Quantification of live and dead probiotic bacteria in lyophilised product by real-time PCR and by flow cytometry

Abstract

The basic requirement for probiotic bacteria to be able to exert expected positive effects is to be alive; therefore, appropriate quantification methods are crucial. Due to disadvantages of conventional microbiological methods, the bacterial quantification based on the nucleic acid detection is increasingly used. The objective of this study was to evaluate the possibility to use propidium monoazide (PMA) in combination with real-time polymerase chain reaction (PCR) method or LIVE/DEAD BacLight viability kit in combination with flow cytometry (FCM) for determination of probiotic bacteria in a lyophilised product containing Lactobacillus acidophilus LA-5 and Bifidobacterium animalis ssp. lactis BB-12. In addition, the viability of probiotic bacteria in lyophilised product during 3 months storage was investigated. In the product, the results of real-time PCR quantification of PMA-treated cells did not differ significantly from those of non-treated cells, which indicate that most of the bacterial cells retained the membrane integrity although they have lost the culturability. The results obtained by FCM analysis were comparable with those by PMA real-time PCR. In conclusion, the PMA real-time PCR and FCM determination of the viability of probiotic bacteria could complement the plate count method which considers only the culturable part of the population.

Probiotics - What They Are, Their Benefits and Challenges

“…Thus, the RNA content of the cell detected by fluorescent probes cannot be regarded as reliable indicator of cellular viability (Vives-Rego et al, 2000). Also, real time PCR detects both viable and non-viable bacteria, thus does not provide information on the condition of the cells and results in an overestimation of metabolically active cells (Kramer et al, 2009;Masco et al, 2007).…”

“…Treatment with DNA-binding dyes and subsequent PCR analysis uses membrane integrity as the criterion in determining viability of cells. Live cells are able to exclude DNA-binding dyes such as ethidium monoazide (EMA) and propidium monoazide (PMA), while dead cells or those whose membrane integrity has been compromised are able to pick-up these stains (Kramer et al, 2009). These dyes form covalent bonds with DNA upon exposure to visible bright light and thus inhibit subsequent PCR amplification.…”

Viable quantitative PCR for assessing the response of Candida albicans to antifungal treatment

“…The DNA of dead cells is expected to remain stable over prolonged periods of time (Josephson et al 1993); thus, DNA techniques may include the dead population and overestimate the microorganisms present in a sample. To overcome this, selective nucleic acid intercalating dyes, including ethidium monoazide (EMA) and propidium monoazide 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 5 (PMA), have been successfully used in conjunction with qPCR (herein called viableqPCR) for a large spectrum of microorganisms: bacteria (Agustí et al 2010;Chang et al 2009;Elizaquível et al 2012;Kralik et al 2010;Kramer et al 2009;Takahashi et al 2011), fungi (Andorrà et al 2010;Rawsthorne and Phister 2009;Vesper et al 2008), protozoans (Brescia et al 2009;Fittipaldi et al 2011;Thomas et al 2012), and viruses Parshionikar et al 2010;Sánchez et al 2012). …”

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products

“…Among the resuspension buffers, ¼ Ringer solution with or without cysteine (0,05 %), peptone physiological solution (0.1% wt/vol peptone, 0.85% wt/vol NaCl) or water are used most often (Temmerman et al, 2003;Masco et al, 2005;Masco et al, 2007;Kramer et al, 2009;Bogovic Matijasic et al, 2010). For the preparation of mesophilic cultures for qPCR analysis, which present similar medium as probiotic formulations, Friedrich and Lenke (2006) used PBS and sodium citrate (1% wt/vol).…”