Germination is the process by which the dormant spore form of a bacteria become active and develop. Once germinated, the Bacillus organism can grow, produce metabolites and benefit gut health and performance of poultry.
Germination is essential for the full expression a probiotic’s potential.
The capacity of most Bacillus strains to germinate and grow in vivo is unknown. Due to the complexity of the germination process especially in a very complex environment, namely the digestive lumen content, in vivo confirmation of germination remains a challenging task.
Adisseo, an expert in animal sciences, and Novozymes, a leader in biological solutions, have demonstrated and visualized the germination of their Alterion® technology in detail – scientifically proving their probiotic is an effective and consistent solution for poultry.
The partnership has used a variety of methods to carry out an in-depth investigation of the germination and growth of Alterion®. These include – in vitro time course, fluorescent technologies and metabolite production in vivo.
Alterion®’s germination is proven in vitro and more importantly, visualized in vivo. Alterion® is the only probiotic available on the market where germination has been visualized inside birds!
The Alterion® strain has been selected based on its superior germination capabilities in both gut-like and feed conditions.
These photos show germination of Alterion® spores at 0, 4 and 6 hours.
Visualization of in vitro germination and growth of Alterion®
The germination kinetics of Alterion® was compared in vitro with other Bacillus strains, under similar conditions.
This graph clearly demonstrates the superior germination of Alterion® and the wide variation in germination kinetics; between the other Bacillus subtilis strain and a Bacillus licheniformis.
Visualization of in vivo germination
Observation of Alterion®’s germination has been made possible by the development of a unique method. The innovative fluorescent dual-reporter system tracks the germination process within the bird’s gut:
* Fluorescent markers are integrated into the Alterion® genome
* One gene encodes Green Fluorescent Protein (GFP) - expressing a green signal for a dormant spore
* Another encodes Red Fluorescent Protein (dsRed) - delivering a red signal after germination and growth
* Once the variant was created, fluorescence was verified by microscopy analysis.
In-vitro fluorescence study
These photos show the fluorescence of Alterion® spores and vegetative cells observed by microscopy at three time points
A- At t0, the Alterion® dormant spores fluoresce green
B- Upon germination (t1) , green fluorescence rapidly disappears
C- At t2, the germinated cells fluoresce red
Visualization of germination in the intestine of a chicken
To verify the germination of Alterion® in vivo conditions, broiler chicks were either fed a control or a treatment diet to which dual-labelled Alterion® reporter spores were added.
Samples of the ileal contents were analyzed and processed via InCell Analyzer to check fluorescence in the samples.
In these photos, the predominant red colour in the Alterion® group indicates that most spores have germinated. This active growth will support health effects in broiler chickens.
In vivo metabolite production
A complementary approach to investigate the in vivo activity of Alterion® after germination and growth is to monitor its production of metabolites.
Alterion®’s metabolites were first studied in vitro and three metabolites were identified as produced in high and consistent quantities (niacin, pantothenate, hypoxanthin). The concentration of those metabolites was then measured in digestive contents of birds receiving Alterion®.
The study clearly showed that the concentration of the metabolites was strongly related to the number of Bacilli detected.
Clearly, these data support the fact that Alterion® is active within the gut of the birds.
The metabolic activity of Alterion® in the gut supports its beneficial effects on host performance, health and microbiota (Jacquier et al. 2019). Once germinated and active, Alterion® sustains animal resilience acting through three intimately connected lines of defence:
The unique features of the Bacillus strain enable the timely germination Alterion®, which becomes metabolically active where it matters most – in the animal’s gut. Here it produces metabolites and creates the three lines of defence via the microbiota, the gut mucosa and inflammatory responses to sustain animal resilience.
This science proven probiotic provides significant and consistent improvements in gut health, growth rate and feed utilization.
Thereby saving resources and improving the sustainability of animal production.