Nonculturable forms of bacteria in lyophilized probiotic preparations

Abstract

Background: Nonculturable cells are formed under stress. These viable but nonculturable (VBNC) cells retain the ability to revert to active growth and division when conditions become favorable, or after treatment with resuscitating factors. Information about the possible presence of VBNC in bacterial lyophilized probiotic preparations, foodstuffs, live vaccines, etc., indicates that human as well as animal intestines are a significant area for research.Methods: Samples were stored for different periods of time (up to 30 years) according to the manufacturers' manuals. Total counts were conducted using the Goryaev-Thoma counting chamber and actual viability was assessed by luminescence microscopy after staining with Live/Dead® (Baclight™). CFU/ml counts were made using solid or semisolid media. Viable cells that lacked the ability to form colonies were considered VBNC. Results:We studied 11 batches of commercial probiotics (Russia) from different sources, containing lyophilized E. coli, lactobacilli, or bifidobacteria, in ampoules or vials. In E. coli preparations, depending on storage periods, the amounts of VBNC varied from 4.1% (3 years) to 99.7% (30 years) and showed different total viability (52.2 -91.3%), as well as the percentage of VBNC cells. A different sample that had been expired for 11 years was 79.5% NC. It is also noteworthy that the 5-dose vials, 4 years past expiration, from yet another source, showed a higher amount of VBNC cells (85.5%). Two different batches that had been expired for three years contained 4.1 and 21.3% VBNC cells. 4 of the 5-dose vials of lyophilized lactobacilli were not expired and contained 58.8 -80.4% VBNC cells. Total viability varied from 92.9 to 100%, and there was an unmistakable positive correlation between total viability and culturability. The last batch, which had expired 6 years earlier, has 23.7% viable cells and about 98% VBNC. Nonexpired bifidobacterial samples contained 70.7 and 95.5% of viable cells and were 50 and 100% culturable. Conclusion:We demonstrated the presence of VBNC cells in lyophilized probiotic preparations

Assessment of shelf-life and metabolic viability of a multi-strain synbiotic using standard and innovative enumeration technologies

“…Accordingly, the hypothesis risen is that AFU cells are likely representative of the VBNC and/or dormant population plus the subgroup of cultivable cells (CFU). Consequently, VBNCs may constitute a significant portion of live cells that are consumed by the end user with potential health benefits (Blinkova et al, 2014). In this paper, we strengthen the combined use of PC and FC enumeration adding pH acidification as third pillar for monitoring functional stability (i.e., the capability of the product to acidify over time), in order to evaluate if cellular metabolism was more preferentially descriptive of FC or PC enumeration data.…”

Microbiological Testing of Probiotic Preparations

“…These are a form of resting bacteria which can survive in unfavourable environmental conditions [67]. The VBNC cells are characterised by lack of growth on culture media but preserve cell integrity and metabolic activity [48,67,68]. The factors that may trigger the conversion of bacterial cells to the VBNC state may be, for example, inadequate acidity or osmolality of the environment, temperature changes, or a deficiency of certain nutrients [68].…”

Detection of Viable but Nonculturable Microbial Cells in Chicken Mince

“…The environment, in which many microorganisms exist, can stimulate their growth, or on the contrary, limit it or contribute to the cessation of cell reproduction. The latter process is most often associated with emerging stress due to non-optimal values of temperature (Chaisowwong et al, 2012) and pH (Cunningham et al, 2009), osmotic pressure (Asakura et al, 2002), oxygen concentration and other gases (Oh et al, 2015), UV and radiation (Zhang et al, 2015), pulsed light and pulsed energy fields (Kramer, B., & Muranyi, P., 2014), lyophilization (Blinkova et al, 2014), antibiotics (Hu, & Coates 2012), chemical preservatives, disinfectants, metal ions, nutrient deficiencies (Zhao et al, 2017), etc.…”