Synthesis, characterization and optimization of chicken bile–mediated silver nanoparticles: a mechanistic insight into antibacterial and antibiofilm activity

The fast-growing urbanization and slow progress in the field of waste management have led to the accumulation of large quantities of animal wastes. The present work focused on the synthesis of low-cost and eco-friendly chicken bile juice–mediated silver nanoparticles (BJ-AgNP). Results reveal that bile juices have enough potentiality towards the synthesis of almost uniform sizes (average size < 50 nm) of BJ-AgNPs which remains stable for more than 6 months. Response surface methodology (RSM) successfully demonstrated the optimised condition of BJ-AgNP synthesis. Factors like concentration of salt and bile extract and temperature are significantly responsible for nanoparticle synthesis. The synthesis of nanoparticle was further characterized using UV-Vis, TEM, FESEM, XRD, FTIR, TGA, and EDS. The synthesised nanoparticle showed excellent bactericidal activity against both Gram positive and Gram negative bacteria with MIC and MBC of 40 and 50 μg/mL for Bacillus subtilis (MTCC-441) and 60 and 60 μg/mL for Eschecheria coli (MTCC-1687) respectively. The synthesised nanoparticle also exhibited as an antibiofilm activity against B. subtilis, with ~89% biofilm inhibition efficacy at 4 X MIC, having optimal bacterial concentration of 106 CFU/mL. Therefore, the present findings clearly demonstrated that an absolute animal waste could be a valuable ingredient in the field of therapeutic nanoscience.


Introduction
In recent years, synthesis of nanostructured materials has drawn the attention of scientists due to their 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 the various modes of nanoparticle 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 de 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 literatures 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 a vital role in reducing the metals and stabilising the nanoparticles (Das et al. 2017;Jang et al. 2019). Animalderived waste products including bile are 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 search 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. , 2021. Therefore, there is an urgent need to find a novel precursor for biological synthesis of silver nanoparticle that can overcome the abovementioned 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 microorganisms, 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 optimization of process parameter for the synthesis of silver nanoparticles and to increase the yield based on the Surface Plasmon Resonance (SPR) (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; Mondal et al. 2021). Several methods are available for optimization such as Artificial Neural Network (ANN)-based , Taguchi optimization (Ghosh and Mondal 2019), Response Surface Methodology (RSM) 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 the 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 response surface methodology. 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.

Experimental design
The present work is distinctly focused on two major applications. Internal parts of chicken was collected from slaughter house in Burdwan town (Latitude 99° 23′15′ 22″ N Longitude 87°50′58″ E) West Bengal, India, and bile juice was extracted from gallbladder. Then approximate metal salt solution was mixed with bile juice to prepare BJ-AgNP. The optimization of nanosynthesis was performed through response surface methodology and characterization were performed by various analytical techniques. Finally, antibacterial and antibiofilm activity of BJ-AgNP were performed.

Reagents
Chemicals like silver nitrate (AgNO 3 ) was procured from Sigma-Aldrich (ACS reagent, ≥99.0%) and methylene blue and other culture media were purchased from HiMedia Laboratories (Mumbai, India). Throughout the experiment, Milli-Q ultrapure water was used. Before conducting the experiments, glassware were cleaned in aqua regia and autoclaved.

Preparation of silver nanoparticle (BJ-AgNP)
Bile-induced silver nanoparticles were prepared by using bile juice, collected from slaughterhouse gallbladders were pierced with sterile micro tip and juice extracted in sterile test tube (Borosil Pvt. Ltd, Gujrat, India). Different concentrations of bile juice was prepared through dilution technique by using Milli-Q water, with constant stirring followed by filtration (Whatman No 1, USA). Then the filtrate was added dropwise in freshly made silver salt solution (100 mM) in different ratios. The bile juice was used both as reducing and capping agent towards the synthesis of silver nanoparticles. Then the absorbance was evaluated through UV-Vis analysis. Further, the synthesized BJ-AgNP solution was lyophylized using (Biobase Ltd.) lyophylizer. The dried powder was used for further study.

Optimization of BJ-AgNP synthesis
Optimization study was performed by using Design-Expert (Version 7.0.0) statistical software. The design of the experiment (DOE) was constructed with three parameters (temperature, bile concentration and salt concentration). Number of experimental runs was suggested by face centred central composite design (FCCCD) at three levels. Response indicates the absorbance intensity as SPR for yield attributes to response having wavelength ranging between 410 and 430 nm for evidence of the BJ-AgNP synthesis. The detail study is shown in the supplementary section.

Characterization of the BJ-AgNP
The spectral signature of the synthesised BJ-AgNP was recorded by using UV-visible spectroscopy (Optizen-POP). The active functional group was assessed by Fourier transform infrared (FTIR) spectral analysis (Cary 630, Agilent Technology). BJ-AgNP solution was lyophilized using BIOBASE, China. Surface morphology and characterization were performed by TEM (JEOL JEM 1200, assisted with Gatan Image Filter) at an accelerating voltage of 120 KV, and FESEM (GeminiSEM 360 ZEISS). Elemental quantification of BJ-AgNP was estimated using EDS (Gemi-niSEM 360 ZEISS). Dynamic light scattering (DLS) (Malven Instruments) was employed to know the hydrodynamic size, aggregation and polydispersity of the BJ-AgNP. X-ray diffraction (XRD) analysis for BJ-AgNP was captured with K-beta filter having radius 1.54056 Å. The thermal stability for biogenic AgNP-BJ was examined through thermo gravimetric analyser (PerkinElmer, TGA 8000), whereas temperature range into subambient to 1200 °C, 10 °C/min in nitrogen atmosphere with sampling rate of 20 mL/min.

Culture media and strains used in the study
The antibacterial activity of BJ-AgNP was assessed against two bacterial strains, Escherichia coli (MTCC-1687) and Bacillus subtilis (MTCC-441) collected from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India. Nutrient Agar (N.A) and Nutrient Broth (N.B) of bacteriological grade were used as culture medium. Both the Gram positive and Gram negative bacteria were grown in liquid broth at 37°C and 120 rpm for incubating overnight and then sub-cultured in NB using a culture having OD (optical density) = 1, approximately 10 5 CFU/mL.

Agar diffusion method
Agar well diffusion experiment was carried out using the bacterial strain of E .coli and B. subtilis. 100 μL inoculum for both the overnight grown culture were well spread in NA plates and left to dry for 5 min. Wells (diameter 11 mm) were punctured using sterile cork borer (Himedia, Mumbai). Each well was marked and different concentrations of BJ-AgNP were added along with a negative control and raw bile juices were loaded in respective wells. Suspensions of nanoparticles were sonicated in a bath sonicator before loading into the well. After overnight incubation, the zone of inhibition around the wells was measured using a scale. The experiments were conducted in triplicates for each bacterial strain, and the mean and standard deviation were calculated in the results section (Thi Lan Huong and Nguyen 2021).

Broth dilution method
Broth dilution method was employed to determine the minimum inhibitory concentration (MIC) and maximum bactericidal concentration (MBC) of BJ-AgNP against Escherichia coli (MTCC 1687) and Bacillus subtilis (MTCC 441). Bacterial inoculum (1% v/v) was added in 200 μL NB broth, which was taken in 96-micro well plate reader (Tecan Infinite M200 Pro) and the absorbance was recorded at 600 nm.

Minimum inhibitory concentration (MIC) and Maximum bactericidal concentration (MBC) determination
MIC and MBC for both E coli and B. subtilis against BJ-AgNP were estimated by following the standard micro broth dilution assay. In short, 1% v/v of bacterial culture was inoculated in 200 μL of LB broth taken in each microtitre well.
BJ-AgNP was added in increasing concentration and the plate is incubated overnight at 37°C and 120 rpm. To check the growth of the treated bacterial cell, OD was taken at 600 nm. The lowest concentration of BJ-AgNP showing OD 600 < 0.1 was reinoculated without treatment into fresh LB broth and allowed to incubate further for 12 h at 37°C and 120 rpm. The lowest concentration of AgNP-BJ that completely killed the bacteria (i.e., wells with OD 600 < 0.1 value) was considered as the MBC value.

Time-dependent in vitro killing assay (growth kinetics)
The growth kinetics of the bacteria under the influence of nanoparticles was assessed through time-dependent in vitro killing assay. Bacterial growth pattern in the presence of the treatment (BJ-AgNP) can be observed following the experiment. 200 μL cell suspensions (10 6 CFU/mL) were taken in 96 microwell plate and treated with various concentration of BJ-AgNP at 37°C for 12 h. Absorbance (600 nm) was monitored at an interval of 30 min using microwell plate reader (Tecan Infinite M200 Pro). Data analysis and graph plotting were done using Origin 9.1 software.

Measurement of intracellular Glutathione (GSH) activity
Glutathione (GSH) acts as an antioxidant and protects cell from oxidative stress related damage. GSH upon oxidation, converts to oxidized glutathione (GSSG) which fails to protect cells from oxidative damage, eventually oxidizes vital cellular components like protein, nucleic acid and lipid , finally leads to cell death (Ulrich and Jakob 2019). Intracellular GSH activity was measured following standard Ellman's assay, as reported in previous literature (Parandhaman et al. 2015). The E. coli and B. subtilis (10 6 CFU/mL) were treated with increasing concentrations of AgNP-BJ for 6 h at 37° C and 200 rpm. Bacterial cells suspensions were centrifuged at 4000 rpm for 10 min, washed with PBS and finally lysed, supernatant was collected after re-centrifugation and mixed with 50 mM Tris-HCl and 100 mM 5, 5-dithiobis (2-nitrobenzoic acid). The mixture was kept in dark place for 30 min incubation. Absorbance analysis was done at 412 nm using UV−visible spectrometer. The loss of glutathione was compared with control using the following formulae

Measurement of metabolic activity
The metabolic activity was estimated by Alamar Blue assay (1993). In this assay, the cells convert a pink fluorescent dye resofurin from purple resazurin (nonfluorescent). E. coli and B. subtilis cells were grown under the exposure of MIC to 4 X MIC concentration of nanoparticle for 6 h at 37°C and 200 rpm. The cells were further incubated with 25 μL of 50 μg/ mL resazurin solution for 1 h at 37°C. The metabolic conversation of pink coloured resofurin from resazurin was measured spectrophotometrically at 571 nm (Rampersad 2012).

Measurement of respiratory dehydrogenase enzyme (LDH) activity
The LDH enzyme activity was measured using Pierce LDH Cytotoxicity Assay Kit. Both control and treated bacterial cells of E. coli and B. subtilis were incubated for 6 h at 37 °C and 200 rpm. Cells were centrifuged at 5000 rpm for 5 min and supernatant was discarded, pellet was washed with PBS and LDH reaction solution was added. Then 30 min incubation in dark was followed for red formazan production. The reaction was terminated by adding a stop solution and absorption was measured at 490 nm. Results were obtained after comparing the % LDH activity of the treated cell with that of control (Roy et al. 2019).

Protein Leakage Assay
E. coli and B. subtilis cells (10 6 CFU/mL) were treated with increasing concentrations of BJ-AgNP for 6 h at 37 °C and 200 rpm. The bacterial cells were pelleted at 5000 rpm for 5 min and the supernatant was collected to measure the amount of protein leaked by following the standard Bradford assay (Kim et al. 2017;Roy et al. 2019).

Antibiofilm study
To know the efficacy of the synthesised BJ-AgNP against the biofilm formation, the experiment was conducted using E. coli (MTCC-1687). Biofilm were grown with 48 h culture in a 96 microwell plate with increasing dose of BJ-AgNP treatments including a control. The media was decanted after complete incubation and washed with sterile PBS (Phosphate buffer saline) solution and air dried. Excess stains were solubilised using 95% ethanol and OD was taken at 590 nm using a microwell plate reader (Tecan Infinite M200 Pro). During the inhibition calculation, the following formula was followed

Synthesis and optimization of BJ-AgNP
A wine red colour solution was formed after mixing bile juice with 100 mM of silver nitrate solution in the ratio of %Inhibition = Control absorbance 575nm − Treatment absorbance 575nm Control absorbance 575nm × 100 1:9. This is perhaps due to the surface plasmon resonance (SPR) of silver nanoparticle (Mondal et al. 2021). The synthesis of nanoparticle was monitored after varying factors like temperature, amount of bile and concentration of silver nitrate solution, which is shown in Fig. 1, and the optimized data was recorded from RSM study. From Fig. 1a, it is also seen that varying bile concentration and temperature interfered the BJ-AgNP synthesis. Moreover, bile and salt concentration are responsible factors for nanoparticle formation which is clearly showed in Fig. 1b. The interaction effect of salt concentration and temperature during nanoparticle synthesis is shown in Fig. 1c. Therefore, results revealed that bile concentration is the regulatory factor that largely affects the yield of silver nanoparticles. This may be due to its composition, including bilirubin and fats (cholesterol, fatty acids, and lecithin) which collectively helps towards the reduction of Ag + ions (Mandal et al. 2002). On the other hand, the synthesis of AgNPs was recorded highest at higher temperature (near boiling point). At higher temperature bile molecules undergo thermal degradation which helps to make the availability of free electron in the medium and subsequently reduce Ag + to Ag 0 . Therefore, temperature plays an important role in nanoparticle synthesis (Jiang et al. 2010). Moreover, the synthesised nanoparticles were protected from agglomeration by bile containing biomolecules. The outcomes of optimization study were depicted in Table S1. From Table S1, it is clear that the model is significant (P < 0.001) with very high F value (97.57). Modelling study also revealed that bile juice-mediated nano synthesis is strongly influenced by temperature (P < 0.0001) and bile concentration (P < 0.0001). On the other hand, higher desirability (1.00) was achieved under the following conditions: temperature, (99.73°C) bile concentration (5.70 μL/mL (v/v)) and salt concentration (0.30 mg/L) (Fig. 1d).

UV-Vis study
In order to investigate the optical response of bile juice-mediated AgNPs which is the results of intense surface plasmon resonance in the visible region (Sharifi-Rad et al. 2021). The distinct colour change of the mixture was observed when the silver nitrate solution was mixed with chicken bile juice. This colour change is a clear indication for the formation of the silver nanoparticles which is again confirmed from the shift of λ max peak in the UV-Vis spectrum ( Fig. 2a) (Saad et al. 2021). In the present study, two sharp spectral bands were recorded at 430 and 400 nm for BJ-AgNPs and raw bile juice, respectively (Fig. 2a). Moreover, symmetrical spectral also revealed that the synthesised AgNPs are uniform in shape (Yılmaz Öztürk et al. 2020).

FESEM, EDX, TEM and DLS study
Field Emission Scanning Electron Microscopy (FESEM) clearly revealed the crystalline spherical structure of BJ-AgNP (Fig. 3a). Figure 3a also showed that the synthesised BJ-AgNP, are well dispersed and mostly spherical in shape. Moreover, from Fig. 3a, it is also clear that about 25.76% ± 3.77 particles are within 40 nm. The size distribution patterns of nanoparticles are depicted in Fig. 3b. The EDX study demonstrates the clear signature of silver metal (Fig. S2). Similarly, dynamic light scattering (DLS) study also supports the above results. The dimension of particles is depicted in Fig. 2c. The nanoparticles size ranges from 30 to 45 nm with average particle size of 40 ±1.55 nm. The polydispersity index was recorded 0.181 and Zeta potential (ζ) ranges from −14.74 to −41.79 mV. This high value of Zeta potential clearly suggests the higher stability of synthesised nanoparticles. Present findings are in accordance with previous literature (Hebeish et al. 2013;Haque et al. 2017). Transmission electron microscopy (TEM) study also supports the above outcome (Fig. 3b). Selected area electron diffraction (SAED) pattern again demonstrated the polycrystalline structure of the BJ-AgNP, with miller indices (111),

FTIR, XRD and TGA analysis
The FTIR spectral signature of both raw bile juice and bile mediated AgNPs were recorded for the identification of functional group (Garibo et al. 2020). The possible chemical profile of bile revealed that the presence of alkyl halide, aldehyde, amines and aromatic compounds with transmission peaks at 1250, 2863, 1053 and 1405 cm −1 , respectively (Fig. 3a). However, bile mediated silver nanoparticles (BJ-AgNP) showed three distinct peaks at 3735 cm −1 , 1650 cm −1 , and 1405 cm −1 which corresponds to the stretching frequencies of free hydroxyl -OH, -C=O and C=C-(aromatic), respectively. These functional group could be responsible for the synthesis of nano particle as well as capping of Ag o (Kleiner et al. 2002). This explores the dual nature of bile juice. Figure 3a also  . Previous reports also support that those peaks are responsible for the deformation of the β-(1,4)-glycosidic bond of the peptidoglycan layer of the bacterial cell wall, leading to the inhibition of the bacterial growth (Nikonenko et al. 2000;Feo and Aller 2001;Slavin et al. 2017). The spectral signature of BJ-AgNP formation is remarkably changed when BJ-AgNP interacted with formed biofilms (Fig. 2b). Another analytical technique, XRD analysis revealed that the synthesised BJ-AgNP has distinct six measured d-  (222) and (300), respectively (JCPDS # 01-087-0719) (Fig. 3c) (Yılmaz Öztürk et al. 2020). The average size of the synthesized BJ-AgNP was also calculated by following the Scherrer equation , and the average size of 41.81±1.66 nm.
Similarly, TGA is one of the important characterization tool which confirmed the presence of organic capping layer over silver nanoparticles. Weight loss depends on moisture loss and organic compounds decomposed that exist in the BJ-AgNP compound (Paterlini et al. 2021). The current results of BJ-AgNP and bile juice were shown in Fig. 3d. Moreover, from Fig. 3d, it has been found that the synthesised BJ-AgNP, losses 15.39% weight when temperature increased from 400 to 600°C. Similarly raw bile loosed 37.46% weight at 500°C. Therefore, TGA study clearly revealed that the synthesised BJ-AgNP are moderately stable. This is perhaps due to the presence of strong organic capping agents (Thi Lan Huong and Nguyen 2021).

Antimicrobial activity
Primarily, agar cup diffusion method was applied to evaluate the antimicrobial activity of synthesised BJ-AgNP. Diameters of the zone of inhibition were measured after 24 h of incubation at 37°C and the results are depicted in Fig. S4. The zone of inhibition obtained with 2X MIC doses of BJ-AgNP, were 2.5 and 2.1 cm for E. coli and B. subtilis, respectively and non-significant zone of inhibition was recorded for raw bile juice (Fig. S4). The Zone of inhibition is probably due to damage of bacterial cell walls through penetration of silver nanoparticles (Liao et al. 2019). The diameter of the zone of inhibition is very less and marginal difference was observed between 1X MIC and 2X MIC concentration. This may happened due to the aggregation of the BJ-AgNP in the agar medium.
Similarly, results of MIC and MBC value of BJ-AgNP against E. coli and B. subtilis were depicted in Table S4. The MIC and MBC values of BJ-AgNP against E. coli were recorded as 60 and 60 μg/mL and for B. subtilis as 40 and 50 μg/mL respectively (Table 1). These results clearly revealed that BJ-AgNP have lower bactericidal efficiency against E.coli compared to B. subtilis. Almost similar findings was recorded by (Bruna et al. 2021) Antibacterial potentiality was also examined by MBC/MIC ratio and it was recorded as ≤ 1.2 for both E. coli and B. subtilis which clearly suggests the potentiality BJ-AgNP is an active bactericidal agent (Table 1) (Roy et al. 2019). The study also highlighted the time dependent growth inhibition and complete reduction was observed at 9 th hour of exposure (Fig. S3).
The growth kinetics of E. coli and B. subtilis were shown in Fig. 5a and b respectively. From Fig. 5a and b, it is clear that complete inhibition occurred at 90 and 80 μg/mL of BJ-AgNP for E. coli and B. subtilis respectively. Antibacterial efficacy of BJ-AgNP against E. coli was also determined after 12 h of exposure and results shows following trend of reduction with respect to control as: 20 mg/L (61.66%) > 40 mg/L (51.45%) > 60 mg/L (12.33%) >80 mg/L (5.39%) > 100 mg/L (1.29%). Similarly, for B. subtilis showed that the growth reduction trend as; 20 mg/L (31.29%) > 40 mg/L (30.39%) > 60 mg/L (1.22%) > 80 mg/L (3.59%) > 100 mg/L (0.79%) after 12 h. This information also suggested that the growth curves gradually declines with increasing the concentration of BJ-AgNPs. This is possibly due to direct intervention of BJ-AgNP resulting in the degradation of enzyme, DNA and inactivation of cellular proteins (Nikaido 2003). The recent study suggested that nanoparticle-induced cell death is due to generation of reactive oxygen species (ROS) which subsequently damages protein and other biomolecules (Rohde et al. 2021).

Metabolic arrest
In the present study, by observing the colour change of non-fluorescent purple dye resazurin to resofurin (pink fluorescent) which is the indicator of metabolically active cells load, depicted in Fig. 6(a). From Fig. 6(a), it was clearly observed that metabolic activity of both the bacterial strains (E. coli and B. subtilis) are greatly hampered under different concentrations of BJ-AgNP. At 4 X MIC, E. coli and B. subtilis showed 53.44% and 62.26% reduction in metabolic activity respectively. Therefore, it is clearly established that BJ-AgNP upon interacting with bacterial cells impeded the cellular respiration leading to metabolic arrest and ultimately cell death occurs (Rampersad 2012).

Inhibition of lactate dehydrogenase activity (LDH)
The LDH activities under different concentration of nanoparticle treatment against two bacterial species (E.coli and B. subtilis) were presented in Fig. 6(b). Fig 6(b) highlighted that reduction of LDH% increases from 33.26 to 72.34% with increasing the MIC concentration from 1× to 4× for E. coli. On the other hand, Bacillus subtilis also exhibited similar reduction of LDH% (55.73%) level at 4× MIC. Results clearly depict that BJ-AgNP treatment on bacterial cells has greatly affected the LDH activity. Extensive membrane damage under nano treatment could be the possible cause that inhibited the respiratory enzyme (Roy et al. 2019).

Intracellular glutathione oxidation
In the present study, percent loss of GSH activity was evaluated with respect to control and it is presented in Fig. 6(c). Figure 6(c) indicate a progressive loss of GSH activity for both E. coli and B. subtilis with 25.79% and 22.79% at 4× MIC treatment of BJ-AgNP respectively. This phenomenon can be attributed to the fact that the entry of BJ-AgNP inside the bacterial cell is due to the small size of BJ-AgNP. Disruption of lipid bilayer occurs which may be the additional cause of ROS generation along with the membrane damage (Fahey et al. 1978). Therefore, it can be said that BJ-AgNP possesses an overall improved antibacterial efficacy.

Protein leakage
The percentage of protein leakage was recorded from the two target species (E. coli and B. subtilis) under nano treatment and control conditions were depicted in Fig. 6d. The result revealed that maximum protein leakage was recorded at 4× MIC for E. coli. On the other hand, nearly 35% protein leakage was enhanced when B. subtilis cells treated with 2× MIC concentrations with respect to MIC concentration. On the other hand, TEM images ( Fig. 4e and f), clearly supported that the synthesised BJ-AgNP is responsible for damaging the cell. TEM images also demonstrated that fluid dispersion and protein leakage is due to the damage of the peptidoglycan layer (Roy et al. 2019). Therefore, from all the activities (LDH, GSH and protein leakage), it has been observed that BJ-AgNPs exhibited better bactericidal activity against E. coli than B. subtilis. This is possibly due to different structural pattern of the two tested bacteria, specially the thickness of the peptidoglycan layer. Almost similar antibacterial activity for GO/Ag nanocomposite against Gram positive (S. epidermidis) and Gram negative (P. aeruginosa) bacteria was reported by Jang et al. (2020).
Finally, trypan blue exclusion assay has been introduced to observe the cell viability under different concentration of BJ-AgNP and the microscopic results were shown in Fig. 4. The live cells for both bacteria under controlled experiment were depicted in Fig. 4a and b. The cells of E. coli and B. subtilis were taken in glass slides and it was observed that 2× MIC concentration is effective to kill the cells ( Fig. 4c and d).

Biofilm inhibition assay
The result of the biofilm inhibitory assay with respect to E. coli was demonstrated in Fig. 5c and d. Present result revealed that BJ-AgNP is effective for 49.73%, 65.24%, 89% inhibition at MIC, 2×MIC, 4×MIC respectively with respect to the control. The results also highlighted that BJ-AgNP successfully crossed the extracellular polymeric metrix of biofilm to ultimately invade bacterial cells by causing membrane damage. The intensity of the stained preformed biofilm plate gradually decreases with the increasing the concentration of the nanoparticles. This clearly supported that the synthesised BJ-AgNP have a strong antibiofilm property and it was absolutely in dose-dependent manner. The antibacterial activity of silver nanoparticles synthesised from various biogenic sources has been presented in Table 2. From Table 2, it is clear that the size of the nanoparticle is directly related to the minimum inhibitory concentration (MIC) of the nanoparticles. Data also revealed that the smaller the size of nanoparticles, the lower is the MIC value (7.8-40 μg/ml). This is probably due to higher level of silver ions rereleased from smaller size of AgNPs (Sotiriou and Pratsinis 2010) (

Conclusions
According to the current study, BJ-AgNPs were successfully prepared using chicken bile juice as waste material, exhibiting spherical shape, high crystallinity and average size of 40 nm with high stability. For nanoparticle synthesis, statistical optimization was successfully applied. Synthesised nanoparticles are highly effective against Gram positive (B. subtilis) and Gram negative bacteria (E. coli) with MIC values of 40 and 60 μg/mL, respectively. Results also revealed that synthesized BJ-AgNP kills bacteria through metabolic arrest, protein leakage, intracellular glutathione oxidation, and inhibition of lactate dehydrogenase activity. In these studies, bacterial membrane damage, oxidative damage, and a loss of metabolic activity are clearly demonstrated. BJ-AgNP inhibited biofilm formation almost to 90% at 4× MIC concentration, as demonstrated by the biofilm inhibition assay. Therefore, from the present findings, it can be concluded that BJ-AgNP can act as a promising antimicrobial agent against both Gram positive and Gram negative pathogenic bacteria. Moreover, it can be used as a potent antibiofilm agent. Finally, it can be concluded that the bile juice could be an effective ingredient towards synthesis of noble metal nano synthesis. However, further research should be focussed in the area of other animal waste towards biomedical applications.

Competing interests
The authors declare no competing of interests.