Paraprobiotics retain their ability to counteract diet-induced dysbiosis during shelf-life: an efficacy study correlating fecal metabolomic and microbiome changes

The increasing interest in paraprobiotics has raised concerns about their stability and performance throughout their shelf life. This study investigated whether Bifidobacterium animalis BB-12 ( BB )-derived paraprobiotics ( BB P) retain their ability to restore gut microbiota (GM) balance and fecal metabolites in rats fed a high-fat diet (HFD) after various storage times. BB P was produced through ohmic heating (OH) processing, followed by freeze-drying and storing at −20°C, 4°C, and 25°C for 4 months. Flow cytometry (FC) was used to analyze BB P stability, while their biochemical, metabolomic, and microbiomic functionality were studied in vivo. Results showed that BB Ps stored at −20°C in powder form exhibited significantly higher stability. Administration of both fresh and stored BB Ps alleviated HFD-induced metabolic disorders. Compared with the HFD group, BBP administration at the start of storage (IP) declined serum total cholesterol and triglycerides by approximately 28% and 20%, respectively, and raised HDL by 22%. Furthermore, it significantly decreased serum AST and ALT activities by 16% and 38%, respectively. Additionally, it alleviated HFD-induced dysbiosis in rats by enhancing GM microbial diversity and the abundance of beneficial bacteria, such as Bacteroides and Akkermansia , even after prolonged storage. 1 H-NMR spectroscopy revealed enhanced levels of amino acids and short-chain fatty acids (SCFAs), with acetate and propionate concentrations increasing by up to 81% and 157%, respectively, while also indicating decreased fecal sugars and alcohols, confirming improved gut metabolic activity. Overall, BB Ps retained substantial bioactivity and partially maintained their ability to restore HFD-induced dysbiosis throughout storage, although functionality declined slightly over time.