ABSTRACT
CONTEXT: Coated whey protein micro-beads may improve probiotic protection and provide delayed cell-release mechanisms. OBJECTIVE: Lactobacillus rhamnosus GG was encapsulated in whey protein micro-beads by droplet extrusion with coating via electrostatic deposition: primary-polysaccharide and secondary-whey protein. MATERIALS AND METHODS: Storage studies were performed in cranberry and pomegranate juice (pH 2.4; 28 days; 4 and 25°C) followed by simulated ex vivo porcine gastric (pH 1.6) and intestinal (pH 6.6) digestion. RESULTS AND DISCUSSION: After storage and simulated gastro-intestinal digestion, free cells, cells suspended in protein and cells encapsulated in alginate micro-beads, illustrated complete probiotic mortality, while coated micro-beads enhanced probiotic viability after juice storage (8.6 ± 0.1 log(10)CFUmL(-1)). Beads also showed significant binding of hydrophobic molecules. Coated micro-beads illustrated high gastric survival (9.5 ± 0.1 log(10)CFUmL(-1)) with 30 min delayed intestinal release relative to non-coated micro-beads. CONCLUSIONS: Micro-bead coatings could be applied in delayed cell-release for targeted intestinal probiotic delivery.
Subject(s)
Beverages , Drug Compounding , Food Preservation , Fruit , Gastric Mucosa/metabolism , Milk Proteins , Probiotics , Animals , Digestion , Electrophoresis, Polyacrylamide Gel , Flow Cytometry , Spectroscopy, Fourier Transform Infrared , Swine , Whey ProteinsABSTRACT
The aim of this study was to demonstrate that flow cytometry (FACS) could potentially be employed for rapid viability assessment of probiotic bacteria immobilized or encapsulated in complex matrices. Lactobacillus rhamnosus GG was immobilized within six different protein environments using whey protein isolate (WPI) and yoghurt matrices and encapsulated within protein micro-beads, all of which ranged in structural complexity. Following a series of environmental-stress trials, survival of the strain was examined using FACS compared to traditional plate count techniques. Cell extraction and digestive pre-treatments were designed to release cells and reduce the protein background, respectively, which represent compositional obstacles for efficient FACS analysis. Physico-chemical properties of protein-probiotic components revealed the mechanism necessary for efficient cell delivery during FACS analysis. This assay required 40 min sample preparation and distinct functional populations were discriminated based on fluorescent properties of thiazole orange (TO) and propidium iodide (PI). This assay yielded 45-50 samples/h, a detection range of 10(2)-10(10)cfu/ml of homogenate and generated correlation coefficients (r) of 0.95, 0.92 and 0.93 in relation to standard plate counts during heat, acid and storage trials, respectively. In conclusion, this methodology provides impetus for dynamic progression of FACS for rapid viability assessment of live bacteria immobilized/encapsulated within complex protein systems.
Subject(s)
Flow Cytometry/methods , Lacticaseibacillus rhamnosus/chemistry , Microbial Viability , Milk Proteins/chemistry , Probiotics/chemistry , Staining and Labeling/methods , Cells, Immobilized/chemistry , Cells, Immobilized/physiology , Lacticaseibacillus rhamnosus/cytology , Lacticaseibacillus rhamnosus/isolation & purification , Probiotics/isolation & purification , Whey Proteins , Yogurt/microbiologyABSTRACT
This study investigated cell immobilization of Lactobacillus rhamnosus GG in three separate protein products: native, denatured and hydrolysed whey protein isolate (WPI). Treatments were assessed for their ability to enhance probiotic survival during storage, heat stress and ex vivo gastric incubation. Spatial distribution of probiotic cells within immobilized treatments was evaluated by atomic force and confocal scanning laser microscopy, while cell viability was enumerated by plate count and flow cytometry (FACS). Microscopic analysis of denatured treatments revealed an oasis of immobilized cells, phase-separated from the surrounding protein matrix; an environmental characteristic analogous to hydrolysed networks. Cell immobilization in hydrolysed and denatured WPI enhanced survival by 6.1+/-0.1 and 5.8+/-0.1 log10 cycles, respectively, following 14 day storage at 37 degrees C and both treatments generated thermal protection at 57 degrees C (7.3+/-0.1 and 6.5+/-0.1 log(10) cfu/ml). Furthermore, denatured WPI enhanced probiotic protection (8.9+/-0.2 log(10) cfu/ml) following 3h gastric incubation at 37 degrees C. In conclusion, hydrolysed or denatured WPI were the most suitable matrices for cell immobilization, while native protein provided the weakest safeguard against thermal and acid stress, thus making it possible to envision whey protein gel networks as protective substrates for cell immobilization applications.
