By-products contribute substantially towards industrial pollution. However, it is possible to turn by-products into valuable items by using appropriate management systems. Industrial pollution is not always from harmful, toxic ingredients, or heavy metals, but it can also result from ecofriendly products. For example, in the silk industry, water pollution occurs when wastewater from the degumming process runs out through drains. Worldwide, it is estimated that 400,000 tons of dry cocoons produce 50,000 tons of sericin each year. However, sericin is mostly discarded in silk processing wastewater and results in a high chemical oxygen demand (COD) and biological oxygen demand (BOD) levels in the degumming wastewater . Therefore, the recovery and reuse of discarded sericin are beneficial because of its economic, social, and environmental advantages.
Silk sericin is a type of water-soluble globular protein derived from the silkworm Bombyx mori, and it represents a family of proteins whose molecular mass ranges from 10 to 310 kDa [2,3,4,5,6]. Silk sericin has many unique properties, including biodegradability, nontoxicity, oxidation resistance, antimicrobial activity, ultraviolet (UV) resistance, and moisture absorption [1, 2, 7]. The physico-chemical properties of molecules are responsible for numerous applications in biomedicine and are influenced by the extraction method and silkworm lineage, which can lead to variations in the molecular weight and amino acid concentration of sericin. The silk sericin has been widely used in biomaterial applications due to its biocompatibility, biodegradability, and anti-oxidative and bioactive activities . The presence of highly hydrophobic amino acids and their antioxidant potential makes it possible for sericin to be applied in the food and cosmetic industry. Silk sericin membranes are good bandage materials, and their film has adequate flexibility and tensile strength. Due to its substantial biocompatibility and infection-resistant nature, it is a novel wound coagulant material. Additionally, its flexibility and water absorption properties enable its use as a smooth cure for defects in the skin and do not cause any peeling of the skin under regeneration when detached from the skin . Zhoarigetu et al. mentioned that sericin can be used as a raw material for making contact lenses: oxygen permeable membranes comprising fibroin and sericin with 10–16% water are used for making contact lenses and as artificial skin . Masahiro et al. reported that the consumption of sericin enhances the bioavailability of Zn, Fe, Mg, and Ca in rats and suggested that sericin is a valuable natural ingredient for the food industry . Recently, sericin has been used as a reducing and stabilizing agent in the synthesis of metal nanoparticles . The primary requirement for the synthesis of metal nanoparticles (NPs) is reducing the biological agents and other constituents present in the cells acting as stabilizing and capping agents, so there is no need to add capping and stabilizing agents from outside [11,12,13,14,15]. He et al. developed a novel, simple, one-step biosynthesis method to prepare a sericin-silver nanoparticle composite in situ in solution. Sericin served as the reductant of silver ion, the dispersant and stabilizer of the prepared sericin-silver nanoparticle composite . Tahir et al. (2020) evaluated the antibacterial activity of sericin-conjugated silver NPs synthesized using sericin as a reducing and capping agent [13, 17]. Aramwit et al.  synthesized silk sericin (SS)-capped silver nanoparticles (AgNPs) under alkaline conditions (pH 11) using SS as a reducing and stabilizing agent instead of toxic chemicals. Most relevant studies stated that SS-capped AgNPs have substantial potential for use as antibacterials. For this reason, our interest has grown to extract SS from the cocoon of a very popular B. mori variety “Rajshahi Silk” and to introduce a green synthesis approach for the extraction of AgNPs in an aqueous neutral condition. The green synthesis of silver nanoparticles is primarily concerned with the selection of solvent medium, reducing agent, and nontoxic substances for stability [18, 19]. In this context, it is essential to mention that the synthesis of silver nanoparticles using biological systems makes nanoparticles more biocompatible and environmentally benign.
AgNPs have strong antibacterial action towards gram-positive, or gram-negative bacteria are reported in many past studies. Sondi and Salopek-Sondi first observed that AgNPs accumulate in the cellular membrane and form pits in the bacterial cell wall of Escherichia coli which led to an augmented permeability of the cell wall and ultimately the cell death . Shrivastava et al. revealed that AgNPs create disorder in the integrity of the bacterial cell wall and membrane, supporting the permeability of the membrane and the leakage of the cell constituents, and eventually induce cell death . Afterward, many advanced studies have been reported that DNA and protein of bacterial cell were destroyed by peroxidation, dephosphorylation, etc., reaction of Ag+ ions in aerobic conditions [21,22,23,24,25]. Even then, some antibacterial mechanisms of AgNPs on multidrug-resistant bacteria remain unknowable. Nowadays, a group of pathogens namely ESKAPE (i.e., Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have become cause of several important nosocomial infections and already resistant to the last-line of antibiotics [26,27,28,29]. Yet again, Szmolka and Nagy were shown the multidrug resistivity of Escherichia coli in non-clinical sources . In our study, we used Escherichia coli MZ20, Pseudomonas aeruginosa MZ2F, and Pseudomonas aeruginosa MZ4A. Previously, Zulkar et al. have reported that all bacteria are multidrug-resistant, e.g., Escherichia coli MZ20 is resistant to penicillin G (P), tetracycline (TE), cotrimoxazole (COT), erythromycin (E), kanamycin (K), streptomycin (S), ciprofloxacin (CIP), ceftazidime (CAZ), nalidixic acid (NA), colistin (CL), ceftriaxone (CTR), doxycycline (DO), amoxicillin (AMX); Pseudomonas aeruginosa MZ2F is resistant to P-TE-COT-E-K-S-CAZ-NA-DO-AMX, and Pseudomonas aeruginosa MZ4A is resistant to P-TE-COT-E-K-S-CAZ-NA-CL-DO-AMX .
In the present work, silver nanoparticles were synthesized using a simple, effective, and ecofriendly method using silk sericin. Silk sericin solution was extracted from B. mori silk cocoons and used as a reducing and stabilizing agent. The synthesized silver nanoparticles (AgNPs) were characterized using UV-visible spectroscopy, Fourier-transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy, X-ray diffraction (XRD), and transmission electron microscopy (TEM). Additionally, we explored the antibacterial activity of biosynthesized silver nanoparticles (AgNPs) against multidrug-resistant Escherichia coli MZ20, Pseudomonas aeruginosa MZ2F, and Pseudomonas aeruginosa MZ4A.
Silk sericin is a protein that is extracted from silkworm cocoons. Silkworm cocoons were collected from the Bangladesh Sericulture Research & Training Institute, Rajshahi, 6207. Silver nitrate Anhydrous (extra pure) was brought from MOLYCHEM, India. All the analytical grade reagents used in this study were purchased from Merck, Germany.
Escherichia coli MZ20, Pseudomonas aerugiosa MZ2F, and Pseudomononas aeruginosa MZ4A were collected from the Department of Biotechnology and Genetic Engineering of our university. Bacterial strains were collected from different sources, such as hospital waste material and poultry litters. All bacterial strains were resistant to multiple drugs .
Extraction of silk sericin
The silkworm cocoons were cut into small pieces and boiled at 90°C in demineralized water for 90 min at a ratio of 1 part cocoon to 40 parts water. Silk fibroin was separated using polyester filter cloths followed by Whatman filter paper. The SS solution was refluxed to remove volatile matters and concentrated until the desired concentration was achieved. The prepared SS colloidal solution (60 ml obtained from 5g of cocoons) was frozen at −20°C in a deep freeze (BIO Memory 175L) for about 12 h. Then, the frozen SS was lyophilized in a freeze dryer (Yamato Freeze Dryer DC 401) for about 24 h until dry. Then, the percentage of the solid content of silk sericin was calculated and found to have an average value of 17wt% sericin with respect to the weight of the silk cocoons.
Synthesis of silk sericin-capped silver nanoparticles
Aqueous silver nitrate solution was prepared for AgNP synthesis. The lyophilized silk sericin was placed in a conical flask containing an aqueous AgNO3 solution. Two molar ratios of SS to AgNO3 were used: 1:6 and 1:8. The silver ions were reduced to silver nanoparticles (AgNPs) within a few minutes at 65°C with continuous stirring. The color of the solution changed from yellow to brown. The SS-AgNPs synthesized using different molar ratios were characterized using UV-visible spectroscopy, FTIR-ATR spectroscopy, XRD, and TEM measurements.
Sericin and SS-AgNPs were characterized using UV-1700 predominantly for accurate quantification of sericin protein and SS-AgNPs. The instrument can automatically calculate sample concentrations based on a standard curve using the K factor method.
Fourier-transform infrared-attenuated total reflection spectroscopy
FTIR (ATR) spectra of the sericin and SS-AgNPs were measured using an FTIR-ATR Instrument manufactured by SHIMADZU (IRAffinity-1S) with a resolution of 4 cm−1 in the wavenumber range 700–4000 cm−1.
Transmission electron microscopy
The size and shape of SS and SS-AgNPs were determined using TEM (model number is JEM-2100 F JEOL Japan) with ultra-high regulation FETEM operating at 200 kV.
The crystalline behavior of SS-AgNPs was examined using an X-ray diffractometer (Rigaku Ultima IV) operating at a voltage of 40 kV and a current of 40 mA with CuKα (λ = 1.5406 Å) radiation and a programmable divergent slit. The samples were scanned in the 2θ range 10°–70° with a scanning speed and step size of 3° min−1 and 0.02°, respectively.
The well diffusion method [32, 33] was used to test the antibacterial activities of synthesized silk sericin-silver nanoparticles (SS-AgNPs) according to the National Committee for Clinical Laboratory Standards (NCCLS) . Three selected multidrug-resistant bacterial isolates (Escherichia coli MZ20, Pseudomonas aerugiosa MZ2F, Pseudomononas aeruginosa MZ4A were grown in Luria–Bertani (LB) for 18–20 h. The bacterial inoculums were prepared by maintaining a turbidity of 0.5 McFarland standard (equal to 1.5×108 colony-forming units (CFU)/ml). Mueller Hinton agar (MHA) plates (150-mm diameter) were prepared, and bacterial suspensions were spread over the surface. The wells (9-mm diameter) were made using a cork borer in MHA plates. Each well was loaded with 70 μl of different concentrations (10 μg/ml, 20 μg/ml, 30 μg/ml, 40 μg/ml, and 50 μg/ml) of SS-AgNPs. Plates were incubated at 37 °C for 24 h. The diameters of the zones of inhibition around the wells were measured in millimeters (mm). The experiment was performed in triplicate. Data were expressed as the mean ± standard deviation.