Lactobacillus bulgaricus, 1901

Naseer, Q. A., Xue, X., Wang, X., Dang, S., Din, S. U. & Jamil, J., 2022, Synthesis of silver nanoparticles using Lactobacillus bulgaricus and assessment of their antibacterial potential, Brazilian Journal of Biology (e 232434) 82, pp. 1-8 : 3-5

3.1. Identification and characterization of Lactobacillus bulgaricus

Bacterial strain Lactobacillus bulgaricus was isolated from raw milk for extracellular synthesis of AgNPs. The morphological characteristics showed that the colony was creamy grey, round in shape, and had a raised elevation and entire margin ( Figure 1 ). Biochemical tests showed that Lactobacillus bulgaricus was positive for mannitol fermentation and negative for catalase, citrate, indole, and urease ( Table 1 ).

3.2. Synthesis and characterization of AgNPs

The biosynthesis of AgNPs was successfully achieved by inoculating 1 mL of inoculum in 10 mL of 1 mM silver nitrate solution (1:10). The positive result for the biosynthesis of AgNPs was observed by color changes from yellow to dark brown, while intensity increasing during the time of incubation. Whereas, both controls were observed unchanged ( Figure 2 ), for accuracy in result, different suspension solution of AgNO 3 with varying concentration (1-5%) was added to Lactobacillus suspension. The characteristics of synthesized nanoparticles are greatly affected by adding different concentrations of AgNO 3 to the supernatant. Maximum synthesis of nanoparticle was observed at 4 mM AgNO 3 ( Figure 3 ).

3.2.1. Determination of AgNPs using spectrophotometer

The production of AgNPs was confirmed by UV-VIS absorption. AgNPs from Lactobacillus bulgaricus was observed by UV-VIS at visible range 410 to 430 nm ( Figure 4 ) which is presumed peak for AgNPs reported in previously by Sikder et al., (2018).

3.2.2. SEM confirmation of AgNPs

SEM analysis of AgNPs was performed to determine the size and shapes of AgNPs and dispersed AgNPs with a diameter ranges 30-100 nm and different shapes were observed ( Figure 5 ). The characteristics of synthesized nanoparticles are greatly affected by adding varying concentration of AgNO 3 to the supernatant. It was observed in this study that the best concentration for synthesis of nanoparticles was 4 mM AgNO 3.

3.2.3. Energy dispersive-X-ray spectroscopy

Energy Dispersive-X-ray Spectroscopy (EDS) was carried out to find out overall compositions of elements in reaction mixture of Lactobacillus bulgaricus and silver nitrate after completion of reactions. The EDS analysis showed the significant amount of AgNPs presence, furthermore along with AgNPs other elements were also detected which might be present in media composition ( Figure 6 ).

3.3. Comparative antibacterial activity of antibiotics and AgNPs

The antibacterial activity of AgNPs from Lactobacillus bulgaricus were evaluated against three selected bacterial strains including Staphylococcus aureus , Staphylococcus epidermidis and Salmonella typhi . An AgNO 3 solution (4 mM) and some antibiotics were found to be effective against selected strains.It was found that the antimicrobial activities of biologically synthesized AgNPs were not uniform across bacterial strains.The mean value of zones of inhibition noted for selected bacterial strains were 15-mm for Staphylococcus aureus , 17-mm for S. epidermidis , and Salmonella typhi respectively.The effects of antibiotics against selected bacterial strains were also evaluated according to Birmingham Children’s Hospital (2014) guidelines.

We found that the antibiotic activities against selected bacterial strains were not uniform.Furthermore, bacterial strains were found highly sensitive to AgNPs in comparison to tested antibiotics ( Table 2 and Figure 7 ).

Figures

Figure 1. Morphological and microscopic properties of Lactobacillus bulgaricus. (a) Lactobacillus stained with gram stain under light microscope (X100); (b) Lactobacillus on MRS agar medium, 37 °C, 48 h.

Figure 2. Lactobacillusbulgaricus synthesis of Ag-NPs:(a) AgNO3 (control); (b) Reaction mixture before synthesis; (c) Reaction mixture after synthesis.

Figure 3. Results of suspension at different AgNO concentrations of Lactobacillus bulgaricus.

Figure 4. Absorption spectrum of AgNPs synthesized by Lactobacillus bulgaricus having clear peak at 410 nm.

Figure 5. SEM of silver nanoparticles synthesized by Lactobacillus bulgaricus. The shape of AgNPs was spherical and size between (30- 100 nm), magnification (16053×, 17876×, 15568×, and 9742 ×), the voltage (5 kV) and spot size 4 for (a), (b), (c) and (d) respectively.

Figure 6. EDS Analysis of AgNPs synthesized by Lactobacillus bulgaricus.

Figure 7. Antibacterial effect of Ag-NPs and antibiotics against (a) Staphylococcus epidermis (b) Salmonella (c) Staphylococcus aureus.