Whole-Genome Sequence and Interaction Analysis in the Production of Six Enzymes From the Three Bacillus Strains Present in a Commercial Direct-Fed Microbial (Norum™) Using a Bliss Independence Test

Abstract

The three Bacillus strains present in Norum™ were initially selected by their excellent to good relative enzyme activity (REA) production score for amylase, protease, lipase, phytase, cellulase, β-glucanase, and xylanase. Further studies confirmed that the three isolates also showed an antibacterial activity, Gram-positive and Gram-negative poultry pathogens. Norum™ (Eco-Bio/Euxxis Bioscience LLC) is a Bacillus spore direct-fed microbial (DFM). The Bacillus isolates were screened and selected based on in vitro enzyme production profiles. Moreover, in chickens fed high non-starch polysaccharides, this DFM demonstrated to reduce digesta viscosity, bacterial translocation, increase performance, bone mineralization, and balance the intestinal microbiota. In the present study, we present the whole-genome sequence of each of the three isolates in Norum™, as well as the synergistic, additive, or antagonistic effects on the enzyme production behavior of the three Bacillus strains and their combinations when grown together vs. when grown individually. The whole-genome sequence identified isolate AM1002 as Bacillus subtilis (isolate 1), isolate AM0938 as Bacillus amyloliquefaciens (isolate 2), and isolate JD17 as Bacillus licheniformis (isolate 3). The three Bacillus isolates used in the present study produce different enzymes (xylanase, cellulase, phytase, lipase, protease, and β-glucanase). However, this production was modified when two or more Bacillus strains were combined, suggesting possible synergistic, antagonistic, or additive interactions. The Bliss analysis suggested (p < 0.05) that the combination of Bacillus strains 1–2 and 1–2–3 had intermediate effects and predicted that the combination of Bacillus strains 2–3 could have better effects than the combination of all the three Bacillus strains. In summary, the current study demonstrated the need of selecting Bacillus strains based on quantitative enzyme determination and data analysis to assess the impacts of combinations to avoid antagonistic interactions that could limit treatment efficacy. These results suggest that using Bacillus strains 2–3 together could lead to a new generation of DFMs with effects superior to those already examined in Bacillus strains 1–2–3 and, therefore, a potential alternative to growth-promoting antibiotics. More research utilizing poultry models is being considered to confirm and expand the existing findings.

Probiotic-Fermented Distillers Grain Alters the Rumen Microbiome, Metabolome, and Enzyme Activity, Enhancing the Immune Status of Finishing Cattle

“…The microbiota produces various digestive enzymes, such as protease, lipase, and cellulase [74]. We analyzed the rumen digestive enzymes and found that the activities of protease, lipase, and cellulase in the rumen fluid of finishing cattle were significantly increased after feeding with PFDG, which was consistent with the results of previous studies showing that feeding with probiotics and probiotic fermentation products can promote the animal digestive enzyme activity and improve the efficiency of the digestion and absorption of nutrients [75,76].…”

Probiotics as Alternatives to Antibiotics for the Prevention and Control of Necrotic Enteritis in Chickens

“…The authors found that combination of B. amyloliquefaciens and B. licheniformis had more beneficial effects related to their enzyme activity and antibacterial ability when compared all three strains combined. These observations suggested that that while the above mentioned bacterial species are beneficial, additional research is needed to use mixtures of probiotic bacterial species to evaluate their combined synergistic effects to maximize gut health as well as antimicrobial benefits in chickens [81].…”

Effect of fenugreek seeds and Bacillus-based direct-fed microbials on the growth performance, blood biochemicals, and intestinal histomorphology of broiler chickens

“…Flavonoid glycosides are natural compounds that have antioxidant and anti-inflammatory properties, but they can also induce oxidative stress and organ damage when ingested in excess. The Bacillus -based DFM used in this study, Norum™ (Eco Bio/Euxxis Bioscience LLC, Fayetteville, AR), is a spore-based DFM culture consisting of three Bacillus strains: Bacillus subtilis , B. amyloliquefaciens , and B. licheniformis with a stable Bacillus spore content of ~3 × 10 11 spores/g ( 22 ). These Bacillus strains can produce various enzymes (xylanase, cellulase, phytase, lipase, protease, and β-glucanase) that can improve the digestibility and nutrient availability of the feed, as well as modulate the intestinal microbiota and enhance the gut health and immunity of poultry ( 31 , 32 ).…”

“…Norum™ was sourced from Eco-Bio/Euxxis Bioscience LLC and added to feed at 0.1 g/kg for experimental groups. The Bacillus strains within Norum™ were identified through whole-genome sequencing as Bacillus subtilis (AM1002), Bacillus amyloliquefaciens (AM0938), and Bacillus licheniformis (JD17) ( 22 ).…”