Optimizing Fermentation-Derived Probiotics in Cambodian Fish Sauce

Thesis Abstract

This study addresses the quality, safety, and functional potential of fermentation-derived probiotics integrated into Cambodian fish sauce (nuoc mam) to enhance dietary probiotic intake and product value. The problem stems from limited standardization of fermentation practices, inconsistent probiotic communities, and a lack of robust evidence linking probiotic viability and functional traits to sensory quality and shelf stability in traditional fish sauce systems. The aim is to optimize probiotic production, viability, and activity during fermentation and storage, while maintaining organoleptic acceptability and regulatory compliance. Specific objectives are (1) to characterize baseline microbial communities and probiotic candidates in artisanal and semi-industrial Cambodian fish sauces; (2) to optimize fermentation parameters (temperature, salinity, salt-to-protein ratio, and starter culture composition) to maximize viable probiotic counts (log CFU/mL) and functional properties (gas production, acidification rate, bile and acid tolerance) over a 180-day shelf period; (3) to evaluate sensory profiles and consumer acceptability of optimized formulations; (4) to quantify probiotic transfer and persistence in a simulated meal context; and (5) to model relationships between process variables, probiotic viability, and sensory quality using multivariate analyses. The study adopts a mixed-methods design combining experimental fermentation trials with sensory evaluation and statistical modeling. A laboratory experimental framework based on an explanatory sequential design will be employed. The population comprises Cambodian fish sauce producers and consumer panels, with samples collected from five representative production sites across Kampot, Preah Sihanouk, and Kampong Cham provinces. A factorial experimental design investigates three starter cultures (Lactobacillus plantarum, Halomonas sp., and Pediococcus pentosaceus) in combination with gradients of temperature (25–35°C), salinity (10–25% w/w NaCl), and protein-to-salt ratios (11 to 13). Primary data consist of probiotic viability (log CFU/mL measured by plate counts on de Man, Rogosa and Sharpe agar), community profiling (16S rRNA amplicon sequencing), and functional assays (bile salt tolerance, gastric tolerance, auto-aggregation, and inhibition of common spoilage organisms). Secondary data include physicochemical parameters (pH, salt content, amino nitrogen), and sensory attributes (descriptive analysis by a trained panel, 9-point hedonic scale). Instruments include a high-accuracy incubator shaker, salinity-controlled fermentation vessels, qPCR for rapid strain verification, and Illumina MiSeq for amplicon sequencing. Data collection spans three fermentation runs with triplicate batches per treatment, plus a 180-day shelf study with sampling at 0, 30, 90, and 180 days. Validity and reliability are addressed through calibration of microbial assays, standard operating procedures, and cross-validation of sequencing data with culture-based counts. Statistical analyses will utilize ANOVA and factorial regression to assess main and interaction effects on probiotic viability and functional traits; principal component analysis (PCA) will summarize multivariate sensory and physicochemical data; regression tree models will identify key predictors of shelf stability. A generalized linear model will evaluate perceived acceptability as a function of probiotic load and sensory descriptors. Theoretical framing integrates the Food Safety and Quality Assurance theory and the Probiotic Functionality framework, with explicit reference to the Theory of Planned Behavior to interpret consumer acceptance outcomes. Ethical considerations follow established guidelines for human sensory testing, including informed consent and data confidentiality. Expected findings include identification of optimal starter-culture combinations and process conditions that sustain probiotic viability above 6 log CFU/mL throughout storage, with maintained sensory acceptability (mean overall score ?6.5 on a 9-point scale) and demonstrable probiotic resistance to simulated gastric and bile challenges. The study will establish a robust correlation between fermentation parameters and probiotic persistence, enabling predictive modeling of shelf performance. The contribution to knowledge lies in providing a scientifically validated, scalable approach to integrating fermentation-derived probiotics into traditional Cambodian fish sauce, informing standardization efforts, quality control protocols, and regulatory guidance. Practical implications include enhanced product value, potential health claims grounded in quantified viability and functionality, and a framework for similar fermented condiments in Southeast Asia. The main conclusion anticipates that controlled selection of starter cultures and fermentation conditions can reliably augment the probiotic content of Cambodian fish sauce without compromising sensory quality, offering actionable recommendations for producers, policymakers, and researchers, and suggesting avenues for future work on long-term health outcomes and regional market expansion.