95%+ Survival Through Simulated GI Conditions — By Design, Not by Chance

Full Genomic Safety Screening and Strain-Specific Prebiotic Matching for the JBD Probiotic Family

Taxonomy

L. reuteri JBD301, L. plantarum JBD302 and L. paracasei JB305 were genetically classified as the respective species by high quality 16S rRNA gene sequencing. Additionally, whole genome sequencing was conducted combining state of the art short and long read sequencing techniques followed by comparative genomics, confirming the species of the three JBD strains.

Acid/bile tolerance

The generally accepted definition of probiotics is "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host".34 While not explicitly stated, this definition implies that a probiotic should survive passage through the GI tract.35 The most important trait for surviving GI tract passage, is tolerance to the highly acidic conditions present in the stomach and to the concentrations of bile salts found in the small intestine.

Internal in vitro acid and bile tolerance testing was conducted to characterize the rate of survival of L. reuteri JBD301, L. plantarum JBD302 and L. paracasei JB305 through the GI tract. The three JBD strains demonstrate exceptional acid tolerance (≥95% viability after exposure to HCl at pH3 for 30 min and to 0.3% pepsin at pH2 for 30 min at 37°C) as well as outstanding bile tolerance (≥95% viability in medium containing 0.3% bile salts for 48 hours at 37°C).

Safety aspects

All JBD strains were evaluated for genetic safety using whole genome sequencing. The genome of L. reuteri JBD301, L. plantarum JBD302 and L. paracasei JB305 was analyzed for antibiotic resistances, virulence factors, and biogenic amines; according to the criteria proposed by the European Food Safety Authority (EFSA statement on the requirements for whole genome sequence analysis of microorganisms intentionally used in the food chain, 2021).

The analysis confirmed the safety of the strains as no genes for antibiotic resistances, virulence factors, or biogenic amines such as histamine, were detected.

Because some species of bacteria can produce hemolysins, substances that cause the destruction of red blood cells, in vitro testing on blood agar was conducted to verify the absence of harmful hemolytic activity of the strain. No virulent β-hemolysis36 was observed, indicating phenotypically that the three JBD strains do not possess the ability to lyse red blood cells. Although JBD302 and JBD305 grown on blood agar showed α-hemolysis, α-hemolysis does not indicate harmful destruction of red blood cell membranes.37

Fermentation profile of different prebiotics

Optimizing the growth and metabolic activity of JBD strains in the intestinal environment represents an additional strategy for enhancing their health benefits. Probiotic bacteria can produce beneficial metabolites such as lactate and short-chain fatty acids (SCFAs) including acetate, which contribute to intestinal health through pH reduction, pathogen inhibition, and cross-feeding of other commensal bacteria.38,39 To identify optimal prebiotic substrates for supporting JBD strains' growth and metabolite production, we conducted a comprehensive in vitro fermentation screening, cultivating the JBD strains with 4 distinct prebiotics, namely inulin, resistant starch, partially hydrolyzed guar gum (PHGG), and galactooligosaccharides (GOS) and measured the lactate and SCFA concentrations in the culture supernatants at the late logarithmic growth phase of the bacteria.

The data revealed distinct substrate preferences and metabolic profiles across the three JBD strains:

  • L. reuteri JBD301 exhibited poor growth on resistant starch and robust proliferation with PHGG. Lactate production was highest with inulin and PHGG, while acetate generation was maximized with GOS supplementation.
  • L. plantarum JBD302 demonstrated optimal growth with GOS and PHGG, with moderate performance on inulin. Both lactate and acetate production followed similar patterns, with GOS yielding slightly superior metabolite levels.
  • L. paracasei JBD305 showed strongest growth on inulin, though PHGG and GOS more effectively stimulated both acetate and lactate production, indicating a dissociation between biomass accumulation and metabolic output.

These findings suggest that GOS and PHGG represent the most suitable prebiotic candidates for a synbiotic formulation aimed at maximizing both, the colonization potential and the metabolic functionality of the JBD probiotic family. The observed strain-specific variations in substrate utilization also emphasize the potential for tailored prebiotic selection to optimize specific metabolic outcomes.

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Innobiotic

Innobiotic

26 Oct 2026

3 min read

Sources

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