Artificial Neural Network (ANN) based (Sen and Mondal 2020), Response Surface Methodology (RSM) (Sen et al. 2020; Azmi et al. 2021) etc. However, the RSM is a potent tool due to its easy operational procedure, good performance for low input data etc. (Sen et al. 2019).
In present study, silver nanoparticles have synthesised from bile juice as a novel agent and the synthesised nanomaterial was characterized by advanced analytical techniques and optimization of nano synthesis was performed by RSM study. Finally, the efficacy of BJ-AgNP was evaluated through antibacterial activity against both the Gram positive (Bacillus subtilis) and Gram negative (Escherechia coli) bacterial strains. Metabolic activity, respiratory dehydrogenase enzyme activity, intracellular glutathione activity, protein leakage were observed to identify the therapeutic potential of the synthesised nanomaterial. Live dead assay was further carried out to monitor the viable and dead cell in the BJ-AgNP exposure. Lastly, the synthesised nanomaterial was also used as a biofilm inhibiting agent.