Browse > Article
http://dx.doi.org/10.9721/KJFST.2021.53.2.204

Chemical changes in resazurin by probiotics and its application for evaluating living bacterial cell counts and their reduction potentials  

Lee, Hyowon (Division of Applied Food System, College of Natural Science, Seoul Women's University)
Oh, Yeong Ji (Department of Food and Animal Biotechnology, Center for Food and Bioconvergence, Seoul National University)
Hong, Jungil (Division of Applied Food System, College of Natural Science, Seoul Women's University)
Publication Information
Korean Journal of Food Science and Technology / v.53, no.2, 2021 , pp. 204-212 More about this Journal
Abstract
Resazurin, an oxidized blue dye, is reduced to resorufin, showing a peak absorbance change and emitting fluorescence due to the metabolic activity of living cells. In this study, the growth of Lactobacillus rhamnosus GG (LGG) and the redox potential of living probiotic bacteria were evaluated based on changes in the absorbance or fluorescence of resazurin. Fluorescence analysis is a more accurate and sensitive method for quantifying viable LGG than the colorimetric absorbance measurement of resazurin change. Fluorescence measurement could detect LGG of 6.5-9.5 log CFU/mL within 30 min with R2=0.99. No significant effect of further reduction of resorufin to dihydroresorufin by LGG was observed. Various probiotics showed different resazurin-reducing activities, and L. kimchicus had the highest reducing activity among the six probiotics tested. These findings suggests that fluorescence measurement in a resazurin-based assay is useful for analyzing bacterial growth and the redox potential of living probiotics.
Keywords
resazurin; probiotics; resorufin; Lactobacillus rhamnosus GG; Lactobacillus kimchicus;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Caplice E, Fitzgerald GF. Food fermentations: role of microorganisms in food production and preservation. Int. J. Food Microbiol. 50: 131-149 (1999)   DOI
2 Cohen Y. Bioremediation of oil by marine microbial mats. Int. Microbiol. 5: 189-193 (2002)   DOI
3 Devi SM, Archer AC, Halami PM. Screening, characterization and in vitro evaluation of probiotic properties among lactic acid bacteria through comparative analysis. Probiotics Antimicro. 7: 181-192 (2015)   DOI
4 Erb RE, Chlers MH. Resazurin reducing time as an indicator of bovine semen fertilizing capacity. J. Dairy Sci. 33: 853-864 (1950)   DOI
5 Fleming A. On the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation of B. influenzae. Brit. J. Exp. Pathol. 10: 226-236 (1929)
6 Glick BR. Phytoremediation: synergistic use of plants and bacteria to clean up the environment. Biotechnol. Adv. 21: 383-393 (2003)   DOI
7 Grela E, Kozlowska J, Grabowiecka A. Current methodology of MTT assay in bacteria-A review. Acta Histochem. 120: 303-311 (2018)   DOI
8 Guerin TF, Mondido M, McClenn B, Peasley B. Application of resazurin for estimating abundance of contaminant-degrading microorganisms. Lett. Appl. Microbiol. 32: 340-345 (2001)   DOI
9 Jung DS, Kim SY, Oh YJ. Food microbial experiment for HACCP application. Gyomoonsa, Gyeonggi, Korea. pp. 47-48 (2019)
10 Jung MY, Lee J, Park B, Hwang H, Sohn SO, Lee SH, Lee JH. Applicability of a colorimetric method for evaluation of lactic acid bacteria with probiotic properties. Food Microbiol. 64: 33-38 (2017)   DOI
11 Ladisch MR, Kohlmann KL. Recombinant human insulin. Biotechnol. Progr. 8: 469-478 (1992)   DOI
12 Lin MY, Yen CL. Antioxidative ability of lactic acid bacteria. J. Agr. Food Chem. 47: 1460-1466 (1999)   DOI
13 Peeters E, Nelis HJ, Coenye T. Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J. Microbiol. Meth. 72: 157-165 (2008)   DOI
14 Mannanov RN, Sattarova RK. Antibiotics produced by Bacillus bacteria. Chem. Nat. Compd. 27: 117-123 (2001)   DOI
15 Mariscal A, Lopez-Gigosos RM, Carnero-Varo M, Fernandez-Crehuet J. Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm. Appl. Microbiol. Biot. 82: 773-783 (2009)   DOI
16 Min TG, Kang WS. Simple, quick and nondestructive method for Brassicaceae seed viability measurement with single seed base using resazurin. Hortic. Environ. Biote. 52: 240-245 (2011)   DOI
17 O'brien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267: 5421-5426 (2000)   DOI
18 Patel RM, Denning PW. Therapeutic use of prebiotics, probiotics, and postbiotics to prevent necrotizing enterocolitis: what is the current evidence?. Clin. Perinatol. 40: 11-25 (2013)   DOI
19 Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Bio. Med. 26: 1231-1237 (1999)   DOI
20 Ros RP, Morgan S, Hill C. Preservation and fermentation: past, present and future. Int. J. Food Microbiol. 79: 3-16 (2002)   DOI
21 Sarker SD, Nahar L, Kumarasamy Y. Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods 42: 321-324 (2007)   DOI
22 Wang Y, Wu Y, Wang Y, Xu H, Mei X, Yu D, Li W. Antioxidant properties of probiotic bacteria. Nutrients 9: 521 (2017)   DOI
23 Sobko T, Huang L, Midtvedt T, Norin E, Gustafsson LE, Norman M, Lundberg JO. Generation of NO by probiotic bacteria in the gastrointestinal tract. Free Radical Bio. Med. 41: 985-991 (2006)   DOI
24 Speck ML. Enumeration of vjable Lactobacillus acidophilus organisms in dairy products. J. Food Protect. 42: 135-137 (1978)   DOI
25 Vega-Avila E, Pugsley MK. An overview of colorimetric assay methods used to assess survival or proliferation of mammalian cells. P. W. Pharmacol. Soc. 54: 10-14 (2011)
26 Wijtzes T, Van't Riet K, in't Veld JH, Zwietering MH. A decision support system for the prediction of microbial food safety and food quality. Int. J. Food Microbiol. 42: 79-90 (1998)   DOI
27 Barnes S, Spenney JG. Stoichiometry of the NADH-oxidoreductase reaction for dehydrogenase determinations. Clin. Chim. Acta 107: 149-154 (1980)   DOI
28 Blois MS. Antioxidant determinations by the use of a stable free radical. Nature 181: 1199-1200 (1958)   DOI
29 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 (1976)   DOI
30 Brugger SD, Baumberger C, Jost M, Jenni W, Brugger U, Muhlemann K. Automated counting of bacterial colony forming units on agar plates. PloS One e33695 (2012)   DOI