Antibacterial efficacy of greenly synthesized silver nanoparticles using nanocurcumin and silver nanoparticle gel on bovine mastitis

This study summarizes the synthesis of AgNPs using nanocurcumin as a reducing and stabilizing agent (CurAgNPs). The stability of CurAgNPs after 12 months of storage and their antimicrobial activity against four bacteria causing mastitis in cows were investigated. Ultraviolet—visible (UV–vis) spectroscopy of the dark brownish-red stabilized CurAgNPs solution decating the surface plasmon resonance peak of the sample was observed at 438 nm. Images of the spherical CurAgNPs were obtained using transmission electron microscopy (TEM), which showed a mean particle size distribution of 15–58 nm, with a mean size of 32 nm. The influence of CurAgNPs on four microorganisms that cause mastitis in cows, Streptococcus agalactiae (S. agalactiae), Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and Eschericia coli (E. coli), was studied, and the minimum inhibitory concentration (MIC) was from 1.6 to 6.25 ppm. From this, the MBC of CurAgNPs were observed at 3.15 ppm for S. agalactiae, and 6.25 ppm for S. aureus, P. aeruginosa and E. coli, respectively. The formulated homogeneous gel containing 100 ppm CurAgNPs, 1.5% carboxymethyl cellulose (CMC), and distilled water was developed with a viscosity of 141 ± 7.55 cP, pH of 6.72 ± 0.11, and homogenized. The zeta potential of CurAgNPs gel after 6 months of storage is almost constant. In addition, the improved CurAgNPs gel demonstrated significant antimicrobial activity compared with tetracycline at a concentration of 100 ppm.


Introduction
Bovine mastitis is one of the most common and costly diseases affecting livestock, particularly in the dairy industry in particular. The main bacterial pathogens causing bovine mastitis, including E. coli, S. uberis, S. aureus, and P. aeruginosa have been described in the literature [1]. Cases of mastitis in cows are increasing in many countries, including Vietnam, but prevention is ineffective and mainly treated with antibiotics [2,3]. The treatment of cows with mastitis using antibiotics via intramuscular injection or injection into the udder [4,5]. This method of treatment is highly effective; however, after the injection of antibiotics, it is necessary to abandon cow's milk, since it contains antibiotics that do not meet the standard criteria for milk. On the other hand, antimicrobial resistance to available antibiotics is one of the biggest global health problems, making it difficult to treat infections and increasing the risk of disease spread, serious illness and death [6]. This has increased the need for new antibiotics with improved antibacterial activities. Silver is one of the most commonly used metals to address these issues because of its significant antimicrobial activity, catalytic activity, good electrical conductivity, and chemical stability [7]. Silver nanoparticles (AgNPs) are among the most attractive nanomaterials in biomedicine owing to their unique physicochemical properties [8]. These materials are Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. zero-dimensional, with a size range of 1-100 nm and characteristic morphologies [9,10]. Owing to their small size, AgNPs exhibit significant antimicrobial and fungicidal activities as well as biocompatible surface properties [9][10][11][12][13]. Our recent studies have also confirmed the antimicrobial activities of AgNPs as reported in the literature [14][15][16]. Due to their unique properties, AgNPs are applied in many fields, such as livestock performance and health [17], veterinary hospitals [18], veterinary medicine [19], and in safer environmental practices [20]. The biosynthesized AgNPs gel healed wounds more efficiently than the silver ion formulations available in the market [21]. The intrinsic biological activity of AgNPs can be enhanced in combination with curcumin through the synergistic effect of the combination of the two materials [22].
Curcumin, a natural polyphenol, is extracted from turmeric (Curcuma longa L.) [23]. It has many beneficial effects such as pharmacological properties, including anti-inflammatory, antioxidant, and wound healing effects [24]. Curcumin has antibacterial properties against a wide range of bacteria, including gram-positive and gramnegative bacteria [25]. Furthermore, curcumin can be considered a potential and sustainable antibacterial drug candidate to address the problem of antibiotic resistance [26]. Several studies have confirmed its significant antibacterial activity. Adebayo-Tayo's research team showed that green synthetic curcumin-silver nanoparticles (Cur-AgNPs) can be effectively used as potent antibacterial agents for food and meat preservation owing to their small size and homogeneity [27]. Balouiri et al, 2016 reported that CurAgNPs with a size of 25-35 nm was synthesized using curcumin, a phytochemical [28]. However, the use of a combination of AgNPs and curcumin in the treatment of bovine mastitis is limited. Therefore, this study focused on the gel formulation of AgNPs biosynthesized using nanocurcumin and evaluated their antimicrobial activity against bacteria causing mastitis in cows.

Biosynthesis of AgNPs using nanocurcumin
The biosynthesis of silver nanoparticles using nanocurcumin (CurAgNPs) was reported in a preliminary study [14]. The procedure for the biosynthesis of CurAgNPs shown in figure 1 was modified from the method of Khan et al [29]. Briefly, a solution of 15 μg ml −1 nanocurcumin was added to 150 ml of AgNO 3 (0.02 M) in 250 ml beakers. The resulting mixture was homogeneously mixed at 30°C for 20 min with an ultrasonic device. The obtained CurAgNPs had a dark brownish-red color, and their properties are described below by UV-vis, TEM and DLS analysis.
Spherical CurAgNPs with a face-centered cubic crystal structure (size range: 5-22 nm), average size of 10.9 nm, absorption peak at 406 nm, and zeta potential of −27.2 mV were successfully synthesized at ambient temperature. The CurAgNPs prepared using this method exhibited a higher dispersion stability in the aqueous phase.

Stability of CurAgNPs after 12 months of storage
The stability of the CurAgNPs after 12 months of storage was also investigated. These nanoparticles (NPs) were characterized by visual observation and UV-vis (SHIMADZU Corp., Kyoto, Japan) [30] and TEM (JEOL, Ltd, Tokyo, Japan) analyses [31].

Formulation of CurAgNPs gel
CurAgNPs gel was prepared according to the method of Dhase et al [32], with some modifications. Briefly, 500 ml CurAgNPs solution, 2% glycerol (99.0%; Sigma Aldrich), CMC (99.0%; Sigma Aldrich) at different concentrations (0.5, 1%, 1.5%, and 2%), and distilled water up to 1000 ml. Mixture was continuously stirred at 500 rpm dissolve for 10 min at 50°C, then increase the speed to 5000 rpm for 60 min at 50°C to form a gel. Finally, the obtained CurAgNPs gel was cooled and stored in the dark at room temperature.

Evaluation of CurAgNPs gel
The formulations produced with CurAgNPs gels were examined for their physical (pH, color, homogeneity, and spreadability) and rheological properties [33].
2.4.1. Determination of the pH pH of the prepared formula was measured using digital pH meter.

Viscosity measurements
A synchroelectric viscometer (Brookfield, MA, USA) was used to measure the viscosity (cP) of CurAgNPs gel formulations. The spindle was rotated at 2.5 rpm. Samples of the gel were allowed to settle for 30 min at the test temperature (25 ±1°C) before measurements were performed [34].

Determination of CurAgNPs gel spreadability
2mL of CurAgNPs gel were pressed evenly between the two glass slides. The two plates were compressed to uniform thickness under tension by placing a weight (500 g) on the top plate to provide a uniform film. The top plate (with a hook) was then subjected to a 50 g pull using a cord attached to a hook. The time required for the top slide to cover a specific distance is recorded. Spreadability (S) of the gel formulation was calculated as follows: where M(g) is the weight of the upper glass slide, L (cm) is the length of the glass slide, and t (s) is the time required.

Zeta potential measurements
The zeta potential of the CurAgNPs solution and CurAgNPs gel was evaluated using DLS techniques to confirm their stability. The electrophoretic mobility of different particle suspensions was measured via laser Doppler velocimetry using the Zetasizer Nano ZS (Malvern, UK). Measurements were conducted at 25°C [35].

Antibacterial activity potential of CurAgNPs and CurAgNPs gel
A total of 72 milk samples were collected from 24 cows with mastitis from livestock households in the Lam Dong Province, Vietnam. Isolation was performed using a medium containing Tryptic Soy Agar (TSA; BK046HA, Biokar Diagnostic). Pure strains will stain gram, test for oxidase, indole, catalase, and finally using MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry) with the Bruker MALDI-TOF Biotyper system (Bruker Daltonik, Bremen, Germany) to identify a target plate by comparing the mass spectral protein detection pattern with reference patterns in a database. A score of >2.0 on MALDI-TOF MS was accepted as reliable evidence. Finally, four strains of bacteria were identified: two gram-positive bacteria (Streptococcus agalactiae and Staphylococcus aureus) and two gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli), which were stored at the Faculty of Animal Science and Veterinary Medicine, Hue University of Agriculture and Forestry, Vietnam.
The antibacterial activity of the CurAgNPs solution and the CurAgNPs gel was evaluated by an agar well diffusion test [36] against S. agalactiae, S. aureus, P. aeruginosa and E. coli. The bacterial stock-cultures were streaked on an assay plate (90 × 15 mm) containing TSA agar and incubated overnight at 37°C. One colony from each overnight culture was used to prepare bacterial suspension by shaking in 20 ml of Tryptic Soy Broth (TSB; BK046HA, Biokar Diagnostic) for 4 h at 37°C and 160 rpm. The bacterial suspension was prepared in solution of sterile saline (0.85%) and adjusted to a 0.5 McFarland standard (∼1.5 × 10 8 CFU/mL). Sterile swabs were used to inoculate TSA plates, dipping the tip of the swab into the bacterial suspension, and sweeping the wet swab evenly over the entire surface of the plate. Then seven 6 mm diameter holes were punched aseptically with a sterile cork borer. Subsequently, 20 μl of the CurAgNPs gel solution diluted to various concentrations of 100, 50, 25, 12.5, 6.25 ppm was introduced into the appropriate wells. Equal volumes of distilled water and the standard antibiotic tetracycline were used as the negative and positive controls, respectively. The agar plates were incubated at 37°C for 24 h. The diameter of the inhibition zone was measured in millimeters and the antimicrobial effect of the CurAgNPs solutions was analyzed. The experiments were performed in triplicate.

Determination of the MIC and MBC
The MIC and MBC values of the CurAgNPs were determined using a broth microdilution protocol against S. agalactiae, S. aureus, P. aeruginosa and E. coli. Briefly, CurAgNPs were serially diluted with Tryptic Soy Broth (TSB) medium at concentrations of 25, 12.5, 6.25, 3.15, 1.6 and 0.8 ppm. Next, 200 μl of each dilution was added to each well of the 96-well microplate. The tested bacteria were incubated in the appropriate wells at a final concentration of approximately 10 8 CFU/mL. Each CurAgNPs dilution in TSB was used as the blank. The wells containing TSB and bacteria served as positive controls, whereas wells containing only TSB served as negative controls. The Optical density was measured using the SpeciD5 apparatus at 0, 2, and 4 h after incubation at 37°C. The concentration of the CurAgNPs solution that yields the lowest absorbance indicates the MIC of the CurAgNPs solution for that organism [37]. In addition, the lowest concentration of CurAgNPs solution at which the pathogenic bacterial colonies showed no growth when inoculated into TSA plates was dedicated to the MBCs value.

Statistical analysis
Statistical analysis was performed using analysis of variance with SAS 9.0 statistical software. The results are presented as mean ± standard deviation. Duncan's multiple range test was performed at a significant level of P < 0.05.

Results and discussion
3.1. The stability of CurAgNPs solution after 12 months of storage The potential changes in the physicochemical properties of CurAgNPs after 12 months of storage were investigated by UV-vis and TEM analyses as well as visual inspection. Accordingly, the surface plasmon resonance (SPR) peak of the CurAgNPs solution was observed at 438 nm after 12 months of storage, consistent with its onset time (figure 2). Absorption occurs because of the surface plasmon resonance caused by the electrons present on the nanoparticle surface. These results agree with those reported by [38][39][40], where the maximum surface plasmon resonance of spherical AgNPs occurs at 400-500 nm. In addition, neither the size nor the form of the particles showed significant changes. After 12 months of storage, CurAgNPs had a spherical shape and a size range of 8-94 nm, in which the nanoparticles were mainly distributed from to 15-58 nm, with a mean size of 32 nm ( figure 3(B)). At the time of manufacturing, the nanoparticles were distributed in the range of 8-58 nm, with an average size of 27 nm. Differences that may be related to agglomeration appear after storage or are statistically marginal, and do not affect the properties or application efficiency of materials in general. This also shows that the CurAgNPs solution achieved a high level of stability even after 12 months of storage.

Antibacterial activity of the CurAgNPs
The MIC of Cur AgNPs against the tested microorganisms ranged from 1.6 to 6.25 ppm. While the MBC of CurAgNPs were observed at 3.15 ppm for S. agalactiae, and 6.25 ppm for S. aureus, P. aeruginosa and E. coli, respectively (table 1). The activity of CurAgNPs against S. agalactiae, S. aureus, P. aeruginosa, and E. coli confirmed their potent antimicrobial activity against bovine mastitis bacteria, ranging from 7.8-9.7 mm. S. aureus had the highest susceptibility, followed sequentially by S. agalactiae, E. coli, and P. aeruginosa with inhibition zone diameters of 9.68 ± 0.42, 9.17 ± 0.51, 8.94 ± 0.31, 7.82 ± 0.16 mm. The broad-spectrum inhibitory activity of CurAgNPs confirmed that they have great potential as antimicrobial agents against pathogenic microorganisms.
The antibacterial activity of CurAgNPs against all tested bacterial pathogens is consistent with the work of [41] who reported the antibacterial activity of silver nanoparticles of Penicillium atramentosum. This antibacterial activity may be due to loss of activity during DNA replication. It has been hypothesized that the expression of ribosomal subunit proteins and other cellular proteins involved in ATP synthesis are inactivated [42]. The antibacterial potential of the environmentally friendly synthesized CurAgNPs showed that the nanoparticles were effective as antimicrobial agents for controlling pathogenic bovine mastitis.

Formulations of the CurAgNPs gels
The CurAgNPs gels were formulated with different CMC concentrations of 0.5%, 1.0%, 1.5%, and 2%. Their unique physical properties such as pH, viscosity, and homogeneity were studied (table 2). The pH of the CurAgNPs gel did not change significantly with different CMC concentrations and was in the range of 6.65, similar to the natural pH of the cow skin [43]. This acidic milieu renders skin resistant to harsh chemicals and pathogenic bacteria. The viscosity of the gel increased with increasing CMC concentrations. The viscosities of all four formulations were 52 ± 5.02, 93 ± 4.30, 141 ± 7.55, and 176 ± 10.40 cP, respectively. Similar to previous research [44,45], the authors suggested that CMC be used to increase viscosity and stabilize emulsions. CMC also acts as a binder and thickener [46,47]. From the data obtained in table 2, it can be said that an increase in CMC concentration leads to lower spreadability of the gel produced. The high viscosity of CMC may be responsible for this decrease in spreadability.
In terms of homogeneity, the CurAgNPs gel formulations with different CMC concentrations were uniform, and which the gels formed with 1.5% and 2% CMC concentrations were excellent. Visual observation of the CurAgNPs gels formed by the addition of CMC at different concentrations is shown in figure 4. However, after  six months storage (figure 5), the CurAgNPs samples with 1.0% and 1.5% CMC showed aggregation and sedimentation at the bottom. Meanwhile, the gel sample with 2% CMC showed a local caking phenomenon, the gel became more denser, which resulted in a state of inhomogeneity in addition. Spreadability was poor compared to the initial time, impairing skin penetration and spreading when applied to the skin. The CurAgNPs gel with 1.5% CMC, which still retained the original physicochemical properties, was used for the subsequent antibacterial experiments. The stability of the CurAgNPs solution and CurAgNPs gel with 1.5% CMC was evaluated through Zeta potential by DLS measurements. The zeta potential of the CurAgNPs solution (A) and CurAgNPs gel with 1.5% CMC at the initial time (B) and after 6 months of storage (C) were compared and found to be −12.4, −16.3, and −16.5 mV, respectively (figure 6). The zeta potential of CurAgNPs was lower in the presence of CMC, indicating  that the CurAgNPs gel had a higher degree of stability than CurAgNPs solution. This was due to the shift of the slipping plane towards the bulk solution, which was consistent with the studies of Elzbieta Grzadka et al [48] and Rossi et al [49]. Furthermore, the zeta potential of CurAgNPs gel with 1.5% CMC at the initial time (B) was almost unchanged compared to that after 6 months of storage (C), again demonstrating the stability of CurAgNPs in the mixture with 1.5% CMC.

Antibacterial activity of CurAgNPs gel
The CurAgNPs gel demonstrated antimicrobial activity against four studied pathogenic bacteria, including two gram-positive (Streptococcus agalactiae and Staphylococcus aureus) and two gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli). Table 3 and figure 7 show the zones of inhibition exhibited by CurAgNPs gel against each bacterium. The inhibition zone was calculated by measuring the clear zone surrounding the wells in millimeters (mm). Clear zones indicated the inability of bacteria to grow or multiply around the well loaded with CurAgNPs gel.
The results showed that 25 ppm CurAgNPs inhibited E. coli. Meanwhile, the 12.5 ppm CurAgNPs gel began to inhibit the growth of S. agalactiae, S. aureus, P. aeruginosa the diameter of the inhibition zone including the good diameter was found to be 2.42 ± 0.48, 1.16 ± 0.66, 0.42 ± 0.13, mm; respectively. In addition, S. agalactiae and S. aureus were more susceptible than P. aeruginosa and E. coli. The antibacterial effect of the CurAgNPs gel on all tested bacteria was enhanced with increasing concentrations of CurAgNPs gel. Interestingly, for P. aeruginosa alone, the zone of inhibition of the 100 ppm CurAgNPs gel was larger than that of the standard antibiotics (tetracycline 100 ppm). The other three bacterial strains showed the opposite trend. Although the  mode of action of the CurAgNPs gel on bacterial growth inhibition is still unclear, it can be explained that AgNPs in the medium adhere to the bacterial cell wall and consequently penetrate it, leading to membrane damage, leakage of cellular contents, and cell death. Another explanation is that Ag + ions are released and react with sulfhydryl groups in proteins and enzymes, resulting in the antibacterial effect of Ag + ions [37,50]. Gram-positive bacteria (S. agalactiae and S. aureus) showed a higher zone of inhibition than did gramnegative bacteria (P. aeruginosa and E. coli) at the same concentration of CurAgNPs gel (figure 7). This difference could be explained by variations in the cell wall composition of Gram-positive and Gram-negative bacteria. Although Gram-positive bacteria have a thicker cell wall made of peptidoglycan, Gram-negative bacteria have an extra outer membrane. This leads to a multilayer membrane of gram-negative bacteria, and a higher selectivity leads to greater difficulty in the penetration of AgNPs [51].

Conclusion
The advancement of bio-nanomaterials, with applications in the livestock sector in general, and in the treatment of bovine mastitis in particular, has opened avenues for extensive exploitation and study of their impact. AgNPs biosynthesized using curcumin were monitored for stability after 12 months of storage by visual dispersion, UV-vis, and TEM characterization, as well as for their antimicrobial activity against four pathogenic bacteria that cause mastitis in cows. The CurAgNPs gel was successfully formulated with the addition of 1.5% CMC. It exhibited good homogeneity, a viscosity of 141 ± 7.55 cp, pH of 6.72 ± 0.11, and zeta potential of −16.5 mV. Remarkably, it also showed antimicrobial activity against the bacteria tested. Future clinical trials are needed to demonstrate the potential of CurAgNPs against bovine mastitis.