Beyond the billion: dose-response immunophenotyping and machine learning classification of live vs. heat-treated gram-positive probiotic strains in human peripheral blood mononuclear cells—a single-donor proof-of-concept study
- 1Probiotical Research Srl
Abstract
Probiotic research is constrained by three pervasive yet insufficiently challenged assumptions: the requirement for a minimum of 1 billion colony-forming units for efficacy, the necessity for gut colonization, and the inherent superiority of live over heat-treated preparations. This study addresses these gaps through a fully factorial experimental design evaluating 10 Gram-positive probiotic strains in both viable (Active Fluorescent Units, AFU) and heat-treated (Total Fluorescent Units, TFU) forms across three flow cytometry-verified concentrations (10 ⁵ , 10 ⁶ , and 10 ⁷ cells/well per ISO 19344:2015) in primary human peripheral blood mononuclear cells (PBMC) from a single healthy male Caucasian donor (58 years), with simultaneous quantification of 17 cytokines by BioPlex suspension array. Viable preparations induced substantially greater absolute cytokine responses than heat-treated preparations across 16 of 17 analytes, heat-treated preparations demonstrated stronger dose-response correlations (mean within-strain Spearman ρ up to 1.00) for 13 of 17 cytokines, a finding we attribute to the uncontrolled proliferation of live bacteria during 24-h co-culture compressing the effective concentration range. Six of ten viable strains exhibited monotonically increasing profiles; two strains displayed non-monotonic bell-shaped kinetics with peak activity at 10 ⁶ AFU/well and significant attenuation at 10 ⁷ , directly falsifying the assumption that dose escalation uniformly increases immunological activity. MCP-1 was the sole cytokine showing no significant difference between viability states under the paired analysis ( q = 0.98, geometric-mean ratio 0.99, 95% CI 0.89–1.12, excluding any difference beyond approximately 11% in either direction), providing an internal methodological control. In this single-donor model, unsupervised hierarchical clustering identified three immunological phenotype clusters, requiring multi-donor validation before these groupings can be treated as generalizable biological phenotypes, with Random Forest classification achieving 83.3% internal partition-recovery consistency (95% CI 66.4–92.7%; clusters derived from the same data; not an estimate of generalization to novel strains) vs. 33.3% chance; In this single-donor experiment, IL-12p70, IL-13, and IFN-γ, not IL-6 or IL-1β, were the primary discriminators of strain identity; generalizability of this ranking requires multi-donor validation. Heat-treated preparations achieved ≥70% functional equivalence relative to viable preparations at 10 ⁷ TFU/well in 80 of the 140 evaluable strain × analyte combinations, 57.1% of those evaluable (Functional Equivalence Dose, FED ⁷⁰ ), while one strain showed strongly attenuated activity in heat-treated form across all concentrations, a finding subject to the caveat that no positive control stimulus was included to formally verify PBMC functional competence on the experimental day. These findings establish a methodological framework integrating flow cytometric standardization, multiplex immunophenotyping, and machine learning for evidence-based dose characterization, postbiotic functional equivalence assessment, and data-driven strain classification in probiotic research (all p -values are descriptive within a single-donor experimental context).
Related
- Personalized Nutrition for Immune Optimization: A Nutrigenetic Approach to the Peripheral Blood Mononuclear Cell Response
- Single-cell RNA sequencing delineates compositional and transcriptional changes of porcine peripheral blood mononuclear cell populations following Salmonella I 4,[5],12:i:- inoculation
- Novel human long noncoding RNA responses to Candida auris and its cell-wall components in peripheral blood mononuclear cells
- Peripheral blood mononuclear cells as a novel source for mitochondrial transplantation therapy in peripheral nerve injury repair
- Effect of bacteriophages on cytokine production by human peripheral blood mononuclear cells and their subpopulations
BibTeX
@article{deusebio2026beyond,
title = {{Beyond the billion: dose-response immunophenotyping and machine learning classification of live vs. heat-treated gram-positive probiotic strains in human peripheral blood mononuclear cells—a single-donor proof-of-concept study}},
author = {Giovanni Deusebio and Annalisa Visciglia and Angela Amoruso and Marco Pane},
journal = {Frontiers in Microbiology},
year = {2026},
month = oct,
doi = {10.3389/fmicb.2026.1943891},
url = {https://www.frontiersin.org/articles/10.3389/fmicb.2026.1943891/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Beyond the billion: dose-response immunophenotyping and machine learning classification of live vs. heat-treated gram-positive probiotic strains in human peripheral blood mononuclear cells—a single-donor proof-of-concept study
AU - Giovanni Deusebio
AU - Annalisa Visciglia
AU - Angela Amoruso
AU - Marco Pane
PY - 2026
DA - 2026/10/26
JO - Frontiers in Microbiology
DO - 10.3389/fmicb.2026.1943891
UR - https://www.frontiersin.org/articles/10.3389/fmicb.2026.1943891/full
AB - Probiotic research is constrained by three pervasive yet insufficiently challenged assumptions: the requirement for a minimum of 1 billion colony-forming units for efficacy, the necessity for gut colonization, and the inherent superiority of live over heat-treated preparations. This study addresses these gaps through a fully factorial experimental design evaluating 10 Gram-positive probiotic strains in both viable (Active Fluorescent Units, AFU) and heat-treated (Total Fluorescent Units, TFU) forms across three flow cytometry-verified concentrations (10 ⁵ , 10 ⁶ , and 10 ⁷ cells/well per ISO 19344:2015) in primary human peripheral blood mononuclear cells (PBMC) from a single healthy male Caucasian donor (58 years), with simultaneous quantification of 17 cytokines by BioPlex suspension array. Viable preparations induced substantially greater absolute cytokine responses than heat-treated preparations across 16 of 17 analytes, heat-treated preparations demonstrated stronger dose-response correlations (mean within-strain Spearman ρ up to 1.00) for 13 of 17 cytokines, a finding we attribute to the uncontrolled proliferation of live bacteria during 24-h co-culture compressing the effective concentration range. Six of ten viable strains exhibited monotonically increasing profiles; two strains displayed non-monotonic bell-shaped kinetics with peak activity at 10 ⁶ AFU/well and significant attenuation at 10 ⁷ , directly falsifying the assumption that dose escalation uniformly increases immunological activity. MCP-1 was the sole cytokine showing no significant difference between viability states under the paired analysis ( q = 0.98, geometric-mean ratio 0.99, 95% CI 0.89–1.12, excluding any difference beyond approximately 11% in either direction), providing an internal methodological control. In this single-donor model, unsupervised hierarchical clustering identified three immunological phenotype clusters, requiring multi-donor validation before these groupings can be treated as generalizable biological phenotypes, with Random Forest classification achieving 83.3% internal partition-recovery consistency (95% CI 66.4–92.7%; clusters derived from the same data; not an estimate of generalization to novel strains) vs. 33.3% chance; In this single-donor experiment, IL-12p70, IL-13, and IFN-γ, not IL-6 or IL-1β, were the primary discriminators of strain identity; generalizability of this ranking requires multi-donor validation. Heat-treated preparations achieved ≥70% functional equivalence relative to viable preparations at 10 ⁷ TFU/well in 80 of the 140 evaluable strain × analyte combinations, 57.1% of those evaluable (Functional Equivalence Dose, FED ⁷⁰ ), while one strain showed strongly attenuated activity in heat-treated form across all concentrations, a finding subject to the caveat that no positive control stimulus was included to formally verify PBMC functional competence on the experimental day. These findings establish a methodological framework integrating flow cytometric standardization, multiplex immunophenotyping, and machine learning for evidence-based dose characterization, postbiotic functional equivalence assessment, and data-driven strain classification in probiotic research (all p -values are descriptive within a single-donor experimental context).
SN - 1664-302X
ER -