Abstract
BIFICO is a main microecological drug that the main ingredient is Bifidobacterium longum ATCC BAA-2753. It’s necessary to detect the number of Bifidobacterium longum ATCC BAA-2753 for quality control during the production of BIFICO. Plate count assay (PCA) is the most commonly used method for counting microbial cells. However, not all microorganisms can be counted by PCA. (e.g. uncultured microorganisms under specific growth conditions). In this study, a method of fluorescent techniques in combination with flow cytometry (FCM) was established for the counting of Bifidobacterium longum BAA-2753. Using SYTO9 and PI stain, flow cytometric analysis could easily differentiate live bacteria and dead bacteria. The number of live bacteria determined by FCM was about fourfold higher than that counted by PCA. Therefore, the FCM method could be a practical tool for the quality control of Bifidobacterium longum BAA-2753 in the industrial production of BIFICO.
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References
Amor KB, Breeuwer P, Verbaarschot P, Rombouts FM, Akkermans AD, De Vos WM, Abee T. Multiparametric flow cytometry and cell sorting for the assessment of viable, injured, and dead bifidobacterium cells during bile salt stress. Appl. Environ. Microbiol. 68: 5209–5216 (2002)
Breeuwer P, Abee T. Assessment of viability of microorganisms employing fluorescence techniques. Int. J. Food Microbiol. 55: 193–200 (2000)
Bukharin OV, Perunova NB, Ivanova EV. Interaction of Bifidobacterium bifidum with members of normal microflora in human intestine microsymbiocenosis. Zh Mikrobiol Epidemiol Immunobiol. 4: 51–56 (2012)
Hornbaek T, Dynesen J, Jakobsen M. Use of fluorescence ratio imaging microscopy and flow cytometry for estimation of cell vitality for Bacillus licheniformis. Fems Microbiol. Lett. 215: 261–265 (2002)
Huang NN, Wang GZ, Wang JF, Yuan YX. Risk factors for neonatal nosocomial enteric infection and the effect of intervention with BIFICO. Eur. Rev. Med. Pharmacol. Sci. 20: 3713–3719 (2016)
Joux F, Lebaron P. Use of fluorescent probes to assess physiological functions of bacteria at single-cell level. Microbes Infect. 2: 1523–1535 (2000)
Kell DB, Kaprelyants AS, Weichart DH, Harwood CR, Barer MR. Viability and activity in readily culturable bacteria: a review and discussion of the practical issues. Antonie Van Leeuwenhoek. 73: 169–187 (1998)
Laflamme C, Ho J, Veillette M, de Latremoille MC, Verreault D, Meriaux A, Duchaine C. Flow cytometry analysis of germinating Bacillus spores, using membrane potential dye. Arch Microbiol. 183: 107–112 (2005)
Liu XL, Li ML, Ma WX, Xia SL, Xu BL. Clinical trial on the prevention of diarrhea by oral BIFICO for infants aged 1–6 years. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 27: 277–279 (2013)
Shapiro HM. Microbial analysis at the single-cell level: tasks and techniques. J. Microbiol. Methods. 42: 3–16 (2000)
Shin JH, Nam MH, Lee H, Lee JS, Kim H, Chung MJ, Seo JG. Amelioration of obesity-related characteristics by a probiotic formulation in a high-fat diet-induced obese rat model. Eur. J. Nutr. 60: 1–10 (2017)
Sivan A, Corrales L, Hubert N, Williams JB, Aquino-Michaels K, Earley ZM, Benyamin FW, Lei YM, Jabri B, Alegre ML, Chang EB, Gajewski TF. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy. Science. 350: 1084–1089 (2015)
Snyder A, Pamer E, Wolchok J. Immunotherapy. Could microbial therapy boost cancer immunotherapy? Science. 350: 1031–1032 (2015)
Stiefel P, Schmidt-Emrich S, Maniura-Weber K, Ren Q. Critical aspects of using bacterial cell viability assays with the fluorophores SYTO9 and propidium iodide. BMC Microbiol. 15: 36 (2015)
Stocks SM. Mechanism and use of the commercially available viability stain, BacLight. Cytometry A. 61: 189–195 (2004)
Suller MT, Lloyd D. Fluorescence monitoring of antibiotic-induced bacterial damage using flow cytometry. Cytometry. 35: 235–241 (1999)
Williams NT. Probiotics. Am. J. Health Syst. Pharm. 67: 449–458 (2010)
Yu HJ, Liu W, Chang Z, Shen H, He LJ, Wang SS, Liu L, Jiang YY, Xu GT, An MM, Zhang JD. Probiotic BIFICO cocktail ameliorates Helicobacter pylori induced gastritis. World J. Gastroenterol. 21: 6561–6571 (2015)
Zhao L, Zhao Y, Liu R, Zheng X, Zhang M, Guo H, Zhang H, Ren F. The transcription factor DAF-16 is essential for increased longevity in C. elegans exposed to bifidobacterium longum BB68. Sci. Rep. 7: 7408 (2017)
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We wish to thank for BD Biosciences technical assistance and Dr. Xiao-fen Xu for preparation of the manuscript. We would also like to thank Dr. Ning-yun Sun for his helpful support.
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Gao, Y., Yu, Hj. & Wen, B. The use of fluorescent techniques in combination with flow cytometry for fast counting of Bifidobacterium longum ATCC BAA-2753 in BIFICO capsule. Food Sci Biotechnol 27, 1405–1410 (2018). https://doi.org/10.1007/s10068-018-0388-z
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DOI: https://doi.org/10.1007/s10068-018-0388-z