Strain-specific quantification of probiotic Bacillus subtilis in feed by imaging high-performance thin-layer...

Area of Science:

• Microbiology
• Analytical Chemistry
• Animal Science

Background:

• European Union ban on antibiotic growth promoters increases reliance on probiotics in animal feed.
• Rising use of probiotics necessitates robust quality control systems for accurate quantification in feed.
• Existing methods struggle to differentiate specific probiotic strains from similar bacteria.

Purpose of the Study:

• To develop and validate a strain-specific quantification method for the probiotic Bacillus subtilis DSM 29784 in animal feed.
• To identify a unique metabolite produced by the target probiotic strain for accurate detection.
• To establish a reliable quality control system for probiotic feed additives.

Main Methods:

• Utilized a two-step cultivation procedure to induce the production of a strain-specific metabolite.
• Employed hyphenated chromatography techniques: normal-phase HPTLC coupled with RP-HPLC-DAD and HR-MS for metabolite identification.
• Developed a quantification method based on the identified strain-specific metabolite and validated its performance.

Main Results:

• Identified a strain-specific metabolite from Bacillus subtilis DSM 29784 with antimicrobial properties, indicating probiotic activity.
• Preliminary molecular formula assignment of the metabolite: C35H44N6O2 (580.3527 Da).
• The developed HPTLC-based method demonstrated good selectivity, linearity, detection limit, recovery, and precision for quantifying probiotics in feed.

Conclusions:

• A novel, validated method enables accurate quantification of Bacillus subtilis DSM 29784 in animal feed using a strain-specific metabolite.
• This quality control approach supports the effective and reliable use of probiotics in animal production.
• The method addresses the need for precise probiotic enumeration following regulatory changes in feed additives.