{
  "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).",
  "abstract_shown": true,
  "affiliations": [
    "Probiotical Research Srl"
  ],
  "article_type": null,
  "arxiv": null,
  "author_total": 4,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Giovanni Deusebio"
    },
    {
      "affs": [
        0
      ],
      "name": "Annalisa Visciglia"
    },
    {
      "affs": [
        0
      ],
      "name": "Angela Amoruso"
    },
    {
      "affs": [
        0
      ],
      "name": "Marco Pane"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/184dz404c3gfd.bib",
  "cited_by": 0,
  "doi": "10.3389/fmicb.2026.1943891",
  "fulltext_url": "https://www.frontiersin.org/articles/10.3389/fmicb.2026.1943891/full",
  "id": "184dz404c3gfd",
  "issns": [
    "1664-302X"
  ],
  "landing_url": "https://www.frontiersin.org/articles/10.3389/fmicb.2026.1943891/full",
  "language": "en",
  "licence": "CC-BY-4.0",
  "licence_label": "CC BY 4.0",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "lcc",
      "QR1-502"
    ],
    [
      "openalex",
      "1106|Probiotics and Fermented Foods"
    ]
  ],
  "openalex": "W7223702140",
  "pdf_url": "https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2026.1943891/pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-26",
  "publisher": "Frontiers Media SA",
  "ris": "https://research.draconitic.com/paper/184dz404c3gfd.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:19:12Z",
      "native_id": "10.3389/fmicb.2026.1943891",
      "source": "crossref",
      "url": "https://www.frontiersin.org/articles/10.3389/fmicb.2026.1943891/full"
    },
    {
      "first_seen": "2026-10-30T13:47:29Z",
      "native_id": "47abdbe0047146d283b6017fac2845fe",
      "source": "doaj",
      "url": "https://doi.org/10.3389/fmicb.2026.1943891"
    }
  ],
  "subjects": [
    {
      "field": "biology",
      "origin": "lcc:QR1-502",
      "subfield": "microbiology"
    }
  ],
  "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",
  "title_text": "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",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T13:47:29Z",
  "url": "https://research.draconitic.com/paper/184dz404c3gfd",
  "venue": "Frontiers in Microbiology",
  "versions": []
}