Nanoparticles have been prepared through a diverse range of synthesis approaches over the last decades and different fundamental principles of synthesis procedures have been investigated to obtain nanoparticles of desired sizes, shapes, and functionalities. Their unique size-dependent thermal, electrical, chemical, and optical properties have enabled their use in fields as medicine and chemical analysis .
The green synthesis approach is a promising synthesis procedure in the research and development of materials science and technology due to the biosynthetic pathway of nanoparticles preparation, potentially eliminating the usage of chemicals and making the nanoparticles more biocompatible. Some basic principles of “green synthesis” can be explained by prevention or minimization of waste, reduction of pollutant derivatives, and the use of safer solvent as well as renewable feedstock . The biomaterials extracted from several parts of the plant are mixed with metal precursor solutions at different reaction conditions for nanoparticle synthesis. The biomaterials play several roles such as reducing, capping, and stabilizing agents in the nanoparticle synthesis process. Nanoparticles are synthesized within a few minutes or hours depending upon the type and concentration of biochemicals arising from plant sources. The plant extracts have been proven to possess high efficiency as stabilizing and reducing agents for the synthesis of nanoparticles but detailed investigation on the role of reaction parameters in synthesis is still needed to overcome existing problems in ‘green’ synthesis .
Capping agents significantly modify the properties of colloidal suspensions of nanoparticles. The efficacy of colloidal NPs application is linked to high suspension stability. Small changes to the solution (e.g., background salt concentration, temperature, pH), or changes to the nanoparticles (e.g., surface coating or concentration) can substantially affect stability. There are several parameters that affect the green synthesis of nanoparticles, including pH of the solution, temperature, the concentration of the plant material used, reaction time, and above all the protocols that are used for the synthesis process. The change in the type and characteristic of the synthesized nanoparticles is especially affected by the type of plant material used in the synthesis process . The morphology and surface property of nanoparticles are important parameters for their applications. The better antibacterial action may be attributed to their smaller size and surface charge to come in contact with the microbial cells . An ideal synthesis method should be able to allow reliable adjustment of particle distribution, size, and composition .
There are different phases of iron oxides as hematite (α-Fe2O3), maghemite (γ-Fe2O3), goethite (α-FeOOH), and magnetite (Fe3O4). The size scale affects the optical, electrical, and biocompatibility properties of these materials  . The functional groups of coating materials alter the surface charge, which influences the biological behaviors of iron oxide nanoparticles . The dispersity, size, and surface chemistry of the iron oxide materials are crucial for environmental applications in the aqueous phase due to the porous structure with particle-surface interaction . Iron oxide nanoparticles act as promising antibacterial agents that have high surface areas with crystalline morphologies at a high number of edges and corners and generation oxidative stress by reactive oxygen species .
In this study, we report the green synthesis of iron oxide nanoparticles using biochemicals extracted from Ceratonia siliqua. L. (carob pod). Carob pod is rich in sugars as sucrose (437.3 mg/g dry weight), glucose (395.8 mg/g dry weight), and fructose (42.3 mg/g dry weight). The other content is as total phenolics group (13.51 mg gallic acid equivalents [GAE]/g dry weight), proanthocyanidins (0.36 mg GAE/g dry weight), gallotannins (0.41 catechin equivalents [CE]/g dry weight), and flavanols (3.21 mg CE/g dry weight protein). Gallic acid (3.27 mg/g dry weight) is the most abundant phenolic material. Aspartic acid (18.25 mg/g dry weight protein) is the predominant amino acid in the protein fraction. Major minerals are vitamin K, calcium, potassium, and magnesium .
The effects of reaction parameters on the synthesis of iron oxide nanoparticles were investigated by the Taguchi method in this study. The Taguchi method is used for evaluating the results of matrix experiments to determine the best levels of experimental parameters. It makes it possible to provide an acceptable formulation using minimum raw materials and time . The signal-to-noise (S/N) ratio and the analysis of variance (ANOVA) were employed to analyze the experimental parameters. The parameters including the concentration of plant extract, the concentration of iron ion, reaction temperature, pH of plant extract, stirring rate, and reaction time were evaluated. The polydispersity value of nanoparticles was measured by a dynamic light scattering (DLS) instrument after each synthesis to analyze the effect of chosen experimental parameters. The zeta potential and UV-Vis spectrum was monitored over 3 months period for analysis of colloidal stability. Furthermore, the antibacterial activity of powder gIONPs were tested against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria.