Novel Method for Enumeration of Viable Lactobacillus plantarum WCFS1 Cells after Single-Droplet Drying

Abstract

dSurvival of probiotic bacteria during drying is not trivial. Survival percentages are very specific for each probiotic strain and can be improved by careful selection of drying conditions and proper drying carrier formulation. An experimental approach is presented, comprising a single-droplet drying method and a subsequent novel screening methodology, to assess the microbial viability within single particles. The drying method involves the drying of a single droplet deposited on a flat, hydrophobic surface under well-defined drying conditions and carrier formulations. Semidried or dried particles were subjected to rehydration, fluorescence staining, and live/dead enumeration using fluorescence microscopy. The novel screening methodology provided accurate survival percentages in line with conventional plating enumeration and was evaluated in single-droplet drying experiments with Lactobacillus plantarum WCFS1 as a model probiotic strain. Parameters such as bulk air temperatures and the carrier matrices (glucose, trehalose, and maltodextrin DE 6) were varied. Following the experimental approach, the influence on the viability as a function of the drying history could be monitored. Finally, the applicability of the novel viability assessment was demonstrated for samples obtained from drying experiments at a larger scale. P robiotics are defined as "live microorganisms which when administered in adequate amounts confer a health benefit on the host" (11). Health benefits are usually related to the influence of probiotic bacteria on the microbial balance in the hosts' intestine or via modulation of the gut-associated immune system (6,12,19,21,25). Probiotics are delivered to the gastrointestinal tract as food products or dietary supplements and supplied on the market as fermented food commodities, freeze-dried cultures, or frozen cultures, which enhance their stability and facilitate their implementation in appropriate product formulations (1,16,30). Compared to freeze drying and freezing, spray drying could be an interesting alternative for providing shelf life to probiotic ingredients. Spray drying is more energy and cost efficient and can be operated continuously at higher production capacities (2, 26, 34). The major drawback of spray drying is the limited survival of probiotics (31,33). Several studies have successfully explored approaches to increase the survival percentages after spray drying (8,10,13,23). However, most of the results so far are very specific and difficult to translate between different species. It was, for example, found that a high variability exists between different strains of the same species (14, 24). In addition, the process conditions applied during spray drying (2, 28) and the protective carrier materials added (10, 31) have strong influence on the final viability. Since these parameters need to be optimized for each specific case (different process parameters, different strains or species, and different carrier formulations used during drying), many cost-, time-, and labor-intensive e...

Novel Method for Viability Assessment ofBifidobacterium longumATCC 15707 on Non-dairy Foods during drying

“…The minimum detection limit for this QFM was ∼ 10 7 cells mL -1 for B. longum ATCC 15707 and an ideal cell density between 1 ∼ 3 x 10 8 cells mL -1 gave 50 to 200 cells per image, which is in accordance with the recommended cell density of 10 8 cells mL -1 for Bifidobacterium UCC 35612 (1). A similar study was conducted by measuring viability of L. plantarum WCFS1 in 20 images with 200 to 500 cells per image (2,080 by 1,544 pixels) (19). Another study suggested cell density of 40 – 50 cells per image but the size of the images was not mentioned so comparison of the cell density between different studies is difficult (12).…”

“…An alternative fixation method has been reported by other, bioadhesive slides (Excell AdhesionTM, Fisher Scientific) which showed good cell trapping, but compounds present on the slides caused mortality or affected permeability of the cell membrane, thus inducing mortality of some pure cultures and river water microbes (5). Recently, a low-melting point agarose gel supplemented with Bac Light stains was used to rehydrate and determine viability of dried cell particles deposited on Anopore chips (19). However, Anopore chips are expensive for routine determination of live or dead cells in a product.…”

Підбір Молочнокислих Бактерій, Ізольованих З Природних Еконіш, Для Виготовлення Кисловершкового Масла Геродієтичного Призначення

“…Цей штам є пробіотичним, він детально вивчений і описаний за послідовністю цілого геному. Штам є представником мікробіоценозу людини, підтверджено його вплив на формування імуностатусу організму (Perdana et al, 2012;Bron et al, 2006).…”

Pulsed electric field pre-treatment for drying of living bacteria

“…Approximately 20 images were captured per sample at a magnification of 630x using an Olympus XC30 camera (Olympus, Japan) and CellSens imaging software (Olympus, Japan). Image analysis was performed using a Matlab script to discriminate between red and green cells, as was described earlier by Perdana et al (2012).…”

“…Carrier materials with a high glass transition temperature (Tg) are expected to provide a glassy matrix earlier during droplet drying or soon after drying upon cooling (Perdana et al, 2014b(Perdana et al, , 2012. We estimated the Tg as function of moisture content for every component studied in this work using the Couchman-Karasz theory (Fig.…”