Assessment of shelf-life and metabolic viability of a multi-strain synbiotic using standard and innovative enumeration technologies (original) (raw)
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Frontiers in Microbiology
Probiotics are typically enumerated by agar plate counting (PC) techniques. PC has several limitations including poor specificity, high variability, inability to enumerate dead cells, viable but non-culturable cells and cells in complex matrices. Viability droplet digital polymerase chain reaction (v-ddPCR) is an emerging enumeration technique with improved specificity, precision, and the ability to enumerate cells in varying states of culturability or in complex matrices. Good correlation and agreement between v-ddPCR and PC is well documented, but not much research has been published on the comparison when enumerating freeze-dried (FD) probiotics during storage. In this study, v-ddPCR utilizing PE51 (PE51-ddPCR), a combination of propidium monoazide (PMA) and ethidium monoazide (EMA), was evaluated as alternative enumeration technique to PC on blends of four FD probiotic strains over the course of a 3-month storage study with accelerated conditions. When PMA and EMA are combined (...
Frontiers in Microbiology, 2024
This study introduces an optimized integration of flow cytometry and fluorescence in situ hybridization (Flow-FISH) as an approach for the specific enumeration of gram-positive bacteria in probiotic products, overcoming the limitations of conventional methods. The enhanced Flow-FISH technique synergizes the rapid and automated capabilities of flow cytometry with the high specificity of FISH, facilitating the differentiation of viable cells at the species level within probiotic blends. By analyzing lyophilized samples of Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, and Bifidobacterium animalis subsp. lactis, and a commercial product, the study highlights the optimized Flow-FISH protocol's advantages, including reduced hybridization times to 1.5 h and elimination of centrifugation steps. Comparative evaluations with the widely accepted enumeration methods plate count and Live/Dead (L/D) staining were conducted. The study revealed that Flow-FISH produces higher viable cell counts than plate count, thereby challenging the traditional "gold standard" by highlighting its predisposition to underestimate actual viable cell numbers. Against L/D staining, Flow-FISH achieved comparable results, which, despite the different foundational premises of each technique, confirms the accuracy and reliability of our method. In conclusion, the optimized Flow-FISH protocol represents a significant leap forward in probiotic research and quality control. This method provides a rapid, robust, and highly specific alternative for the enumeration of probiotic bacteria, surpassing traditional methodologies. Its ability to enable a more detailed and reliable analysis of probiotic products paves the way for precise quality control and research insights, underscoring its potential to improve the field significantly.
Journal of Microbiological Methods, 2014
Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. Standard culture techniques are commonly used to quantify probiotic strains, but cell culture only measures replicating cells. In response to the stresses of processing and formulation, some fraction of the live probiotic microbes may enter a viable but non-culturable state (VBNC) in which they are dormant but metabolically active. These microbes are capable of replicating once acclimated to a more hospitable host environment. An operating definition of live probiotic bacteria that includes this range of metabolic states is needed for reliable enumeration. Alternative methods, such as fluorescent in situ hybridization (FISH), nucleic acid amplification techniques such as real-time quantitative PCR (RT-qPCR or qPCR), reverse transcriptase (RT-PCR), propidium monoazide-PCR, and cell sorting techniques such as flow cytometry (FC)/fluorescent activated cell sorting (FACS) offer the potential to enumerate both culturable and VBNC bacteria. Modern cell sorting techniques have the power to determine probiotic strain abundance and metabolic activity with rapid throughput. Techniques such as visual imaging, cell culture, and cell sorting, could be used in combination to quantify the proportion of viable microbes in various metabolic states. Consensus on an operational definition of viability and systematic efforts to validate these alternative techniques ultimately will strengthen the accuracy and reliability of probiotic strain enumeration.
Improving and Comparing Probiotic Plate Count Methods by Analytical Procedure Lifecycle Management
Frontiers in Microbiology, 2021
Probiotics are live microorganisms that confer a health benefit to the host when administered in adequate amounts. This definition links probiotic efficacy to microbial viability. The current gold standard assay for probiotic potency is enumeration using classical microbiology plating-based procedures, yielding results in colony-forming units (CFU). One drawback to plating-based procedures is high variability due to intrinsic and extrinsic uncertainties. These uncertainties make comparison between analytical procedures challenging. In this article, we provide tools to reduce measurement uncertainty and strengthen the reliability of probiotic enumerations by using analytical procedure lifecycle management (APLM). APLM is a tool that uses a step-by-step process to define procedure performance based on the concept that the reportable value (final CFU result) must be fit for its intended use. Once the procedure performance is defined, the information gathered through APLM can be used to...
International Dairy Journal, 2019
Probiotic products are becoming increasingly popular worldwide. Regulators and quality control personnel need a clear path to count viable cells within each product. We have reviewed progress on the enumeration of probiotics in foods and supplements. Today, no single culture medium or combination of culture media can accurately enumerate all probiotics available. Culture-independent techniques can speed the counting process compared with traditional agar plating, but their sophistication makes them unsuitable for routine quality control in many laboratories. The enumeration of probiotics in a given product needs to be tailor-made: the response of each probiotic and starter culture used in the food or supplement to a narrow set of culture media should be assessed first to identify the proper conditions for an accurate enumeration. A standardised procedure should be developed in the near future with the consensus of the scientific, industrial and regulator communities.
Applied and Environmental Microbiology, 2002
Flow cytometry (FCM) is a rapid and sensitive technique that can determine cell numbers and measure various physiological characteristics of individual cells by using appropriate fluorescent probes. Previously, we developed an FCM assay with the viability probes carboxyfluorescein diacetate (cFDA) and TOTO-1 {1′-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3dihydro(benzo-1,3-oxazole)-2-methylidene]-1-(3′-trimethylammoniumpropyl)-pyridinium tetraiodide} for (stressed) lactic acid bacteria (C. J. Bunthof, K. Bloemen, P. Breeuwer, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 67: 2326-2335, 2001). cFDA stains intact cells with enzymatic activity, and TOTO-1 stains membrane-permeabilized cells. Here we used this assay to study the viability of bacterial suspensions in milk, dairy fermentation starters, and probiotic products. To facilitate FCM analysis of bacteria in milk, a commercially available milk-clearing solution was used. The procedure was optimized ...
Flow cytometry as an auxiliary tool for the selection of probiotic bacteria
Beneficial Microbes, 2015
Selection of appropriate bacterial strains is crucial for development of new probiotic preparations. The fundamental prerequisite for potential efficacy of a probiotic preparation for oral application is the selection of appropriate bacterial strains with good gastrointestinal colonisation abilities, antimicrobial activity, and tolerance of conditions in the gastrointestinal tract, resistance to different antimicrobial agents, survival during processing and storage. The strain should be genetically stable, it should have good growth properties, to maintain its high viability at processing and when in storage. Mostly, the properties of promising strains are tested in the first phase in vitro, and only the best ones undergo subsequent in vivo testing. in vitro tests are often performed by classical microbiological cultivation methods which are material and time consuming, and they are not able to distinguish between ‘viable but nonculturable’ and dead bacteria. Flow cytometry is usual...
Frontiers in Microbiology, 2020
Improvements offered by viability droplet digital PCR (v-ddPCR) include increased precision, specificity and decreased time to results making for an attractive alternative method to traditional plate count enumeration of probiotic products. A major hurdle faced in v-ddPCR, however, is distinguishing between live and dead cells. The objective of this study was to evaluate a combination of PMA and EMA (PE51) for viability treatment of freeze-dried probiotic powders. Lactobacillus acidophilus La-14 and Bifidobacterium animalis subsp. lactis Bi-07 were analyzed over a 2-log PE51 concentration gradient to investigate the efficiency across genus and assay targets. Results suggest a need to optimize viability dye concentration based on the genera of the organism, but also the assay target, even when analyzing the same organism. When optimized for PE51 concentration, strain specific v-ddPCR assays for both La-14 and Bi-07 were demonstrated to agree with plate count enumeration results. In conclusion, while these v-ddPCR assays require highly specific optimization, they are better suited for the future of the probiotic industry and are suggested to be implemented in probiotic product testing.
Foods
The flow cytometry method (FCM) is a widely renowned practice increasingly used to assess the microbial viability of probiotic products. Additionally, the measurement of water activity (aw) can be used to confirm the presence of viable cells in probiotic products throughout their shelf lives. The aim of this study was to investigate the correlation between changes in aw and variations in active fluorescent units (AFU), a unit commonly used in flow cytometry method, during the aging of probiotic products containing freeze-dried bacteria. We controlled the stability of probiotic products for bacterial counts (using ISO 19344 method) and aw levels in commercially available capsules containing freeze-dried bacteria such as Lactobacillus sp. or combinations of Lactobacillus sp. and Bifidobacterium sp. in standard conditions (25 ± 2 °C and 60% relative humidity) over a period of 24 months. During this time, the bacterial contents decreased by 0.12 Log10 in the single-strain product, by 0....