Purple rice is a potential source of ferulic acid, which has antimicrobial properties. However, the inhibitory mechanism of ferulic acid on the growth of bacteria, particularly Salmonella and Listeria, has not been elucidated. This study aimed to determine the bioactivity of ferulic acid from purple rice as an antimicrobial agent against Salmonella typhimurium and Listeria monocytogenes using in vitro and in silico analyses. The antimicrobial activity of a purple rice ferulic acid extract was tested using the agar well diffusion method. Its effect on bacterial cells was observed using scanning electron microscopy. Ferulic acid was confirmed to have antimicrobial properties using in silico software to attenuate the binding of bacterial virulence factors (lipoproteins, lipopolysaccharides, and flagellins) to Toll-like receptors (TLRs) and to prevent interactions with peptidoglycans. The purple rice ferulic acid extract inhibited bacterial growth. The inhibitory effects included induction of a biofilm and shrinkage of S. Typhimurium, as well as osmotic lysis of L. monocytogenes. This activity was supported by the ability of ferulic acid to inhibit the binding of bacterial virulence factors with TLRs and block bacterial peptidoglycans. These findings indicate that the purple rice ferulic acid extract acts as antimicrobial, both directly to bacterial cells and indirectly through TLRs. We conclude that ferulic acid from Indonesian purple rice has a biological function as an antimicrobial agent.



[1] Huang, Y.P. and Lai, H.M. 2016. Bioactive compounds and antioxidative activity of colored rice bran. J. Food Drug Anal. 24 (3): 564–574, doi: 10.1016/j.jfda.2016.01.004.

[2] Fatchiyah, F., Sari, D.R.T., Safitri, A. and Cairns, J.R.K. 2020. Phytochemical compound and nutritional value in black rice from Java Island, Indonesia. Syst. Rev. Pharm. 11 (7): 414–421, doi: 10.31838/srp.2020.7.61.

[3] Irakli, M.N., Samanidou, V.F., Katsantonis, D.N., Biliaderis, C.G. and Papadoyannis, I.N. 2016. Phytochemical profiles and antioxidant capacity of pigmented and non-pigmented genotypes of rice (Oryza sativa L.). Cereal Res. Commun. 44 (1): 98–110, doi: 10.1556/0806.43.2015.033.

[4] Mbanjo E.G.N, Kretzschmar, T., Jones, H., Ereful, N., Blanchard, C., Boys, L.A., Sreenivasulu, N. 2020. The genetic basis and nutritional benefits of pigmented rice grain. Front. Genet. 11: 1–18, doi: 10.3389/fgene.2020.00229.

[5] Tamprasit, K., Weerapreeyakul, N., Sutthanut, K., Thukhammee, W., Wattanathorn, J. 2019. Harvest age effect on phytochemical content of white and black glutinous rice cultivars. Molecules. 24: 1–16, doi: 10.3390/molecules24244432.

[6] Zhang, H., Shao, Y., Bao, J., Beta, T. 2014. Phenolic compounds and antioxidant properties of breeding lines between the white and black rice. Food Chem. 172: 630–639, doi: 10.1016/j.foodchem.2014.09.118.

[7] Alam, M.A. 2019. Anti-hypertensive effect of cereal antioxidant ferulic acid and its mechanism of action. Front. Nutr. 6: 1–7, doi: 10.3389/fnut.2019.00121.

[8] Zduńska, K., Dana, A., Kolodziejczak, A., and Rotsztejn, H. 2018. Antioxidant properties of ferulic acid and its possible application. Skin Pharmacol. Physiol. 31 (6): 332–336, doi: 10.1159/000491755.

[9] Boz, H. 2015. Ferulic acid in cereals - A review. Czech J. Food Sci. 33 (1): 1–7, doi: 10.17221/401/2014-CJFS.

[10] Lis, K.A., Boulaaba, A., Binder, S., Li, Y., Kehrenberg, C., Zimmermann, J.L., Klein, G., Ahlfeld, B. 2018. Inactivation of Salmonella typhimurium and Listeria monocytogenes on ham with nonthermal atmospheric pressure plasma. PLoS One. 13 (5): 1–21, doi: 10.1371/journal.pone.0197773.

[11] Johnson, R., Mylona, E., Frankel, G. 2018. Typhoidal Salmonella: Distinctive virulence factors and pathogenesis. Cell. Microbiol. 20 (9): 1–14, doi: 10.1111/cmi.12939.

[12] Lecuit, M. 2020. Listeria monocytogenes, a model in infection biology. Cell. Microbiol. 22 (4): 1–8, doi: 10.1111/cmi.13186.

[13] Kumar, A., Allison, A., Henry, M., Scales, A., Fouladkhah, A.C. 2019. Development of salmonellosis as affected by bioactive food compounds. Microorganisms. 7 (9): 1–18, doi: 10.3390/microorganisms7090364.

[14] Xiang, Y., Li, F., Dong, N., Tian, S., Zhang, H., Du, X., Zhou, X., Xu, X., Yang, H., Xie, J., Yang, C., Liu, H., Qiu, S., Song, H., Sun, Y. 2020. Investigation of a salmonellosis outbreak caused by multidrug resistant Salmonella typhimurium in China. Front. Microbiol. 11: 1–12, doi: 10.3389/fmicb.2020.00801.

[15] Olaimat, A.N., Al-Holy, M.A., Shahbaz, H.M., Al-Nabulsi, A.A., Ghoush, M.H.A., Osaili, T.M., Ayyash, M.M., Holley, R.A. 2018. Emergence of antibiotic resistance in Listeria monocytogenes isolated from food products: A comprehensive review. Compr. Rev. Food Sci. Food Saf. 17 (5): 1277–1292, doi: 10.1111/1541-4337.12387.

[16] Ma, X., Guo, Z., Zhang, Z., Li, X., Liu, Y., Zhao, L., Wang, X. 2020. Ferulic acid protects against porcine parvovirus infection-induced apoptosis by suppressing the nuclear factor- B inflammasome axis and toll-like receptor 4 via nonstructural protein 1. Evid. based Complement. Alternat. Med. 1–9, doi: 10.1155/2020/3943672.

[17] Marini, E., Magi, G., Ferretti, G., Bacchetti, T., Giuliani, A., Pugnaloni, A., Rippo, M.R., Facinelli, B. 2018. Attenuation of Listeria monocytogenes virulence by Cannabis sativa L. essential oil. Front. Cell. Infect. Microbiol. 8: 1–11, doi: 10.3389/fcimb.2018.00293.

[18] Nguyen, M.T., Götz, F. 2016. Lipoproteins of gram-positive bacteria: Key players in the immune response and virulence. Microbiol. Mol. Biol. Rev. 80 (3): 891–903, doi: 10.1128/mmbr.00028-16.

[19] Misra, S., Sharma, V., Srivastava, A.K. 2014. Bacterial polysaccharides: An overview. In: Ramawat K., Mérillon JM. (eds) Polysaccharides. Springer, Cham. 1: 81–108, doi: 10.1007/978-3-319-16298-0_68.

[20] Farhadi, T., Ovchinnikov, R.S., Ranjbar, M.M. 2016. In silico designing of some agonists of toll-like receptor 5 as a novel vaccine adjuvant candidates. Netw. Model. Anal. Health. Inform Bioinform. 5 (1): 1–10, doi: 10.1007/s13721-016-0138-1.

[21] Lu, X., Wang, M., Qi, J., Wang, H., Li, X., Gupta, D., Dziarski, R.2006. Peptidoglycan recognition proteins are a new class of human bactericidal proteins. J. Biol. Chem. 281 (9): 5895–5907, doi: 10.1074/jbc.M511631200.

[22] Chaparro, S.C.V., Salguero, J.T.V., Baquero, D.A.M., Pérez, J.E.R. 2018. Effect of polyvalence on the antibacterial activity of a synthetic peptide derived from bovine lactoferricin against healthcare-associated infectious pathogens. Biomed Res. Int. 1-12, doi: 10.1155/2018/5252891.

[23] Stavova, E., Porizka, J., Stursa, V., Enev, V., Divis, P. 2017. Extraction of ferulic acid from wheat bran through alkaline hydrolysis. MendelNet. 574–579.

[24] Ruiz-Ruiz, J.C., Ramón-Sierra, J., Arias-Argaez, C., Magaña-Ortiz, D., Ortiz-Vázquez, E. 2017. Antibacterial activity of proteins extracted from the pulp of wild edible fruit of Bromelia pinguin L. Int. J. Food Prop. 20 (1): 220–230, doi: 10.1080/10942912.2016.1154572.

[25] Musini, A., Giri, A. 2019. Investigation of mode of action of anti bacterial activity of Salacia oblonga extract against drug resistant pathogen. Braz. Arch. Biol. Technol. 62: 1–11, doi: 10.1590/1678-4324-2019180051.

[26] Kim, Y.J., Yu, H.H., Park, Y.J., Lee, N.K., Paik, H.D. 2020. Anti-biofilm activity of cell-free supernatant of Saccharomyces cerevisiae against Staphylococcus aureus. J. Microbiol. Biotechnol. 30 (12): 1854–1861, doi: 10.4014/jmb.2008.08053.

[27] Hikmawati, V.F., Alam, F.M., Ainnayah, J.S., Fatchiyah, F. 2020. Virtual prediction of phenolic and glucosinolate compounds with Keap1 protein as anti-aging by stimulating Nrf2. J. Exp. Life Sci. 10 (2): 104–112.

[28] Borges, A., Ferreira, C., Saavedra, M.J., Simões, M. 2013. Antibacterial activity and mode of action of ferulic and gallic acids against pathogenic bacteria. Microb. Drug Resist. 19 (4): 256–265, doi: 10.1089/mdr.2012.0244.

[29] Liu, J., Du, C., Beaman, H.T., Monroe, M.B.B. 2020. Characterization of phenolic acid antimicrobial and antioxidant structure–property relationships. Pharmaceutics. 12 (5): 1–17, doi: 10.3390/pharmaceutics12050419.

[30] Mitani, T., Ota, K., Inaba, N., Kishida, K., Koyama, H.A. 2018. Antimicrobial activity of the phenolic compounds of Prunus mume against enterobacteria. Biol. Pharm. Bull. 41 (2): 208–212, doi: 10.1248/bpb.b17-00711.

[31] Aarabi, A., Honarvar, M., Mizani, M., Faghihian, H., Gerami, A. 2016. Extraction and purification of ferulic acid as an antioxidant from sugar beet pulp by alkaline hydrolysis. Ital. J. Food Sci. 28 (3): 362–375, doi: 10.14674/1120-1770/ijfs.v143.

[32] Goufo, P., Trindade, H. 2014. Rice antioxidants: phenolic acids, flavonoids, anthocyanins, proanthocyanidins, tocopherols, tocotrienols, γ -oryzanol, and phytic acid. Food Sci. Nutr. 2 (2): 75–104, doi: 10.1002/fsn3.86.

[33] Greenwood. 1995. Antibiotics susceptibility (sensitivity) test, antimicrobial and chemoteraphy. Mc Graw Hill Company, USA.

[34] Penesyan, A., Nagy, S.S., Kjelleberg, S., Gillings, M.R., Paulsen, I.T. 2019. Rapid microevolution of biofilm cells in response to antibiotics. npj Biofilms Microbiomes. 5 (1): 1–14, doi: 10.1038/s41522-019-0108-3.

[35] Hartmann, M., Berditsch, M., Hawecker, J., Ardakani, M.F., Gerthsen, D., Ulrich, A.S. 2010. Damage of the bacterial cell envelope by antimicrobial peptides gramicidin S and PGLa as revealed by transmission and scanning electron microscopy. Antimicrob. Agents Chemother. 54 (8): 3132–3142, doi: 10.1128/AAC.00124-10.

[36] El-Zayat, S.R., Sibaii, H., Mannaa, F.A. 2019. Toll-like receptors activation, signaling, and targeting: an overview. Bull. Natl. Res. Cent. 43 (1), doi: 10.1186/s42269-019-0227-2.

[37] Cegelski, L., Marshall, G.R., Eldridge, G.R., Hultgren, S.J. 2008. The biology and future prospects of antivirulence therapies. Nat Rev Microbiol. 6 (7): 17–27.

[38] Ford, C.A., Hurford, I.M., Cassat, J.E. 2021. Antivirulence strategies for the treatment of Staphylococcus aureus infections: A mini review. Front. Microbiol. 11: 1–10, doi: 10.3389/fmicb.2020.632706.

[39] Scheffers, D.J., Pinho, M.G. 2005. Bacterial cell wall synthesis: New insights from localization studies. Microbiol. Mol. Biol. Rev. 69 (4): 585–607, doi: 10.1128/MMBR.69.4.585.

[40] Yashin, D.V., Sashchenko, L.P., Georgiev, G.P. 2021. Mechanisms of action of the PGLYRP1/Tag7 protein in innate and acquired immunity. Acta Naturae. 13 (1): 91–101, doi: 10.32607/actanaturae.11102.

[41] Ma, P., Wang, Z., Pflugfelder, S.C., Li, D.-Q. 2010. Toll-like receptors mediate induction of peptidoglycan recognition proteins in human corneal epithelial cells. Exp Eye Res. 90 (1): 130–136, doi: 10.1016/j.exer.2009.09.021.Toll-like.

[42] Li, J., Koh, J.J., Liu, S., Lakshminarayanan, R., Verma, C.S., Beuerman, R.W. 2017. Membrane active antimicrobial peptides: Translating mechanistic insights to design. Front. Neurosci. 11: 1–18, doi: 10.3389/fnins.2017.00073.

[43] Wu, Y., Bai, J., Zhong, K., Huang, Y., Qi, H., Jiang, Y., Gao, H. 2016. Antibacterial activity and membrane-disruptive mechanism of 3-p-trans-coumaroyl-2-hydroxyquinic acid, a novel phenolic compound from pine needles of Cedrus deodara, against Staphylococcus aureus. Molecules. 21 (8): 1–12, doi: 10.3390/molecules21081084.

[44] Epand, R.M., Walker, C., Epand, R.F., Magarvey, N.A. 2015. Molecular mechanisms of membrane targeting antibiotics. Biochim. Biophys. Acta Biomembr. 1858 (5): 980–987, doi: 10.1016/j.bbamem.2015.10.018.

[45] Guo, L., Sun, Q., Gong, S., Bi, X., Jiang, W., Xue, W., Fei, P. 2019. Antimicrobial activity and action approach of the olive oil polyphenol extract against Listeria monocytogenes. Front. Microbiol. 10: 1–8, doi: 10.3389/fmicb.2019.01586.

[46] Zhao, P., Zhang, Y., Deng, H., Meng, Y. 2021. Antibacterial mechanism of apple phloretin on physiological and morphological properties of Listeria monocytogenes. Food Sci. Technol. 1–9, doi: 10.1590/fst.55120.

[47] Sadiq, M.B., Tarning, J., Cho, T.Z.A., Anal, A.K. 2017. Antibacterial activities and possible modes of action of Acacia nilotica (L.) Del. against multidrug-resistant Escherichia coli and Salmonella. Molecules. 22 (1): 1–16, doi: 10.3390/molecules22010047.

[48] Wang, C., Chang, T., Yang, H., Cui, M. 2015. Antibacterial mechanism of lactic acid on physiological and morphological properties of Salmonella enteritidis, Escherichia coli and Listeria monocytogenes. Food Control. 47: 231–236, doi: 10.1016/j.foodcont.2014.06.034.

[49] Cushnie, T.P.T., O’Driscoll, N.H., Lamb, A.J. 2016. Morphological and ultrastructural changes in bacterial cells as an indicator of antibacterial mechanism of action. Cell. Mol. Life Sci. 73 (23): 4471–4492, doi: 10.1007/s00018-016-2302-2.

[50] Hall, C.W., Mah, T.F. 2017. Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria. FEMS Microbiol. Rev. 41 (3): 276–301, doi: 10.1093/femsre/fux010.

[51] Wang, L., Hu, X., Tao, G., Wang, X. 2012. Outer membrane defect and stronger biofilm formation caused by inactivation of a gene encoding for heptosyltransferase I in Cronobacter sakazakii ATCC BAA-894. J. Appl. Microbiol. 112 (5): 985–997, doi: 10.1111/j.1365-2672.2012.05263.x.

[52] Huszczynski, S.M., Lam, J.S., Khursigara, C.M. 2020. The role of Pseudomonas aeruginosa lipopolysaccharide in bacterial pathogenesis and physiology. Pathogens. 9 (1): 1–22, doi: 10.3390/pathogens9010006.

[53] Jaiswal, S., Pati, N.B., Dubey, M., Padhi, C., Sahoo, P.K., Ray, S., Arunima, A., Mohakud, N.K., Suar, M. 2015. The O-antigen negative ΔwbaV mutant of Salmonella enterica serovar Enteritidis shows adaptive resistance to antimicrobial peptides and elicits colitis in streptomycin pretreated mouse model. Gut Pathog. 7 (1): 1–12, doi: 10.1186/s13099-015-0070-4.

[54] Lal, A.F., Singh, S., Franco, F.C., Bhatia, S. 2021. Potential of polyphenols in curbing quorum sensing and biofilm formation in gram-negative pathogens. Asian Pac. J. Trop. Biomed. 11 (6): 231–243, doi: 10.4103/2221-1691.314044.

[55] Kaplan, J.B. 2011. Antibiotic-induced biofilm formation. Int. J. Artif. Organs. 34 (9): 737–751, doi: 10.5301/ijao.5000027.

[56] Rabin, N., Zheng, Y., Opoku-Temeng, C., Du, Y., Bonsu, E., Sintim, H.O. 2015. Biofilm formation mechanisms and targets for developing antibiofilm agents. Futur. Med. Chem. 7 (4): 493–512, doi: 10.2307/j.ctvnwc0d0.18.

[57] Aka, S.T., Haji, S.H. 2015. Sub-MIC of antibiotics induced biofilm formation of Pseudomonas aeruginosa in the presence of chlorhexidine. Braz J. Microbiol. 46 (1): 149–154, doi: 10.1590/S1517-838246120140218.



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