Abstract
Screening of bacteriocin-producing lactic acid bacteria (LAB) is an important aspect for the search of new/novel probiotic strains. Here, a vesicle-based colorimetric assay was compared with conventional inhibition-based antimicrobial assays using 54 isolates of LAB. All isolates demonstrated zone of growth inhibition ranging from 2.5 to 7.5 mm against indicator strain, Micrococcus luteus MTCC106 using point inoculation method. Cell-free supernatant of the isolates showed zone of growth inhibition varying from 14.5 to 25 mm using agar well diffusion assay. These isolates inhibited the growth of indicator strain by 89.56–98.65%. The antimicrobial activity present in cell-free supernatant of different isolates was found to be in the range of 10–160 AU ml− 1. The treatment of polydiacetylene (PDA) vesicles with cell-free supernatant of selected isolates led to blue-red color transition, and presence of protein band on tricine SDS-PAGE confirmed the presence of membrane-acting peptides, bacteriocins. The colorimetric responses (CR%) varied between 0 and 59%, and the assay was found to be more sensitive, faster, and reliable as compared to the other conventional indicator-based methods used. Therefore, the colorimetric assay may be specifically applied for screening of bacteriocin-producing LAB.
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References
Gupta A, Tiwari SK (2015) Probiotic potential of bacteriocin-producing Enterococcus hirae strain LD3 isolated from Dosa batter. Ann Microbiol 65:2333–2342. https://doi.org/10.1007/s13213-015-1075-4
Lv X, Du J, Jie Y, Zhang B, Bai F, Zhao H, Li J (2017) Purification and antibacterial mechanism of fish-borne bacteriocin and its application in shrimp (Penaeus vannamei) for inhibiting Vibrio parahaemolyticus. World J Microbiol Biotechnol 33:1–12. https://doi.org/10.1007/s11274-017-2320-8
Repally A, Perumal V, Dasari A, Palanichamy E, Venkatesan A (2017) Isolation, identification of Lactobacillus mucosae AN1 and its antilisterial peptide purification and characterization. Probiotics Antimicrob Proteins 9:1–12. https://doi.org/10.1007/s12602-017-9341-3
Techo S, Visessanguan W, Vilaichone R, Tanasupawat S (2018) Characterization and antibacterial activity against Helicobacter pylori of lactic acid bacteria isolated from Thai fermented rice noodle. Probiotics Antimicrob Proteins 10:1–11. https://doi.org/10.1007/s12602-018-9385-z
Wannun P, Piwat S, Teanpaisan R (2016) Purification, characterization, and optimum conditions of fermencin SD11, a bacteriocin produced by human orally Lactobacillus fermentum SD11. Appl Biochem Biotechnol 179:572–582. https://doi.org/10.1007/s12010-016-2014-y
Hegarty JW, Guinane CM, Ross RP, Hill C, Cotter PD (2017) Lack of heterogeneity in bacteriocin production across a selection of commercial probiotic products. Probiotics Antimicrob Proteins 9:459–465. https://doi.org/10.1007/s12602-017-9326-2
Dobson A, Cotter PD, Paul Ross R, Hill C (2012) Bacteriocin production: a probiotic trait? Appl Environ Microbiol 78:1–6. https://doi.org/10.1128/AEM.05576-11
Gupta A, Tiwari SK, Netrebov V, Chikindas ML (2016) Biochemical properties and mechanism of action of enterocin LD3 purified from Enterococcus hirae LD3. Probiotics Antimicrob Proteins 8:161–169. https://doi.org/10.1007/s12602-016-9217-y
Kaškonienė V, Stankevičius M, Bimbiraitė-Survilienė K, Naujokaitytė G, Šernienė L, Mulkytė K, Malakauskas M, Maruška A (2017) Current state of purification, isolation and analysis of bacteriocins produced by lactic acid bacteria. Appl Microbiol Biotechnol 101:1323–1335. https://doi.org/10.1007/s00253-017-8088-9
Daba GM, Ishibashi N, Zendo T, Sonomoto K (2017) Functional analysis of the biosynthetic gene cluster required for immunity and secretion of a novel Lactococcus-specific bacteriocin, lactococcin Z. J Appl Microbiol 123:1124–1132. https://doi.org/10.1111/jam.13564
Sahoo TK, Jena PK, Prajapati B, Gehlot L, Patel AK, Seshadri S (2017) In vivo assessment of immunogenicity and toxicity of the bacteriocin TSU4 in BALB/c mice. Probiotics Antimicrob Proteins 9:345–354. https://doi.org/10.1007/s12602-016-9249-3
Moreno I, Marasca ETG, de Sá PBZR, de Souza Moitinho J, Marquezini MG, Alves MRC, Bromberg R (2018) Evaluation of probiotic potential of bacteriocinogenic lactic acid bacteria strains isolated from meat products. Probiotics Antimicrob Proteins 10:1–13. https://doi.org/10.1007/s12602-018-9388-9
Camargo AC, de Paula OAL, Todorov SD, Nero LA (2016) In vitro evaluation of bacteriocins activity against Listeria monocytogenes biofilm formation. Appl Biochem Biotechnol 178:1239–1251. https://doi.org/10.1007/s12010-015-1941-3
Ramu R, Shirahatti PS, Devi AT, Prasad A, Kumuda J, Lochana MS, Zameer F, Dhananjaya BL, Nagendra Prasad MN (2015) Bacteriocins and their applications in food preservation. Crit Rev Food Sci Nutr 8398:00–00. https://doi.org/10.1080/10408398.2015.1020918
Perez RH, Zendo T, Sonomoto K (2014) Novel bacteriocins from lactic acid bacteria (LAB): various structures and applications. Microb Cell Factories 13:S1–S3. https://doi.org/10.1186/1475-2859-13-S1-S3
Kolusheva S, Boyer L, Jelinek R (2000) A colorimetric assay for rapid screening of antimicrobial peptides. Nat Biotechnol 18:225–227. https://doi.org/10.1038/72697
Wei M, Liu J, Xia Y, Feng F, Liu W, Zheng F (2015) A polydiacetylene-based fluorescence assay for the measurement of lipid membrane affinity. RSC Adv 5:66420–66425. https://doi.org/10.1039/C5RA13445E
Yadav MK, Kumar V, Singh B, Tiwari SK (2017) Phospholipid/polydiacetylene vesicle-based colorimetric assay for high-throughput screening of bacteriocins and halocins. Appl Biochem Biotechnol 182:142–154. https://doi.org/10.1007/s12010-016-2316-0
Gupta A, Tiwari SK (2014) Plantaricin LD1: a bacteriocin produced by food isolate of Lactobacillus plantarum LD1. Appl Biochem Biotechnol 172:3354–3362. https://doi.org/10.1007/s12010-014-0775-8
Kumar V, Sheoran P, Gupta A, Yadav JP, Tiwari SK (2016) Antibacterial property of bacteriocin produced by Lactobacillus plantarum LD4 isolated from a fermented food. Ann Microbiol 66:1431–1440. https://doi.org/10.1007/s13213-016-1230-6
Chen Y, Wu H, Kuo C, Chen Y, Ho S, Yanagida F (2017) Leucocin C-607, a novel bacteriocin from the multiple-bacteriocin-producing Leuconostoc pseudomesenteroides 607 isolated from persimmon. Probiotics Antimicrob Proteins 9:1–9. https://doi.org/10.1007/s12602-017-9359-6
Schnaider L, Brahmachari S, Schmidt NW, Mensa B, Shaham-Niv S, Bychenko D, Adler-Abramovich L, Shimon LJW, Kolusheva S, DeGrado WF, Gazit E (2017) Self-assembling dipeptide antibacterial nanostructures with membrane disrupting activity. Nat Commun 8:1365. https://doi.org/10.1038/s41467-017-01447-x
Kaur R, Tiwari SK (2018) Membrane-acting bacteriocin purified from a soil isolate Pediococcus pentosaceus LB44 shows broad host-range. Biochem Biophys Res Commun 498:810–816. https://doi.org/10.1016/j.bbrc.2018.03.062
Cleveland J, Chikindas ML, Montville TJ (2002) Multimethod assessment of commercial nisin preparations. J Ind Microbiol Biotechnol 29:228–232. https://doi.org/10.1038/sj.jim.7000315
Gupta A, Tiwari SK (2014) Probiotic potential of Lactobacillus plantarum LD1 isolated from batter of Dosa, a South Indian fermented food. Probiotics Antimicrob Proteins 6:73–81. https://doi.org/10.1007/s12602-014-9158-2
Tiwari SK, Srivastava S (2008) Characterization of a bacteriocin from Lactobacillus plantarum strain LR/14. Food Biotechnol 22:247–261. https://doi.org/10.1080/08905430802262582
Goh HF, Philip K (2015) Purification and characterization of bacteriocin produced by Weissella confusa A3 of dairy origin. PLoS One 16:1–17. https://doi.org/10.1371/journal.pone.0140434
Carina Audisio M, Torres MJ, Sabaté DC, Ibargurena C, Apella MC (2011) Properties of different lactic acid bacteria isolated from Apis mellifera L. bee-gut. Microbiol Res 166:1–13. https://doi.org/10.1016/j.micres.2010.01.003
Abbasiliasi S, Tan JS, Ibrahim TAT, Ramanan RN, Vakhshiteh F, Mustafa S, Ling TC, Rahim RB, Ariff AB (2012) Isolation of Pediococcus acidilactici Kp10 with ability to secrete bacteriocin-like inhibitory substance from milk products for applications in food industry. BMC Microbiol 12(1):260. https://doi.org/10.1186/1471-2180-12-260
Tiwari SK, Srivastava S (2015) Broad antimicrobial-spectrum of Plantaricin LR14 against Gram-positive and Gram-negative bacteria. Austin J Anal Pharm Chem 2:1036–1032
Kaur R, Tiwari SK (2016) Isolation, identification and characterization of Pediococcus pentosaceus LB44 and Weissella confusa LM85 for the presence of bacteriocin-like inhibitory substances (BLIS). Microbiology 85:540–547. https://doi.org/10.1134/S0026261716050088
Escobar-Zepeda A, Sanchez-Flores A, Quirasco Baruch M (2016) Metagenomic analysis of a Mexican ripened cheese reveals a unique complex microbiota. Food Microbiol 57:116–127. https://doi.org/10.1016/j.fm.2016.02.004
Funding
This work was supported by the Department of Biotechnology (DBT, BT/PR8911/NDB/39/423/2013) and Indian Council of Medical Research (ICMR, 5/9/1117/2013-NUT), New Delhi, India. MKY acknowledges fellowships by the DBT and ICMR.
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Yadav, M.K., Singh, B. & Tiwari, S.K. Comparative Analysis of Inhibition-Based and Indicator-Independent Colorimetric Assay for Screening of Bacteriocin-Producing Lactic Acid Bacteria. Probiotics & Antimicro. Prot. 11, 687–695 (2019). https://doi.org/10.1007/s12602-018-9445-4
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DOI: https://doi.org/10.1007/s12602-018-9445-4