Green Synthesis of Zinc Oxide Nanoparticles Mediated By Lacticaseibacillus Rhamnosus: Evaluation of Their Antioxidant and Hemolytic Activities

Fatima Hamza Alzubaidy; Noor Ismeal Nasser

doi:10.51699/dsep5j70

Authors

Fatima Hamza Alzubaidy Al-Furat Al-Awsat Technical University, Kufa Technical Institute, Iraq
Noor Ismeal Nasser Al-Furat Al-Awsat Technical University, Kufa Technical Institute, Iraq

DOI:

https://doi.org/10.51699/dsep5j70

Keywords:

Green synthesis, Zinc oxide nanoparticles, lacticaseibacillus rhamnosus, Antioxidant activity, Hemolysis

Abstract

Using probiotics to biosynthesize metal oxide nanoparticles is a new, more biocompatible, and environmentally harmless approach compared to traditional physical and chemical synthesis methods. In the current study, we isolated the probiotic Lacticaseibacillus rhamnosus from local dairy products. Instead of directly using the living cells of this bacterium, we extracted its cell-free extracellular supernatant, the clear liquid remaining in the upper layer of the culture medium after the bacterium’s metabolism, and successfully synthesized zinc oxide nanoparticles (ZnO-NPs). The first sign that confirmed the successful synthesis was a distinct color change visible to the naked eye. After preliminary confirmation that the synthesis was complete, we conducted a comprehensive and rigorous set of tests on the physical and chemical properties of these nanoparticles. After observation using Field Emission Scanning Electron Microscopy (FE-SEM), it was found that the synthesized nanoparticles were nearly spherical, with an average diameter of 41.5 nanometers, and the size of all particles fell within the range of 27 to 56 nanometers. The XRD analysis confirmed the crystalline nature of the biosynthesized ZnO nanoparticles, with an average crystallite size of 22.13 nm calculated by Scherrer’s equation. FTIR spectrum showed a characteristic Zn–O stretching vibration at 563 cm⁻¹ and the bands observed in the range of 1448–1580 cm⁻¹ were attributed to carboxylate and amide II groups, indicating the possible involvement of bacterial biomolecules in the capping of nanoparticles. AFM images showed the vertical height values from −0.726 nm to 0.920 nm and the peak to valley height difference of 1.646 nm. The nanoparticles showed strong DPPH radical scavenging activity, and it was found to be concentration-dependent, with 91.89% inhibition at 1000 µg/mL. Moreover, in vitro hemolysis assays with human erythrocytes showed 0% hemolysis at concentrations of 125–500 µg/mL and 1.60% at 1000 µg/mL, indicating low hemolytic activity under the tested in vitro conditions. The present results suggest that the synthesized ZnO nanoparticles derived from L. rhamnosus could be potential potent and non-toxic candidates for biomedical and pharmaceutical applications.

References

[1] B. Ahmed, M. B. Tahir, M. Sagir, and M. Hassan, “Bio-inspired sustainable synthesis of silver nanoparticles as the next generation of nanoproducts in antimicrobial and catalytic applications,” Materials Science and Engineering: B, vol. 301, Art. no. 117165, 2024.

[2] S. Sarwar, D. A. Raja, D. Hussain, M. R. Shah, and M. I. Malik, “Introduction to nanotechnology,” in Handbook of Nanomaterials, vol. 1, M. I. Malik, D. Hussain, M. R. Shah, and D.-S. Guo, Eds. Elsevier, 2024, pp. 447–481.

[3] A. Guleria, S. Neogy, B. S. Raorane, and S. Adhikari, “Room-temperature ionic liquid-assisted rapid synthesis of amorphous Se nanoparticles: Their prolonged stabilization and antioxidant studies,” Materials Chemistry and Physics, vol. 253, Art. no. 123369, 2020.

[4] M. J. Mitchell, M. M. Billingsley, R. M. Haley, M. E. Wechsler, N. A. Peppas, and R. Langer, “Engineering precision nanoparticles for drug delivery,” Nature Reviews Drug Discovery, vol. 20, no. 2, pp. 101–124, 2021.

[5] X. Fu, J. Cai, X. Zhang, W. D. Li, H. Ge, and Y. Hu, “Top-down fabrication of shape-controlled, monodisperse nanoparticles for biomedical applications,” Advanced Drug Delivery Reviews, vol. 132, pp. 169–187, 2018.

[6] K. R. Singh, V. Nayak, J. Singh, A. K. Singh, and R. P. Singh, “Potentialities of bioinspired metal and metal oxide nanoparticles in biomedical sciences,” RSC Advances, vol. 11, no. 40, pp. 24722–24746, 2021.

[7] P. G. Jamkhande, N. W. Ghule, A. H. Bamer, and M. G. Kalaskar, “Metal nanoparticle synthesis: An overview of methods of preparation, advantages, disadvantages, and applications,” Journal of Drug Delivery Science and Technology, vol. 53, Art. no. 101174, 2019.

[8] M. S. Samuel, M. Ravikumar, J. A. John, E. Selvarajan, H. Patel, P. S. Chander, and N. Chandrasekar, “A review on green synthesis of nanoparticles and their diverse biomedical and environmental applications,” Catalysts, vol. 12, no. 5, Art. no. 459, 2022, doi: 10.3390/catal12050459.

[9] N. Mariotti, M. Bonomo, L. Fagiolari, N. Barbero, C. Gerbaldi, F. Bella, and C. Barolo, “Recent advances in eco-friendly and cost-effective materials towards sustainable dye-sensitized solar cells,” Green Chemistry, vol. 22, no. 21, pp. 7168–7218, 2020.

[10] C. Hill, F. Guarner, G. Reid, G. R. Gibson, D. J. Merenstein, B. Pot, L. Morelli, R. B. Canani, H. J. Flint, S. Salminen, P. C. Calder, and M. E. Sanders, “The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic,” Nature Reviews Gastroenterology & Hepatology, vol. 11, no. 8, pp. 506–514, 2014.

[11] P. De Bellis and C. G. Rizzello, “Advances in the use of beneficial microorganisms to improve nutritional and functional properties of fermented foods,” Foods, vol. 13, no. 1, Art. no. 155, 2024.

[12] M. C. Coelho, F. X. Malcata, and C. C. G. Silva, “Lactic acid bacteria in raw-milk cheeses: From starter cultures to probiotic functions,” Foods, vol. 11, Art. no. 2276, 2022.

[13] H. Tang, W. Huang, and Y. F. Yao, “The metabolites of lactic acid bacteria: Classification, biosynthesis and modulation of gut microbiota,” Microbial Cell, vol. 10, no. 3, Art. no. 49, 2023.

[14] S. Seyirt, B. Uymaz Tezel, and P. Şanlıbaba, “Shelf-life dynamics of key probiotic populations in dairy formulations: A temporal stability assessment,” Food Biology, vol. 14, pp. 6–11, 2025, doi: 10.25081/fb.2025.v14.9587.

[15] J. Majumder, K. Pal, W. Chakraborty, P. Karmakar, and R. Gachhui, “Gluconacetobacter kombuchae RG3T, a novel bacterium for AgNP biosynthesis: Characterization and comprehensive evaluation of bioactivity,” Materials Today Communications, vol. 33, Art. no. 104410, 2022.

[16] M. Abd Qasim and L. A. Yaaqoob, “Evaluation of antibacterial activity of iron oxide nanoparticles synthesized by extracellular Lactobacillus against Pseudomonas aeruginosa,” Journal of Medicinal and Chemical Sciences, vol. 6, pp. 1100–1111, 2023, doi: 10.26655/JMCHEMSCI.2023.5.15.

[17] A. A. Mohammed, A. E. Hegazy, and A. Salah, “Novelty of synergistic and cytotoxicity activities of silver nanoparticles produced by Lactobacillus acidophilus,” Applied Nanoscience, vol. 13, no. 1, pp. 633–640, 2023.

[18] A. Król, V. Railean-Plugaru, P. Pomastowski, M. Złoch, and B. Buszewski, “Mechanism study of intracellular zinc oxide nanocomposite formation,” Colloids and Surfaces A: Physicochemical and Engineering Aspects, vol. 553, pp. 349–358, 2018.

[19] H. Mohd Yusof, R. Mohamad, U. H. Zaidan, and N. A. Abdul Rahman, “Microbial synthesis of zinc oxide nanoparticles and their potential application as an antimicrobial agent and feed supplement in the animal industry: A review,” Journal of Animal Science and Biotechnology, vol. 10, Art. no. 57, 2019.

[20] M. Bandeira, M. Giovanela, M. Roesch-Ely, D. M. Devine, and J. da Silva Crespo, “Green synthesis of zinc oxide nanoparticles: A review of the synthesis methodology and mechanism of formation,” Sustainable Chemistry and Pharmacy, vol. 15, Art. no. 100223, 2020.

[21] K. Spyridopoulou, E. Tryfonopoulou, G. Aindelis, P. Ypsilantis, C. Sarafidis, O. Kalogirou, and K. Chlichlia, “Biogenic selenium nanoparticles produced by Lactobacillus casei ATCC 393 inhibit colon cancer cell growth in vitro and in vivo,” Nanoscale Advances, vol. 3, no. 9, pp. 2516–2528, 2021.

[22] H. A. Abdulradha and S. W. Alhadrawi, “Biological and medical efficacy of zinc oxide nanoparticles manufactured using Saccharomyces boulardii against Burkholderia sp. isolated from diabetic foot patients,” International Journal of Advanced Multidisciplinary Research and Studies, vol. 3, no. 3, pp. 703–708, 2023.

[23] R. M. El-Masry, D. Talat, S. A. Hassoubah, N. M. Zabermawi, N. Z. Eleiwa, R. M. Sherif, and A. R. Elbestawy, “Evaluation of the antimicrobial activity of ZnO nanoparticles against enterotoxigenic Staphylococcus aureus,” Life, vol. 12, no. 10, Art. no. 1662, 2022.

[24] Z. Hossain, F. Yasmeen, and S. Komatsu, “Nanoparticles: Synthesis, morphophysiological effects, and proteomic responses of crop plants,” International Journal of Molecular Sciences, vol. 21, no. 9, Art. no. 3056, 2020.

[25] M. Rezvani, M. Manconi, and N. Düzgüneş, “Lactic acid bacteria: From bioprocessing to nanomedicine,” BioChem, vol. 6, no. 1, Art. no. 3, 2026.

[26] J. Jeevanandam, J. K. U. Ling, A. Barhoum, Y. San Chan, and M. K. Danquah, “Bionanomaterials: Definitions, sources, types, properties, toxicity, and regulations,” in Fundamentals of Bionanomaterials, A. Barhoum, J. Jeevanandam, and M. K. Danquah, Eds. Elsevier, 2022, pp. 1–29.

[27] Z. Denkova, P. Zapryanova, R. Denkova-Kostova, B. Goranov, Z. Urshev, Y. Gaytanska, and V. Shopska, “Isolation, identification and investigation of some properties of Lacticaseibacillus rhamnosus 1 for application in the composition of probiotics,” BIO Web of Conferences, vol. 170, Art. no. 02006, 2025.

[28] S. Z. Abedi, S. Yeganeh, F. Moradian, and H. Ouraji, “Isolation and identification of Lactobacillus strains from dairy products and evaluation of carbon source effects on bacterial growth and phytase activity,” Journal of Agricultural Science and Technology, vol. 21, no. 4, pp. 845–855, 2019.

[29] L. A. Minhas, A. S. Mumtaz, M. Kaleem, D. A. Farraj, K. Kamal, M. A. H. Minhas, and R. M. Mahmoud, “Green synthesis of zinc oxide nanoparticles using Nostoc sp. and their multiple biomedical properties,” Catalysts, vol. 13, no. 3, Art. no. 549, 2023, doi: 10.3390/catal13030549.

[30] S. W. Suciyati, P. Manurung, J. Junaidi, and R. Situmeang, “Optical and crystal structure properties of ZnO nanoparticles synthesized through a biosynthesis method for photocatalysis application,” Indonesian Journal of Chemistry, vol. 24, no. 1, pp. 125–140, 2024.

[31] M. H. Shabani, A. Jafari, M. Manteghian, and S. M. Mousavi, “Green synthesis of zinc oxide nanoparticles using Enterobacter cloacae and its application in enhanced oil recovery,” Scientific Reports, vol. 14, Art. no. 29409, 2024.

[32] A. K. Goyal, S. K. Middha, and A. Sen, “Evaluation of the DPPH radical scavenging activity, total phenols and antioxidant activities in Indian wild Bambusa vulgaris ‘Vittata’ methanolic leaf extract,” Journal of Natural Pharmaceuticals, vol. 1, no. 1, pp. 40–45, 2010, doi: 10.4103/2229-5119.73586.

[33] P. S. Chakra, A. Banakar, S. N. Puranik, V. Kaveeshwar, C. R. Ravikumar, and D. Gayathri, “Characterization of ZnO nanoparticles synthesized using probiotic Lactiplantibacillus plantarum GP258,” Beilstein Journal of Nanotechnology, vol. 16, pp. 78–89, 2025, doi: 10.3762/bjnano.16.8.

[34] G. Gahlawat and A. R. Choudhury, “A review on the biosynthesis of metal and metal salt nanoparticles by microbes,” RSC Advances, vol. 9, no. 23, pp. 12944–12967, 2019.

[35] P. Singh, Y. J. Kim, D. Zhang, and D. C. Yang, “Biological synthesis of nanoparticles from plants and microorganisms,” Trends in Biotechnology, vol. 34, no. 7, pp. 588–599, 2016.

[36] H. Agarwal, S. V. Kumar, and S. Rajeshkumar, “A review on green synthesis of zinc oxide nanoparticles—An eco-friendly approach,” Resource-Efficient Technologies, vol. 3, no. 4, pp. 406–413, 2017, doi: 10.1016/j.reffit.2017.03.002.

[37] S. Talam, S. R. Karumuri, and N. Gunnam, “Synthesis, characterization, and spectroscopic properties of ZnO nanoparticles,” International Scholarly Research Notices, vol. 2012, Art. no. 372505, 2012.

[38] W. Muhammad, N. Ullah, M. Haroon, and B. H. Abbasi, “Optical, morphological and biological analysis of zinc oxide nanoparticles using Papaver somniferum L.,” RSC Advances, vol. 9, no. 51, pp. 29541–29548, 2019.

[39] Z. M. Al-Asady, A. H. Al-Hamdani, and M. A. Hussein, “Study of the optical and morphological properties of zinc oxide nanoparticles,” AIP Conference Proceedings, vol. 2213, no. 1, Art. no. 020061, 2020.

[40] F. M. Albarakaty, M. I. Alzaban, N. K. Alharbi, F. S. Bagrwan, A. R. Abd El-Aziz, and M. A. Mahmoud, “Zinc oxide nanoparticles: Biosynthesis, characterization, and their potent photocatalytic degradation and antioxidant activities,” Journal of King Saud University—Science, vol. 35, no. 1, Art. no. 102434, 2023.

[41] A. Sirelkhatim, S. Mahmud, A. Seeni, N. H. M. Kaus, L. C. Ann, S. K. M. Bakhori, H. Hasan, and D. Mohamad, “Review on zinc oxide nanoparticles: Antibacterial activity and toxicity mechanism,” Nano-Micro Letters, vol. 7, no. 3, pp. 219–242, 2015, doi: 10.1007/s40820-015-0040-x.

[42] H. Mohd Yusof, N. A. Abdul Rahman, R. Mohamad, U. H. Zaidan, and A. A. Samsudin, “Biosynthesis of zinc oxide nanoparticles by cell biomass and supernatant of Lactobacillus plantarum TA4 and its antibacterial and biocompatibility properties,” Scientific Reports, vol. 10, Art. no. 19996, 2020.

[43] S. Suba, S. Vijayakumar, E. Vidhya, V. N. Punitha, and M. Nilavukkarasi, “Microbial-mediated synthesis of ZnO nanoparticles derived from Lactobacillus spp.: Characterization, antimicrobial and biocompatibility efficiencies,” Sensors International, vol. 2, Art. no. 100104, 2021.

[44] R. Shanmugam, T. Munusamy, S. Jayakodi, K. A. Al-Ghanim, M. Nicoletti, N. Sachivkina, and M. Govindarajan, “Probiotic-bacteria (Lactobacillus fermentum)-wrapped zinc oxide nanoparticles: Biosynthesis, characterization, and antibacterial activity,” Fermentation, vol. 9, no. 5, Art. no. 413, 2023.

[45] P. Jamdagni, P. Khatri, and J. S. Rana, “Green synthesis of zinc oxide nanoparticles using flower extract of Nyctanthes arbor-tristis and their antifungal activity,” Journal of King Saud University—Science, vol. 30, no. 2, pp. 168–175, 2018.

[46] M. T. El-Saadony, G. Fang, S. Yan, S. S. Alkafaas, M. A. El Nasharty, S. A. Khedr, and S. F. AbuQamar, “Green synthesis of zinc oxide nanoparticles: Preparation, characterization, and biomedical applications—A review,” International Journal of Nanomedicine, vol. 19, pp. 12889–12937, 2024, doi: 10.2147/IJN.S487188.

[47] V. S. Sivasankarapillai, N. Krishnamoorthy, G. E. Eldesoky, S. M. Wabaidur, M. A. Islam, R. Dhanusuraman, and V. K. Ponnusamy, “One-pot green synthesis of ZnO nanoparticles using Scoparia dulcis plant extract for antimicrobial and antioxidant activities,” Applied Nanoscience, vol. 13, no. 9, pp. 6093–6103, 2023.

[48] A. Abdelmoteleb, B. Valdez-Salas, E. Beltran-Partida, V. Mendez-Trujillo, D. González-Mendoza, O. Tzintzun-Camacho, and A. F. Roumia, “Biosynthesis of zinc oxide nanoparticles using garlic peel extract and their antibacterial potential,” Microbiology Research, vol. 15, no. 3, pp. 1655–1669, 2024.

[49] A. Boroumand Moghaddam, M. Moniri, S. Azizi, R. Abdul Rahim, A. Bin Ariff, W. Zuhainis Saad, F. Namvar, M. Navaderi, and R. Mohamad, “Biosynthesis of ZnO nanoparticles by a new Pichia kudriavzevii yeast strain and evaluation of their antimicrobial and antioxidant activities,” Molecules, vol. 22, no. 6, Art. no. 872, 2017, doi: 10.3390/molecules22060872.

[50] B. H. Abbasi, M. Shah, S. S. Hashmi, M. Nazir, S. Naz, W. Ahmad, I. U. Khan, and C. Hano, “Green bio-assisted synthesis, characterization and biological evaluation of biocompatible ZnO NPs synthesized from different tissues of milk thistle (Silybum marianum),” Nanomaterials, vol. 9, no. 8, Art. no. 1171, 2019, doi: 10.3390/nano9081171.

[51] J. Iqbal, B. A. Abbasi, T. Yaseen, S. A. Zahra, A. Shahbaz, S. A. Shah, S. Uddin, X. Ma, B. Raouf, S. Kanwal, T. Mahmood, and P. Ahmad, “Green synthesis of zinc oxide nanoparticles using Elaeagnus angustifolia L. leaf extracts and their multiple in vitro biological applications,” Scientific Reports, vol. 11, Art. no. 20988, 2021, doi: 10.1038/s41598-021-99839-z.